PERSPECTIVE Edited by Milo Glbaldi
UNDERSTANDING AND TREATING SOME GENETIC DISEASES Milo Gibaldi
ADVANCES IN BIOTECHNOLOGY have resulted in intense efforts to better understand and treat inherited disorders. "Although these conditions are individually rare, X-linked and autosomal recessive genetic diseases combined affect more than one child in every 500 live births. These diseases carry a very high social and economic burden... [and] medical treatment remains limited."! This review discusses new replacement therapies for two genetic disorders, hereditary emphysema and Gaucher's disease, and summarizes some current ideas regarding the basis of cystic fibrosis (CF), including new treatment modalities that appear to be promising.
AlpharAntitrypsin: ReplacementTherapyfor Hereditary Emphysema Emphysema affects more than two million people in the US. About two percent of this population has an inherited form of the disease that is the result of a defective gene for synthesizing alphaj-antitrypsin (AAT), a glycoprotein composed of 394 amino acids with three complex carbohydrate side chains. AAT binds to and inhibits the action of proteindigesting enzymes. AAT deficiency is an autosomal recessive disorder, requiring for its development the inheritance of two defective copies of the gene, one from each parent. It is one of the most common lethal hereditary disorders of Caucasians of European descent.P Despite its name, the primary target of AAT is not trypsin but elastase, another protease. Elastase is stored in neutrophils and, as part of the white-blood-cell system, is protective against respiratory infections.' The principal site of AAT gene expression is the liver, which releases about 2 g of AAT into the circulation daily. AAT diffuses into most organs where it protects tissues from attack by the elastase that is released by activated or disintegrating neutrophils. The lungs are particularly in need of adequate levels of AAT. When there are insufficient concentrations of AAT to protect the lower respiratory tract from elastase, progressive destruction of alveoli ensues, culminating in emphysema.' In January 1989, the research news section of Science magazine noted that investigators at a recent symposium on the "Genetic Basis of Human Disease: Molecular Mecha-
MILO GIBALDI, Ph.D., is the Dean, School of Pharmacy, University of Washington, Seattle. WA 98915.
nisms and Strategies for Therapy" claimed to be able to correct the biochemical defect that causes progressive lung degeneration in patients with the hereditary form of emphysema. These investigators found that weekly intravenous injections of purified AAT prepared from human plasma can maintain effective concentrations of the inhibitor in the blood and lungs. Although treatment was not found to reverse existing damage, it did halt progression of the disease.' Based on this and other evidence, the Food and Drug Administration (FDA) quickly approved AAT therapy for patients with the genetic form of emphysema, but required that a national registry of patients receiving AAT be kept to facilitate a retrospective evaluation of its efficacy and safety. Because adult patients with AAT deficiency have a shortened life span, the question of whether AAT therapy prolongs patients' lives is of particular interest. Crystal has noted that "individuals with AAT deficiency known to have emphysema who are at least 18 years old have a 52% chance of being alive at age 50 (compared with a 93% chance for the general population) and a 16% chance of being alive at age 60 (compared with 85%)."3 Hubbard et al. have shown that aerosolized AAT can be recovered from the alveolar surface in an intact, active form, suggesting that inhalation therapy may be effective treatment for hereditary emphysema.' Another possibility for future treatment of this disease is the use of gene therapy to correct more permanently the AAT gene defect. Preclinical studies have shown that cloned human AAT gene can be inserted into mouse fibroblasts, which subsequently can produce the inhibitor protein in culture and in vivo.' Human AAT has been found to persist in mouse plasma and lung epithelial lining fluid for at least one month after transplantation of genetically modified fibroblasts into the peritoneal cavity.' Alternatively, it might be possible to provide augmentation of AAT in the lungs by inserting the human AAT gene into autologous bone marrow precursor cells. Following transplantation and maturation, precursor cells may result in repopulation of alveolar macrophages that are capable of producing AAT in the lower respiratory tract.'
Glucocerebrosidase: ReplacementTherapyfor Gaucher's Disease The breakdown of endogenous cellular components such as membrane lipids is carried out by lysosomes, minute en-
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velopes found in many types of cells that contain highly specific hydrolytic enzymes normally involved in intracellular digestion. Genetic deficiencies in anyone of these enzymes may prevent the breakdown of its substrates and lead to their accumulation. The most common of these disorders is Gaucher's disease, a lipidosis characterized by a deficiency of the enzyme glucocerebrosidase (GCase). Gaucher's disease is also an autosomal recessive disorder; it particularly affects Jews of Eastern European (Ashkenazi) descent. The lack of GCase results in accumulation of the glycolipid glucocerebroside (GC) in the liver, spleen, lymph nodes, alveolar capillaries, and bone marrow. Less common glycolipid storage diseases include Tay-Sachs disease, Fabry's disease, and Niemann-Pick disease.' According to Beutler, "Although GCase deficiency exists in all body cells of patients with Gaucher disease, the disease phenotype is expressed only in the macrophages .... As a consequence, patients with Gaucher disease are burdened by an enlarged liver and spleen and often by painful bone lesions." Gaucher's disease was first described in 1882, but only in the last decade has identification of the lesions that cause this disorder and implementation of successful therapeutic approaches been possible with the application of modern techniques of cellular and molecular biology. The most promising intervention strategy has been enzyme replacement therapy. The idea of correcting the genetic defect in Gaucher's disease by supplying the missing enzyme is not new, but difficulties in producing large amounts of GCase and efficiently delivering it to the target cells (macrophages) have only recently been overcome. A solution to the delivery problem lay in the discovery of mannose receptors on macrophages. With the availability of this information, Genzyme has successfully purified human placental GCase on an industrial scale and has modified it by removing outer sugars to expose additional mannose residues, thereby increasing target specificity without decreasing enzyme activity. Genzyme's modified GCase, known as alglucerase, was approved by the FDA in 1991. The agency considered alglucerase to be an important therapeutic advance and the product received an expedited review. According to a report in an international trade paper, the basis for approval was a pivotal trial in 12 patients with Gaucher's disease who received intravenous infusions of alglucerase 60 units/kg over one to two hours, every two weeks for 9-12 months. Treatment increased hemoglobin and platelet concentrations, decreased acid phosphatase and plasma GC concentrations, and caused significant reductions in liver and spleen volumes. Skeletal improvements also were observed in some patients. The drug's sponsor stated that these improvements were still evident after two years of enzyme replacement therapy.' The world's supply of alglucerase is insufficient to treat all eligible patients. Furthermore, although no problems have yet been reported, there is concern that material prepared from pooled human placental tissue may contain the causative agents of some viral diseases. Genzyme is developing a recombinant form of GCase in hopes of having a sufficient supply of the enzyme, reducing the cost of treatment, and eliminating the risk of viral contamination.' 1590 •
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At the recommended frequency of administration (i.e., 60 units/kg every two weeks), the annual cost per patient for alglucerase is about $380000; this cost is doubled in very sick patients who require weekly infusions of the enzyme.' Beutler has suggested that in some patients a dosage of 2.3 units/kg given three times per week may be as effective as the currently recommended dosage.' If this regimen were widely applicable, costs would fall by more than 75 percent. Even so, an annual drug cost in excess of $50 ()()() is imposing. Alglucerase has received attention from the media not only for its high cost, but also for its seeming profitability. Legislators, business leaders, consumer advocates, and other critics of the drug industry have expressed concerns about windfall profits from new drugs. These concerns are particularly acute with regard to orphan drugs. The Orphan Drug Act, designed to stimulate development of products used for the treatment of rare disorders such as Gaucher's disease, provides market incentives to increase profits and clarifies regulatory issues to decrease the expenses of companies that engage in such development. One of the act's major market incentives is a provision for exclusivity. Exclusive approval is awarded by the FDA to a sponsor for a specific orphan indication of a product. The law precludes FDA approval of the same product developed by a competing sponsor for the same indication for seven years. According to The New York Times, sales of alglucerase, an orphan drug, were $38 million in 19918 - a rather impressive figure when one considers that the drug was first approved in April of that same year. Genzyme told Congress that the research and development costs for alglucerase were in the range of $50-70 million, but filings with the Securities and Exchange Commission indicate that the figure is closer to $30 million. Sometime this year, a senate subcommittee is expected to adopt an amendment to the Orphan Drug Act that will cap the total revenues for an orphan drug at $200 million; sales in excess of this level will result in loss of tax incentives and the remaining period of exclusivity. New and Interesting Aspects in the Treatment o/Cystic Fibrosis
CF is an inherited, fatal disorder that affects children and young adults. It is the most common genetic disease among whites in the US. CF is inherited in an autosomal recessive fashion; heterozygotes, who carry one normal CF gene and one mutant gene, are asymptomatic but are carriers. The child of two carriers has a one-in-four chance of being affected with CF. The prevalence of the disease varies among ethnic groups and is highest among people of Northern European extraction.' Collins has observed that "The clinical features of CF are dominated by involvement of the respiratory tract, where obstruction of the airways by thick, sticky mucus and subsequent infection, especially with Pseudomonas species, predominate. There is also involvement of the gastrointestinal tract ... as a result of obstruction of the pancreatic ducts and subsequent scarring and destruction of exocrine function.?" Until recently, the management of CF was limited to physical therapy to improve the clearance of infected pul-
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monary secretions, antibiotic administration, pancreatic enzyme replacement, and close attention to diet. According to the Merck Manual, "Diet therapy includes (1) sufficient calories and protein to promote normal growth--exceeding usual Recommended Dietary Allowance requirements ... (2) a normal-to-high total fat intake to increase the caloric density of the diet, (3) multivitamins in double the recommended daily allowance, (4) supplemental vitamin E in water miscible form, and (5) salt supplementation during periods of thermal stress and increased sweating."? Increasingly assiduous implementation of this treatment plan has resulted in progressively improved survival over the past 40 years. Collins noted that "an individual born today with CF would be expected to survive about 40 years, even without further advances in therapy.'?" Future prospects for patients with CF are still brighter in light of a recent report describing the development of an animal model for this disease." PREVENTING CHRONIC RESPIRATORY INFECTION
The principal cause of the high morbidity and mortality among patients with CF is chronic pulmonary infection, frequently with Pseudomonas aeruginosa. Once established in the lower respiratory tract, this organism is virtually impossible to eradicate. To determine whether chronic infection with P. aeruginosa is preventable, Valerius et al. studied 26 patients with CF and a positive sputum culture who had never received antipseudomonal therapy. Patients were randomly assigned to groups receiving either no antipseudomonal therapy or oral ciprofloxacin plus aerosol inhalations of colistin, a polymyxin antibiotic, twice daily for three weeks. Treatment was repeated each time the organism was isolated from subsequent monthly sputum samples." During the 27 months of the trial, 58 percent of the untreated patients developed chronic P. aeruginosa infection compared with only 14 percent of the patients given antipseudomonal therapy. No serious adverse effects were reported and no resistance of the organism to ciprofloxacin was observed. The investigators concluded that chronic colonization with P. aeruginosa can be prevented in patients with CF by early institution of a regimen of antipseudomonal chemotherapy that does not interfere substantially with a patient's normal activities.'! This sanguine view, however, must be tempered by the fact that only a small number of patients were evaluated and that this work needs to be repeated in larger numbers of patients over a longer period of time before the prophylaxis suggested by these researchers can be accepted as routine treatment. A P. aeruginosa octavalent 0 -polysaccharide-toxin A conjugate vaccine has been developed and evaluated in healthy human subjects to determine its safety and immunogenicity. The vaccine stimulates the production of antibodies that eradicate the organism by binding and inducing phagocytosis by macrophages and neutrophils." Such antibodies could potentially prevent or retard colonization or disease progression. In order to evaluate its safety and immunogenicity in a high-risk population, Schaad et al. administered this vaccine to children with CF, but with no history of P. aeruginosa infection. These patients were followed for 14-28 months after primary immunization. Adverse reactions were mild or moderate, consisting largely of erythema,
pain, and swelling at the site of injection. No systemic reactions or changes in blood chemistry were observed.'! Immunization uniformly resulted in a significant increase in immunoglobulin G (IgG) antibodies that persisted for at least 12 months. After at least 3 months, however, antibody levels declined progressively. A booster dose given at 12 months resulted once again in a significant increase in IgG antibody concentrations. Before immunization, toxin A neutralizing antibodies were undetectable in most patients (15/22), but thereafter were measured in 20 of 22 patients. 14 The investigators concluded that immunization with P. aeruginosa conjugate A vaccine leads to high levels of binding and functional antibodies, which are long lived. The effectiveness of a booster dose in jogging immunologic memory is also important because patients with CF are at constant risk for P. aeruginosa infection. "That there was no evidence of clinical deterioration or acquisition of Ps aeruginosa in any patient was reassuring .... In view of our fmdings that the vaccine is safe and immunogenic, we believe that a study to evaluate the efficacy of the vaccine in non-colonised patients with CF is warranted.'?" TREATING THE MALABSORPTION PROBLEM
Digestion and absorption of nutrients in the small intestine depends on the secretion of pancreatic enzymes (lipase, protease, and amylase). Fat absorption also requires bile acids. Digestion takes place only if the duodenal contents are maintained at a neutral pH; the existence of a favorable pH depends on the secretion of bicarbonate, largely from the pancreas. "In cystic fibrosis reduced pancreatic bicarbonate secretion and increased gastric acid secretion lower the duodenal pH to a level where endogenous or administered pancreatic enzymes are inactivated, bile acids are precipitated and the whole process of fat absorption is disrupted.i'" Pancreatic enzyme supplements, extracted from hog pancreas, are widely used to treat malabsorption in patients with CF. Successful enzyme replacement relieves the diarrhea, bloating, and cramps associated with fat malabsorption. Two pancreatic enzyme supplements are available in the US: pancreatin and pancrelipase. On a weight basis, pancrelipase has 12 times the lipolytic activity of pancreatin and 4 times the protease and amylase content. Both are inactivated by gastric acid. Pancreatin is available in the form of conventional tablets as well as in capsules containing enteric-coated microspheres. Pancrelipase is available as a powder, conventional tablets and capsules, entericcoated tablets, and capsules containing enteric-coated particulates. Uncoated preparations of pancreatic enzymes are useful in infants and patients who have had a partial gastrectomy or vagotomy and gastroenterostomy. In both groups, the stomach secretes little acid and empties rapidly. Delayedrelease products are less susceptible to acid inactivation than are uncoated products, but enteric-coated tablets have been associated with poor bioavailability and capsules containing subdivided enteric-coated material are preferred." Hendeles et al. have documented treatment failure in three patients with CF who, after a successful response to one product, were switched to a different product. In each
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case, initial treatment with Pancrease, a commercial capsule marketed by McNeil Pharmaceutical and containing enteric-coated microspheres of pancrelipase, was satisfactory. When the prescription was refilled with a seemingly similar product from a different manufacturer, however, symptoms indicative of malabsorption were reported. In two cases, symptoms resolved when the patients were again treated with Pancrease. In the third case, symptoms resolved when the patient was prescribed Creon, a capsule containing enteric-eoated microspheres of pancreatin." When the various products were each exposed to simulated gastric fluid for one hour, the investigators found that there was no loss of lipolytic activity with Pancrease or Creon; conversely, the products that resulted in treatment failure exhibited virtually no activity. These fmdings indicate that the coating on the microspheres of some pancreatic enzyme products may be defective." Based on their results, Hendeles et al. suggested that physicians should mark their prescriptions for pancreatic enzymes "do not substitute," that pharmacists should not substitute one brand for another without advising the prescriber, and that the FDA should "institute meaningful regulation over this group of products including a requirement for documentation of bioequivalence.''" Published comments from the FDA following Hendeles et al. 's report provided the following information: "The FDA's inspection of the firm [manufacturing the ineffective product] revealed that there was no validation for the enteric coating process. Testing conducted by the firm confirmed that the enteric coating was ineffective. All lots were either subpotent or superpotent for one or more active ingredients ... [and] on June 14, 1989, the manufacturer voluntarily instituted a recall of the product. ..."17 In the future, pancreatic enzymes will be treated not as nutritional supplements but as drugs, and such products will be available only by prescription and must adhere to the rigorous standards applied to drug products. MANIPULATING SODIUM AND CHLORIDE TRANSPORT WITH AMILORIDE AND URIDINE TRIPHOSPHATE
The diagnosis of CF is suggested by clinical features and confirmed by demonstrating an excess concentration of salt in sweat. The salt transport defect in sweat glands is also present in the epithelial cells of the respiratory tract of patients with CE An excess of salt in cells leaching water out of mucus could explain why thick mucus accumulates in the lungs of these patients, leading to impaired mucociliary clearance, chronic infection, inflammatory response, and irreversible abnormal enlargement of the bronchi or
bronchioles." In 1989, a gene controlling chloride transport across membranes was identified. It was quickly dubbed the CF gene and the protein encoded by the gene was named the CF transmembrane conductance regulator (CFTR). Patients with CF have an abnormal CF gene and produce a defective CFTR protein resulting in impaired salt trans-
port," The absence of normal CFTR protein results in an abnormal increase in sodium flux into airway epithelial cells. Water moves with salt across the airway epithelium so that increased sodium reabsorption is combined with decreased chloride secretion. Both the increased sodium reabsorption and the decreased chloride secretion can be manipulated. 1592
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Sodium reabsorption can be checked with a sodium-channel blocker such as amiloride and chloride secretion can be stimulated by adenosine triphosphate or uri dine triphosphate (UTP), nucleotides that activate a chloride channel distinct from that affected by CEIS As a result of findings suggesting that the inhalation of amiloride may improve the viscosity, elasticity, and clearance of secretions and improve airflow in patients with CF,19 Knowles et al. designed a double-blind crossover trial to evaluate the safety and efficacy of long-term treatment with aerosolized amiloride for airway disease resulting from CE All patients were first treated for 10-14 days with parenteral tobramycin and ceftazidime to standardize the recent use of antibiotics; they then were randomized to receive either nebulized amiloride or saline four times a day for 25 days. The crossover took place after a two-tofour-week washout period and another course of antibi-
otics." Fourteen patients completed the one-year trial. Amiloride slowed the decline in pulmonary function associated with CE The rate of loss of forced vital capacity was reduced by about 50 percent during the amiloride period compared with the vehicle period. Amiloride also improved measured indices of sputum viscosity and elasticity as well as those of mucociliary and cough clearance. No pulmonary or systemic toxicity was observed during treatment. The investigators concluded that their study "provides preliminary evidence that arniloride may be moderately effective in the treatment of adults with cystic fibrosis and established lung disease.?" According to the Pink Sheet." Glaxo has acquired the rights for the use of an aerosolized formulation of arniloride in CF, an orphan indication. A second potential ion-channel intervention has emerged from experiments designed to improve chloride secretion in patients with CE Agents that stimulate chloride secretion via pathways mediated by cyclic adenosine monophosphate (cAMP) in lung epithelia, such as beta-agonists, are ineffective in patients with CF, suggesting that a normal gene is required to activate these pathways." Attention has therefore been directed to agents that stimulate chloride secretion in epithelia via cAMP-independent pathways. This approach has led to the recognition that extracellular triphosphate nucleotides stimulate chloride secretion when applied to the apical (ciliated) surface of cultured human airway epithelia." As a result of these findings, Knowles et al. set out to determine whether triphosphate nucleotides, specifically adenosine triphosphate (ATP) and UTP, are capable of enhancing chloride secretion in normal subjects and in patients with CE They measured the in vivo effects of ATP and UTP on the transepithelial potential difference (PO) across the nasal mucosa, noting that "in human-airway epithelia, the rate of sodium absorption and of chloride secretion correlates with the absolute magnitude of the transepithelial PD. Changes in the PO in response to pharmacological agents provide evidence of changes in the rate of ion transport and in the types of ions transported." Cultured nasal epithelia from patients with CF and from healthy subjects was used to measure changes in transepithelial bioelectric properties and the permeability of the apical membrane to chloride in response to extracellular UTP. The in vivo experiments showed that ATP and UTP stimulated chloride secretion in both groups of subjects but
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were more effective in patients with CF than in normal subjects. In cell cultures, UTP was again found to effect a greater increase in the transepithelial PO and chloride secretory current, reflecting changes in permeability of the apical membrane to chloride, in nasal epithelial cells from patients with CF versus those from healthy subjects. The investigators noted that the maximally effective concentrations of ATP and UTP can easily be achieved on airway surfaces with the use of current aerosol technology." In both the in vitro and in vivo studies, it was necessary to pretreat the nasal epithelium with amiloride because activation of chloride channels by nucleotides in the absence of amiloride did not induce chloride secretion. In light of these results, Knowles et al. observed: "Because nucleotide therapy in cystic fibrosis would be designed to initiate the secretion of chloride (and water) in the direction of the lumen, a combination of a sodium-channel blocker and a nucleotide will probably be required for therapy."24 DECREASING MUCUS VISCOSITY WITH RECOMBINANT HUMAN DEOXYRIBONUCLEASE
Some investigators are taking a different approach to the treatment of CF and evaluating the benefits of recombinant human deoxyribonuclease (rhDNase). Collins has pointed out that "One of the major clinical problems in CF patients is the high viscosity of the large volumes of mucus produced by CF patients. This mucus is heavily infected with bacterial organisms and contains numerous white blood cells, many of which die and release their DNA, further contributing to the high viscosity.'?" Digestion of the deoxyribonucleic acid (DNA) component of mucus might reduce viscosity and lead to better mucociliary clearance and less airflow resistance. To evaluate the potential clinical utility of rhDNase in treating CF, Shak et al. cloned, sequenced, and expressed the enzyme. They found that small amounts of rhDNase greatly reduced the viscosity of purulent CF sputum, transforming it from a nonflowing viscous gel into a flowing liquid." Later, Hubbard et al. administered rhDNase by means of a jet nebulizer to patients with CF, initially in opendose-ranging and safety studies and later in a placebo-controlled crossover study. Either rhDNase 10 mg or placebo was administered twice a day.26 No significant adverse effects attributable to therapy were observed in the safety study even at dosages of up to 20 mg twice daily. Eleven patients completed the placebocontrolled trial. Nebulized rhDNase, but not placebo, significantly improved forced vital capacity and forced expiratory volume in one second throughout the six-day treatment period." This preliminary study demonstrates that treatment with aerosolized rhDNase is an effective way of improving lung function in patients with CF. "The mechanism of action of the rhDNase in improving lung function in patients with cystic fibrosis is probably related to its ability to cleave high-molecular weight DNA in purulent secretions in the airways .... Because of its mechanism of action, rhDNase is likely to be effective only in patients with purulent sputum."26 In a subsequent letter to the New England Journal of Medicine, responding to a letter from Rubin" criticizing some aspects of their work, Hubbard et al. stated that "Al-
though it is tempting to speculate that the specificity of rhDNase for cleaving DNA improves the rheologic or adhesive properties (or both) of purulent mucus in vivo, we agree with Dr. Rubin that the specific alterations produced in purulent mucus by rhDNase that are critical for obtaining clinical benefit are not known."28 More evidence of aerosolized rhDNase's promise for the short-term management of airflow limitations in patients with CF is found in a recent report by Aitken et al., who also evaluated the safety of rhDNase in normal subjects and in patients with CF. The study design was a repetitive dose escalation of aerosolized enzyme given three times a day for five days followed by a two-day washout
period." Inhalation of rhDNase in daily doses of up to 30 mg was well tolerated by all subjects. A single challenge dose administered 21 days after the last dose of rhDNase had no effect, consistent with the absence of serum anti-DNase antibodies. Although the study was not designed to establish efficacy, an improvement in pulmonary function associated with an improvement in dyspnea was observed in patients with CF.29 The Pink Sheet reported that rhDNase is on a fast track for filing a product licensing application with the FDA's Center for Biologics Evaluation and Research. The drug's sponsor, Genentech, hopes to submit the application in early 1993. A pivotal Phase ill trial initiated in late 1991 now has full enrollment. The trial is designed to determine if the enzyme can reduce the number of days of hospitalization and the need for physical chest therapy and antibiotic treatment, as well as to confirm the effects of rhDNase on pulmonary function." The Pink Sheet also noted that the potential market for rhDNase is much larger than the estimated 15000 patients with CF in the US. Moreover, Genentech is developing the product to reduce the viscosity of sputum in patients with chronic bronchitis. The subset of patients with chronic bronchitis that might benefit from treatment with rhDNase is estimated at 500 000. 30 PROTECTION AGAINST ELASTASE
Earlier, the potential effectiveness of AAT as replacement therapy in hereditary emphysema was described. This enzyme may also be useful in treating CF. Davis has pointed out that neutrophil elastase is also a villain in the lungs of patients with CF.IS It may be the predominant local factor inhibiting host defenses against Pseudomonas. Neutrophil elastase not only injures normal respiratory tissues, but also cleaves immunoglobulins and complement and prevents phagocytosis and killing of Pseudomonas organisms by neutrophils. A range of natural and synthetic inhibitors of elastase are under investigation. Among them is the irreversible elastase inhibitor AAT, the main inhibitor of neutrophil elastase in normal lungs. IS McElvaney et al. administered AAT via aerosol every 12 hours for one week to healthy subjects and to patients with CF. Baseline measurements indicated that AAT concentrations in respiratory epithelial lining fluid (ELF) were similar in normal subjects and those with CF. However, no neutrophil elastase was detectable in normal ELF, but large amounts were detectable in the ELF of CF patients. Treatment significantly elevated ELF AAT concentrations an concentrations increased with dose. Neutrophil elastase ac-
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tivity was completely inhibited when ELF AAT concentrations reached 8 umol/L, Neutrophils exposed to the ELF of patients with CF were not effective in killing Pseudomonas; however, neutrophils exposed to the ELF of patients with CF receiving treatment with AAT retained their effectiveness in clearing the organism." McElvaney et al. concluded that treatment with aerosolized AAT "might effectively prevent damage to the normal respiratory tissues by neutrophil elastase and augment host defence on the respiratory epithelial surface, thus potentially reversing a major problem in cystic fibrosis. The results are sufficiently convincing to support controlled clinical trials of AAT and other anti-neutrophil elastases."31~
References I. Cournoyer D, Scarpa M, Jones SN, Moore KA, Belmont JW, Caskey CT. Gene therapy: a new approach for the treatment of genetic disorders. Clin PharmacolTher 1990;47:1-11. 2. Marx JL. Getting to the heart of genetic disease. Science 1989;243:315-6. 3. Crystal RG. Alphaj-antitrypsin deficiency, emphysema, liver disease: genetic basis and strategies for therapy. 1 Clin invest 1990;85:1343-52. 4. Hubbard RC, Brantly ML, Sellers SE, Michell ME, Crystal RG. Delivery of proteins for therapeutic purposes by aerosolization: direct augmentation of anti-neutrophil elastase defenses of the lower respiratory tract in alphaj-antitrypsin deficiency with an aerosol of alphaj-antitrypsin. Ann Intern Med 1989;111:206-12. 5. Hussar DA. New drugs of 1991. Am Pharm 1992;NS32(4):317-50. 6. Beutler E. Gaucher disease: new molecular approaches to diagnosis and treatment. Science 1992;256:794-9. 7. Genzyme files for Ceredase in Europe. Scrip 1992;(Apr 15):17. 8. Hilts PJ. Seeking limits to a drug monopoly. New York Times 1992 May 12; Sect C:1. 9. Cystic fibrosis. In: Berkow R, ed. Merck manual of diagnosis and therapy. Rahway, NJ: Merck Sharp & Dohme Research Laboratories, 1987: 2055-9. 10. Collins F. Cystic fibrosis: molecular biology and therapeutic implications. Science 1992;256:774-9. 11. Snouwaert IN, Brigman KK, Latour AM, MaloufNN, Boucher RC, Smithies 0, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992;257: 1083-8. 12. Valerius NH, Koch C, Hoiby N. Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment. Lancet 1991;338:725-6. 13. Cryz SJ Jr, Sadoff JC, Cross AS, Furer E. Safety and immunogenicity of a polyvalent Pseudomonas aeruginosa O-polysaccharide-toxin A vaccine in humans. AntibiotChemother 1989;42: 177-83. 14. Schaad VB, Lang AB, Wedgwood J, Ruedeberg A, Que JV, Furer
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E, et al. Safety and immunogenicity of Pseudomonasaeruginosa conjugate A vaccine in cystic fibrosis. Lancet 1991;338:1236-7. 15. Choosing and using a pancreatic enzyme supplement. Drug Ther Bull 1992;30:37-40. 16. Hendeles L, Dorf A, Stecenko A, Weinberger M. Treatment failure after substitution of generic pancrelipase capsules. Correlation with in vitro lipase activity. lAMA 1990;263:2459-61. 17. Rheinstein PH. Regulatory status of pancreatic enzyme preparations.
lAMA 1990;263:2491-2. 18. Davis PB. Cystic fibrosis from bench to bedside. N Engl 1 Med 1991; 325:575-7. 19. Waltner WE, Boucher RC, Gatzy JT, Knowles MR. Pharmacotherapy of airway disease in cystic fibrosis. Trends Pharmacol Sci 1987;8: 316-20. 20. Knowles MR, Church NL, Waltner WE, Yankaskas JR, Gilligan P, King M, et aI. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl 1 Med 1990;322: 1189-94. 21. Aerosolized amiloride plus ATP or UTP for cystic fibrosis. FDC Reports.Pink Sheet 1991;(Aug 26):T&G5-6. 22. Hwang T-C, Zeitlin PL, Gruenert DC, Huganir R, Cuggino WB. C( channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase. Science 1989;244: 1351-3. 23. Mason SJ, Paradiso AM, Boucher RC. Regulation of transepithelial ion transport and intracellular calcium by extracellular adenosine triphosphate in human normal and cystic fibrosis airway epithelium. Br
1 Pharmacoll991;103:1649-56. 24. Knowles MR, Clarke LL, Boucher RC. Activation by extracellular nuc1eotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl 1 Med 1991;325:533-8. 25. Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.
Proc Natl Acad Sci USA 1990;87:9188-92. 26. Hubbard RC, McElvaney NG, Blrrer P, Shak S, Robinson WW, Jolley C, et aI. A preliminary study of aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis. N Engl 1 Med 1992;326:812-5. 27. Rubin BK. Aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis (letter). N Engl 1 Med 1992;327:571. 28. Hubbard RC, Shak S, Crystal RG. Aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis (letter). N Engl 1
Med 1992;327:571. 29. Aitken ML, Burke W, McDonald G, Shak S, Montgomery AB, Smith A. Recombinant human DNase inhalation in normal subjects and patients with cystic fibrosis: a phase I study. lAMA 1992;267: 1947-51. 30. DNase on fast-pace for cystic fibrosis PLA filing: application to FDA planned for early 1993; DNase will be biotech's "next major product," Genentech says. FDC Reports.Pink Sheet 1992;(May 11):9-10. 31. McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, et aI. Aerosol alpha.-antitrypsin treatment for cystic fibrosis. Lancet 1991;337:392-4.
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UNDERSTANDING AND TREATING SOME GENETIC DISEASES Milo Gibaldi
ADVANCES IN BIOTECHNOLOGY have resulted in intense efforts to better understand and treat inherited disorders. "Although these conditions are individually rare, X-linked and autosomal recessive genetic diseases combined affect more than one child in every 500 live births. These diseases carry a very high social and economic burden... [and] medical treatment remains limited."! This review discusses new replacement therapies for two genetic disorders, hereditary emphysema and Gaucher's disease, and summarizes some current ideas regarding the basis of cystic fibrosis (CF), including new treatment modalities that appear to be promising.
AlpharAntitrypsin: ReplacementTherapyfor Hereditary Emphysema Emphysema affects more than two million people in the US. About two percent of this population has an inherited form of the disease that is the result of a defective gene for synthesizing alphaj-antitrypsin (AAT), a glycoprotein composed of 394 amino acids with three complex carbohydrate side chains. AAT binds to and inhibits the action of proteindigesting enzymes. AAT deficiency is an autosomal recessive disorder, requiring for its development the inheritance of two defective copies of the gene, one from each parent. It is one of the most common lethal hereditary disorders of Caucasians of European descent.P Despite its name, the primary target of AAT is not trypsin but elastase, another protease. Elastase is stored in neutrophils and, as part of the white-blood-cell system, is protective against respiratory infections.' The principal site of AAT gene expression is the liver, which releases about 2 g of AAT into the circulation daily. AAT diffuses into most organs where it protects tissues from attack by the elastase that is released by activated or disintegrating neutrophils. The lungs are particularly in need of adequate levels of AAT. When there are insufficient concentrations of AAT to protect the lower respiratory tract from elastase, progressive destruction of alveoli ensues, culminating in emphysema.' In January 1989, the research news section of Science magazine noted that investigators at a recent symposium on the "Genetic Basis of Human Disease: Molecular Mecha-
MILO GIBALDI, Ph.D., is the Dean, School of Pharmacy, University of Washington, Seattle. WA 98915.
nisms and Strategies for Therapy" claimed to be able to correct the biochemical defect that causes progressive lung degeneration in patients with the hereditary form of emphysema. These investigators found that weekly intravenous injections of purified AAT prepared from human plasma can maintain effective concentrations of the inhibitor in the blood and lungs. Although treatment was not found to reverse existing damage, it did halt progression of the disease.' Based on this and other evidence, the Food and Drug Administration (FDA) quickly approved AAT therapy for patients with the genetic form of emphysema, but required that a national registry of patients receiving AAT be kept to facilitate a retrospective evaluation of its efficacy and safety. Because adult patients with AAT deficiency have a shortened life span, the question of whether AAT therapy prolongs patients' lives is of particular interest. Crystal has noted that "individuals with AAT deficiency known to have emphysema who are at least 18 years old have a 52% chance of being alive at age 50 (compared with a 93% chance for the general population) and a 16% chance of being alive at age 60 (compared with 85%)."3 Hubbard et al. have shown that aerosolized AAT can be recovered from the alveolar surface in an intact, active form, suggesting that inhalation therapy may be effective treatment for hereditary emphysema.' Another possibility for future treatment of this disease is the use of gene therapy to correct more permanently the AAT gene defect. Preclinical studies have shown that cloned human AAT gene can be inserted into mouse fibroblasts, which subsequently can produce the inhibitor protein in culture and in vivo.' Human AAT has been found to persist in mouse plasma and lung epithelial lining fluid for at least one month after transplantation of genetically modified fibroblasts into the peritoneal cavity.' Alternatively, it might be possible to provide augmentation of AAT in the lungs by inserting the human AAT gene into autologous bone marrow precursor cells. Following transplantation and maturation, precursor cells may result in repopulation of alveolar macrophages that are capable of producing AAT in the lower respiratory tract.'
Glucocerebrosidase: ReplacementTherapyfor Gaucher's Disease The breakdown of endogenous cellular components such as membrane lipids is carried out by lysosomes, minute en-
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velopes found in many types of cells that contain highly specific hydrolytic enzymes normally involved in intracellular digestion. Genetic deficiencies in anyone of these enzymes may prevent the breakdown of its substrates and lead to their accumulation. The most common of these disorders is Gaucher's disease, a lipidosis characterized by a deficiency of the enzyme glucocerebrosidase (GCase). Gaucher's disease is also an autosomal recessive disorder; it particularly affects Jews of Eastern European (Ashkenazi) descent. The lack of GCase results in accumulation of the glycolipid glucocerebroside (GC) in the liver, spleen, lymph nodes, alveolar capillaries, and bone marrow. Less common glycolipid storage diseases include Tay-Sachs disease, Fabry's disease, and Niemann-Pick disease.' According to Beutler, "Although GCase deficiency exists in all body cells of patients with Gaucher disease, the disease phenotype is expressed only in the macrophages .... As a consequence, patients with Gaucher disease are burdened by an enlarged liver and spleen and often by painful bone lesions." Gaucher's disease was first described in 1882, but only in the last decade has identification of the lesions that cause this disorder and implementation of successful therapeutic approaches been possible with the application of modern techniques of cellular and molecular biology. The most promising intervention strategy has been enzyme replacement therapy. The idea of correcting the genetic defect in Gaucher's disease by supplying the missing enzyme is not new, but difficulties in producing large amounts of GCase and efficiently delivering it to the target cells (macrophages) have only recently been overcome. A solution to the delivery problem lay in the discovery of mannose receptors on macrophages. With the availability of this information, Genzyme has successfully purified human placental GCase on an industrial scale and has modified it by removing outer sugars to expose additional mannose residues, thereby increasing target specificity without decreasing enzyme activity. Genzyme's modified GCase, known as alglucerase, was approved by the FDA in 1991. The agency considered alglucerase to be an important therapeutic advance and the product received an expedited review. According to a report in an international trade paper, the basis for approval was a pivotal trial in 12 patients with Gaucher's disease who received intravenous infusions of alglucerase 60 units/kg over one to two hours, every two weeks for 9-12 months. Treatment increased hemoglobin and platelet concentrations, decreased acid phosphatase and plasma GC concentrations, and caused significant reductions in liver and spleen volumes. Skeletal improvements also were observed in some patients. The drug's sponsor stated that these improvements were still evident after two years of enzyme replacement therapy.' The world's supply of alglucerase is insufficient to treat all eligible patients. Furthermore, although no problems have yet been reported, there is concern that material prepared from pooled human placental tissue may contain the causative agents of some viral diseases. Genzyme is developing a recombinant form of GCase in hopes of having a sufficient supply of the enzyme, reducing the cost of treatment, and eliminating the risk of viral contamination.' 1590 •
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At the recommended frequency of administration (i.e., 60 units/kg every two weeks), the annual cost per patient for alglucerase is about $380000; this cost is doubled in very sick patients who require weekly infusions of the enzyme.' Beutler has suggested that in some patients a dosage of 2.3 units/kg given three times per week may be as effective as the currently recommended dosage.' If this regimen were widely applicable, costs would fall by more than 75 percent. Even so, an annual drug cost in excess of $50 ()()() is imposing. Alglucerase has received attention from the media not only for its high cost, but also for its seeming profitability. Legislators, business leaders, consumer advocates, and other critics of the drug industry have expressed concerns about windfall profits from new drugs. These concerns are particularly acute with regard to orphan drugs. The Orphan Drug Act, designed to stimulate development of products used for the treatment of rare disorders such as Gaucher's disease, provides market incentives to increase profits and clarifies regulatory issues to decrease the expenses of companies that engage in such development. One of the act's major market incentives is a provision for exclusivity. Exclusive approval is awarded by the FDA to a sponsor for a specific orphan indication of a product. The law precludes FDA approval of the same product developed by a competing sponsor for the same indication for seven years. According to The New York Times, sales of alglucerase, an orphan drug, were $38 million in 19918 - a rather impressive figure when one considers that the drug was first approved in April of that same year. Genzyme told Congress that the research and development costs for alglucerase were in the range of $50-70 million, but filings with the Securities and Exchange Commission indicate that the figure is closer to $30 million. Sometime this year, a senate subcommittee is expected to adopt an amendment to the Orphan Drug Act that will cap the total revenues for an orphan drug at $200 million; sales in excess of this level will result in loss of tax incentives and the remaining period of exclusivity. New and Interesting Aspects in the Treatment o/Cystic Fibrosis
CF is an inherited, fatal disorder that affects children and young adults. It is the most common genetic disease among whites in the US. CF is inherited in an autosomal recessive fashion; heterozygotes, who carry one normal CF gene and one mutant gene, are asymptomatic but are carriers. The child of two carriers has a one-in-four chance of being affected with CF. The prevalence of the disease varies among ethnic groups and is highest among people of Northern European extraction.' Collins has observed that "The clinical features of CF are dominated by involvement of the respiratory tract, where obstruction of the airways by thick, sticky mucus and subsequent infection, especially with Pseudomonas species, predominate. There is also involvement of the gastrointestinal tract ... as a result of obstruction of the pancreatic ducts and subsequent scarring and destruction of exocrine function.?" Until recently, the management of CF was limited to physical therapy to improve the clearance of infected pul-
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monary secretions, antibiotic administration, pancreatic enzyme replacement, and close attention to diet. According to the Merck Manual, "Diet therapy includes (1) sufficient calories and protein to promote normal growth--exceeding usual Recommended Dietary Allowance requirements ... (2) a normal-to-high total fat intake to increase the caloric density of the diet, (3) multivitamins in double the recommended daily allowance, (4) supplemental vitamin E in water miscible form, and (5) salt supplementation during periods of thermal stress and increased sweating."? Increasingly assiduous implementation of this treatment plan has resulted in progressively improved survival over the past 40 years. Collins noted that "an individual born today with CF would be expected to survive about 40 years, even without further advances in therapy.'?" Future prospects for patients with CF are still brighter in light of a recent report describing the development of an animal model for this disease." PREVENTING CHRONIC RESPIRATORY INFECTION
The principal cause of the high morbidity and mortality among patients with CF is chronic pulmonary infection, frequently with Pseudomonas aeruginosa. Once established in the lower respiratory tract, this organism is virtually impossible to eradicate. To determine whether chronic infection with P. aeruginosa is preventable, Valerius et al. studied 26 patients with CF and a positive sputum culture who had never received antipseudomonal therapy. Patients were randomly assigned to groups receiving either no antipseudomonal therapy or oral ciprofloxacin plus aerosol inhalations of colistin, a polymyxin antibiotic, twice daily for three weeks. Treatment was repeated each time the organism was isolated from subsequent monthly sputum samples." During the 27 months of the trial, 58 percent of the untreated patients developed chronic P. aeruginosa infection compared with only 14 percent of the patients given antipseudomonal therapy. No serious adverse effects were reported and no resistance of the organism to ciprofloxacin was observed. The investigators concluded that chronic colonization with P. aeruginosa can be prevented in patients with CF by early institution of a regimen of antipseudomonal chemotherapy that does not interfere substantially with a patient's normal activities.'! This sanguine view, however, must be tempered by the fact that only a small number of patients were evaluated and that this work needs to be repeated in larger numbers of patients over a longer period of time before the prophylaxis suggested by these researchers can be accepted as routine treatment. A P. aeruginosa octavalent 0 -polysaccharide-toxin A conjugate vaccine has been developed and evaluated in healthy human subjects to determine its safety and immunogenicity. The vaccine stimulates the production of antibodies that eradicate the organism by binding and inducing phagocytosis by macrophages and neutrophils." Such antibodies could potentially prevent or retard colonization or disease progression. In order to evaluate its safety and immunogenicity in a high-risk population, Schaad et al. administered this vaccine to children with CF, but with no history of P. aeruginosa infection. These patients were followed for 14-28 months after primary immunization. Adverse reactions were mild or moderate, consisting largely of erythema,
pain, and swelling at the site of injection. No systemic reactions or changes in blood chemistry were observed.'! Immunization uniformly resulted in a significant increase in immunoglobulin G (IgG) antibodies that persisted for at least 12 months. After at least 3 months, however, antibody levels declined progressively. A booster dose given at 12 months resulted once again in a significant increase in IgG antibody concentrations. Before immunization, toxin A neutralizing antibodies were undetectable in most patients (15/22), but thereafter were measured in 20 of 22 patients. 14 The investigators concluded that immunization with P. aeruginosa conjugate A vaccine leads to high levels of binding and functional antibodies, which are long lived. The effectiveness of a booster dose in jogging immunologic memory is also important because patients with CF are at constant risk for P. aeruginosa infection. "That there was no evidence of clinical deterioration or acquisition of Ps aeruginosa in any patient was reassuring .... In view of our fmdings that the vaccine is safe and immunogenic, we believe that a study to evaluate the efficacy of the vaccine in non-colonised patients with CF is warranted.'?" TREATING THE MALABSORPTION PROBLEM
Digestion and absorption of nutrients in the small intestine depends on the secretion of pancreatic enzymes (lipase, protease, and amylase). Fat absorption also requires bile acids. Digestion takes place only if the duodenal contents are maintained at a neutral pH; the existence of a favorable pH depends on the secretion of bicarbonate, largely from the pancreas. "In cystic fibrosis reduced pancreatic bicarbonate secretion and increased gastric acid secretion lower the duodenal pH to a level where endogenous or administered pancreatic enzymes are inactivated, bile acids are precipitated and the whole process of fat absorption is disrupted.i'" Pancreatic enzyme supplements, extracted from hog pancreas, are widely used to treat malabsorption in patients with CF. Successful enzyme replacement relieves the diarrhea, bloating, and cramps associated with fat malabsorption. Two pancreatic enzyme supplements are available in the US: pancreatin and pancrelipase. On a weight basis, pancrelipase has 12 times the lipolytic activity of pancreatin and 4 times the protease and amylase content. Both are inactivated by gastric acid. Pancreatin is available in the form of conventional tablets as well as in capsules containing enteric-coated microspheres. Pancrelipase is available as a powder, conventional tablets and capsules, entericcoated tablets, and capsules containing enteric-coated particulates. Uncoated preparations of pancreatic enzymes are useful in infants and patients who have had a partial gastrectomy or vagotomy and gastroenterostomy. In both groups, the stomach secretes little acid and empties rapidly. Delayedrelease products are less susceptible to acid inactivation than are uncoated products, but enteric-coated tablets have been associated with poor bioavailability and capsules containing subdivided enteric-coated material are preferred." Hendeles et al. have documented treatment failure in three patients with CF who, after a successful response to one product, were switched to a different product. In each
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case, initial treatment with Pancrease, a commercial capsule marketed by McNeil Pharmaceutical and containing enteric-coated microspheres of pancrelipase, was satisfactory. When the prescription was refilled with a seemingly similar product from a different manufacturer, however, symptoms indicative of malabsorption were reported. In two cases, symptoms resolved when the patients were again treated with Pancrease. In the third case, symptoms resolved when the patient was prescribed Creon, a capsule containing enteric-eoated microspheres of pancreatin." When the various products were each exposed to simulated gastric fluid for one hour, the investigators found that there was no loss of lipolytic activity with Pancrease or Creon; conversely, the products that resulted in treatment failure exhibited virtually no activity. These fmdings indicate that the coating on the microspheres of some pancreatic enzyme products may be defective." Based on their results, Hendeles et al. suggested that physicians should mark their prescriptions for pancreatic enzymes "do not substitute," that pharmacists should not substitute one brand for another without advising the prescriber, and that the FDA should "institute meaningful regulation over this group of products including a requirement for documentation of bioequivalence.''" Published comments from the FDA following Hendeles et al. 's report provided the following information: "The FDA's inspection of the firm [manufacturing the ineffective product] revealed that there was no validation for the enteric coating process. Testing conducted by the firm confirmed that the enteric coating was ineffective. All lots were either subpotent or superpotent for one or more active ingredients ... [and] on June 14, 1989, the manufacturer voluntarily instituted a recall of the product. ..."17 In the future, pancreatic enzymes will be treated not as nutritional supplements but as drugs, and such products will be available only by prescription and must adhere to the rigorous standards applied to drug products. MANIPULATING SODIUM AND CHLORIDE TRANSPORT WITH AMILORIDE AND URIDINE TRIPHOSPHATE
The diagnosis of CF is suggested by clinical features and confirmed by demonstrating an excess concentration of salt in sweat. The salt transport defect in sweat glands is also present in the epithelial cells of the respiratory tract of patients with CE An excess of salt in cells leaching water out of mucus could explain why thick mucus accumulates in the lungs of these patients, leading to impaired mucociliary clearance, chronic infection, inflammatory response, and irreversible abnormal enlargement of the bronchi or
bronchioles." In 1989, a gene controlling chloride transport across membranes was identified. It was quickly dubbed the CF gene and the protein encoded by the gene was named the CF transmembrane conductance regulator (CFTR). Patients with CF have an abnormal CF gene and produce a defective CFTR protein resulting in impaired salt trans-
port," The absence of normal CFTR protein results in an abnormal increase in sodium flux into airway epithelial cells. Water moves with salt across the airway epithelium so that increased sodium reabsorption is combined with decreased chloride secretion. Both the increased sodium reabsorption and the decreased chloride secretion can be manipulated. 1592
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Sodium reabsorption can be checked with a sodium-channel blocker such as amiloride and chloride secretion can be stimulated by adenosine triphosphate or uri dine triphosphate (UTP), nucleotides that activate a chloride channel distinct from that affected by CEIS As a result of findings suggesting that the inhalation of amiloride may improve the viscosity, elasticity, and clearance of secretions and improve airflow in patients with CF,19 Knowles et al. designed a double-blind crossover trial to evaluate the safety and efficacy of long-term treatment with aerosolized amiloride for airway disease resulting from CE All patients were first treated for 10-14 days with parenteral tobramycin and ceftazidime to standardize the recent use of antibiotics; they then were randomized to receive either nebulized amiloride or saline four times a day for 25 days. The crossover took place after a two-tofour-week washout period and another course of antibi-
otics." Fourteen patients completed the one-year trial. Amiloride slowed the decline in pulmonary function associated with CE The rate of loss of forced vital capacity was reduced by about 50 percent during the amiloride period compared with the vehicle period. Amiloride also improved measured indices of sputum viscosity and elasticity as well as those of mucociliary and cough clearance. No pulmonary or systemic toxicity was observed during treatment. The investigators concluded that their study "provides preliminary evidence that arniloride may be moderately effective in the treatment of adults with cystic fibrosis and established lung disease.?" According to the Pink Sheet." Glaxo has acquired the rights for the use of an aerosolized formulation of arniloride in CF, an orphan indication. A second potential ion-channel intervention has emerged from experiments designed to improve chloride secretion in patients with CE Agents that stimulate chloride secretion via pathways mediated by cyclic adenosine monophosphate (cAMP) in lung epithelia, such as beta-agonists, are ineffective in patients with CF, suggesting that a normal gene is required to activate these pathways." Attention has therefore been directed to agents that stimulate chloride secretion in epithelia via cAMP-independent pathways. This approach has led to the recognition that extracellular triphosphate nucleotides stimulate chloride secretion when applied to the apical (ciliated) surface of cultured human airway epithelia." As a result of these findings, Knowles et al. set out to determine whether triphosphate nucleotides, specifically adenosine triphosphate (ATP) and UTP, are capable of enhancing chloride secretion in normal subjects and in patients with CE They measured the in vivo effects of ATP and UTP on the transepithelial potential difference (PO) across the nasal mucosa, noting that "in human-airway epithelia, the rate of sodium absorption and of chloride secretion correlates with the absolute magnitude of the transepithelial PD. Changes in the PO in response to pharmacological agents provide evidence of changes in the rate of ion transport and in the types of ions transported." Cultured nasal epithelia from patients with CF and from healthy subjects was used to measure changes in transepithelial bioelectric properties and the permeability of the apical membrane to chloride in response to extracellular UTP. The in vivo experiments showed that ATP and UTP stimulated chloride secretion in both groups of subjects but
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were more effective in patients with CF than in normal subjects. In cell cultures, UTP was again found to effect a greater increase in the transepithelial PO and chloride secretory current, reflecting changes in permeability of the apical membrane to chloride, in nasal epithelial cells from patients with CF versus those from healthy subjects. The investigators noted that the maximally effective concentrations of ATP and UTP can easily be achieved on airway surfaces with the use of current aerosol technology." In both the in vitro and in vivo studies, it was necessary to pretreat the nasal epithelium with amiloride because activation of chloride channels by nucleotides in the absence of amiloride did not induce chloride secretion. In light of these results, Knowles et al. observed: "Because nucleotide therapy in cystic fibrosis would be designed to initiate the secretion of chloride (and water) in the direction of the lumen, a combination of a sodium-channel blocker and a nucleotide will probably be required for therapy."24 DECREASING MUCUS VISCOSITY WITH RECOMBINANT HUMAN DEOXYRIBONUCLEASE
Some investigators are taking a different approach to the treatment of CF and evaluating the benefits of recombinant human deoxyribonuclease (rhDNase). Collins has pointed out that "One of the major clinical problems in CF patients is the high viscosity of the large volumes of mucus produced by CF patients. This mucus is heavily infected with bacterial organisms and contains numerous white blood cells, many of which die and release their DNA, further contributing to the high viscosity.'?" Digestion of the deoxyribonucleic acid (DNA) component of mucus might reduce viscosity and lead to better mucociliary clearance and less airflow resistance. To evaluate the potential clinical utility of rhDNase in treating CF, Shak et al. cloned, sequenced, and expressed the enzyme. They found that small amounts of rhDNase greatly reduced the viscosity of purulent CF sputum, transforming it from a nonflowing viscous gel into a flowing liquid." Later, Hubbard et al. administered rhDNase by means of a jet nebulizer to patients with CF, initially in opendose-ranging and safety studies and later in a placebo-controlled crossover study. Either rhDNase 10 mg or placebo was administered twice a day.26 No significant adverse effects attributable to therapy were observed in the safety study even at dosages of up to 20 mg twice daily. Eleven patients completed the placebocontrolled trial. Nebulized rhDNase, but not placebo, significantly improved forced vital capacity and forced expiratory volume in one second throughout the six-day treatment period." This preliminary study demonstrates that treatment with aerosolized rhDNase is an effective way of improving lung function in patients with CF. "The mechanism of action of the rhDNase in improving lung function in patients with cystic fibrosis is probably related to its ability to cleave high-molecular weight DNA in purulent secretions in the airways .... Because of its mechanism of action, rhDNase is likely to be effective only in patients with purulent sputum."26 In a subsequent letter to the New England Journal of Medicine, responding to a letter from Rubin" criticizing some aspects of their work, Hubbard et al. stated that "Al-
though it is tempting to speculate that the specificity of rhDNase for cleaving DNA improves the rheologic or adhesive properties (or both) of purulent mucus in vivo, we agree with Dr. Rubin that the specific alterations produced in purulent mucus by rhDNase that are critical for obtaining clinical benefit are not known."28 More evidence of aerosolized rhDNase's promise for the short-term management of airflow limitations in patients with CF is found in a recent report by Aitken et al., who also evaluated the safety of rhDNase in normal subjects and in patients with CF. The study design was a repetitive dose escalation of aerosolized enzyme given three times a day for five days followed by a two-day washout
period." Inhalation of rhDNase in daily doses of up to 30 mg was well tolerated by all subjects. A single challenge dose administered 21 days after the last dose of rhDNase had no effect, consistent with the absence of serum anti-DNase antibodies. Although the study was not designed to establish efficacy, an improvement in pulmonary function associated with an improvement in dyspnea was observed in patients with CF.29 The Pink Sheet reported that rhDNase is on a fast track for filing a product licensing application with the FDA's Center for Biologics Evaluation and Research. The drug's sponsor, Genentech, hopes to submit the application in early 1993. A pivotal Phase ill trial initiated in late 1991 now has full enrollment. The trial is designed to determine if the enzyme can reduce the number of days of hospitalization and the need for physical chest therapy and antibiotic treatment, as well as to confirm the effects of rhDNase on pulmonary function." The Pink Sheet also noted that the potential market for rhDNase is much larger than the estimated 15000 patients with CF in the US. Moreover, Genentech is developing the product to reduce the viscosity of sputum in patients with chronic bronchitis. The subset of patients with chronic bronchitis that might benefit from treatment with rhDNase is estimated at 500 000. 30 PROTECTION AGAINST ELASTASE
Earlier, the potential effectiveness of AAT as replacement therapy in hereditary emphysema was described. This enzyme may also be useful in treating CF. Davis has pointed out that neutrophil elastase is also a villain in the lungs of patients with CF.IS It may be the predominant local factor inhibiting host defenses against Pseudomonas. Neutrophil elastase not only injures normal respiratory tissues, but also cleaves immunoglobulins and complement and prevents phagocytosis and killing of Pseudomonas organisms by neutrophils. A range of natural and synthetic inhibitors of elastase are under investigation. Among them is the irreversible elastase inhibitor AAT, the main inhibitor of neutrophil elastase in normal lungs. IS McElvaney et al. administered AAT via aerosol every 12 hours for one week to healthy subjects and to patients with CF. Baseline measurements indicated that AAT concentrations in respiratory epithelial lining fluid (ELF) were similar in normal subjects and those with CF. However, no neutrophil elastase was detectable in normal ELF, but large amounts were detectable in the ELF of CF patients. Treatment significantly elevated ELF AAT concentrations an concentrations increased with dose. Neutrophil elastase ac-
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tivity was completely inhibited when ELF AAT concentrations reached 8 umol/L, Neutrophils exposed to the ELF of patients with CF were not effective in killing Pseudomonas; however, neutrophils exposed to the ELF of patients with CF receiving treatment with AAT retained their effectiveness in clearing the organism." McElvaney et al. concluded that treatment with aerosolized AAT "might effectively prevent damage to the normal respiratory tissues by neutrophil elastase and augment host defence on the respiratory epithelial surface, thus potentially reversing a major problem in cystic fibrosis. The results are sufficiently convincing to support controlled clinical trials of AAT and other anti-neutrophil elastases."31~
References I. Cournoyer D, Scarpa M, Jones SN, Moore KA, Belmont JW, Caskey CT. Gene therapy: a new approach for the treatment of genetic disorders. Clin PharmacolTher 1990;47:1-11. 2. Marx JL. Getting to the heart of genetic disease. Science 1989;243:315-6. 3. Crystal RG. Alphaj-antitrypsin deficiency, emphysema, liver disease: genetic basis and strategies for therapy. 1 Clin invest 1990;85:1343-52. 4. Hubbard RC, Brantly ML, Sellers SE, Michell ME, Crystal RG. Delivery of proteins for therapeutic purposes by aerosolization: direct augmentation of anti-neutrophil elastase defenses of the lower respiratory tract in alphaj-antitrypsin deficiency with an aerosol of alphaj-antitrypsin. Ann Intern Med 1989;111:206-12. 5. Hussar DA. New drugs of 1991. Am Pharm 1992;NS32(4):317-50. 6. Beutler E. Gaucher disease: new molecular approaches to diagnosis and treatment. Science 1992;256:794-9. 7. Genzyme files for Ceredase in Europe. Scrip 1992;(Apr 15):17. 8. Hilts PJ. Seeking limits to a drug monopoly. New York Times 1992 May 12; Sect C:1. 9. Cystic fibrosis. In: Berkow R, ed. Merck manual of diagnosis and therapy. Rahway, NJ: Merck Sharp & Dohme Research Laboratories, 1987: 2055-9. 10. Collins F. Cystic fibrosis: molecular biology and therapeutic implications. Science 1992;256:774-9. 11. Snouwaert IN, Brigman KK, Latour AM, MaloufNN, Boucher RC, Smithies 0, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992;257: 1083-8. 12. Valerius NH, Koch C, Hoiby N. Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment. Lancet 1991;338:725-6. 13. Cryz SJ Jr, Sadoff JC, Cross AS, Furer E. Safety and immunogenicity of a polyvalent Pseudomonas aeruginosa O-polysaccharide-toxin A vaccine in humans. AntibiotChemother 1989;42: 177-83. 14. Schaad VB, Lang AB, Wedgwood J, Ruedeberg A, Que JV, Furer
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E, et al. Safety and immunogenicity of Pseudomonasaeruginosa conjugate A vaccine in cystic fibrosis. Lancet 1991;338:1236-7. 15. Choosing and using a pancreatic enzyme supplement. Drug Ther Bull 1992;30:37-40. 16. Hendeles L, Dorf A, Stecenko A, Weinberger M. Treatment failure after substitution of generic pancrelipase capsules. Correlation with in vitro lipase activity. lAMA 1990;263:2459-61. 17. Rheinstein PH. Regulatory status of pancreatic enzyme preparations.
lAMA 1990;263:2491-2. 18. Davis PB. Cystic fibrosis from bench to bedside. N Engl 1 Med 1991; 325:575-7. 19. Waltner WE, Boucher RC, Gatzy JT, Knowles MR. Pharmacotherapy of airway disease in cystic fibrosis. Trends Pharmacol Sci 1987;8: 316-20. 20. Knowles MR, Church NL, Waltner WE, Yankaskas JR, Gilligan P, King M, et aI. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl 1 Med 1990;322: 1189-94. 21. Aerosolized amiloride plus ATP or UTP for cystic fibrosis. FDC Reports.Pink Sheet 1991;(Aug 26):T&G5-6. 22. Hwang T-C, Zeitlin PL, Gruenert DC, Huganir R, Cuggino WB. C( channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase. Science 1989;244: 1351-3. 23. Mason SJ, Paradiso AM, Boucher RC. Regulation of transepithelial ion transport and intracellular calcium by extracellular adenosine triphosphate in human normal and cystic fibrosis airway epithelium. Br
1 Pharmacoll991;103:1649-56. 24. Knowles MR, Clarke LL, Boucher RC. Activation by extracellular nuc1eotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl 1 Med 1991;325:533-8. 25. Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.
Proc Natl Acad Sci USA 1990;87:9188-92. 26. Hubbard RC, McElvaney NG, Blrrer P, Shak S, Robinson WW, Jolley C, et aI. A preliminary study of aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis. N Engl 1 Med 1992;326:812-5. 27. Rubin BK. Aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis (letter). N Engl 1 Med 1992;327:571. 28. Hubbard RC, Shak S, Crystal RG. Aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis (letter). N Engl 1
Med 1992;327:571. 29. Aitken ML, Burke W, McDonald G, Shak S, Montgomery AB, Smith A. Recombinant human DNase inhalation in normal subjects and patients with cystic fibrosis: a phase I study. lAMA 1992;267: 1947-51. 30. DNase on fast-pace for cystic fibrosis PLA filing: application to FDA planned for early 1993; DNase will be biotech's "next major product," Genentech says. FDC Reports.Pink Sheet 1992;(May 11):9-10. 31. McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, et aI. Aerosol alpha.-antitrypsin treatment for cystic fibrosis. Lancet 1991;337:392-4.
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