Reflections Science in the Cultural Context

Reflections Science in the cultural context

he general consumer has little contact with enzymes. solvents. No wonder, then, that the more accessible enzymes TA person may occasionally soak contact lenses in an have long been tried for various medicinal purposes. enzyme-containing solution to remove protein deposits; anoth- er may swallow a lactase capsule before eating dairy food, These Reflections focus largely on one area of clinical enzy- to prevent digestive discomfort; a third may take α-galactosi- mology––the medical uses of the pancreatic enzymes. The dase drops before eating gas-forming foods such as beans. story of these enzymes, used both as crude mixtures and Otherwise, the average person is unaware of enzymes. Yet purified single entities, illustrates the changes in technology enzymes have a considerable history of therapeutic applica- and scientific concepts that have occurred during their long tions, and a growing importance in modern medicine. history. Briefly discussed, also, is the recent leap in technology that has made possible unimagined successes in the field The scientist knows that enzymes are remarkable, not to say of medicinal enzymes. miraculous, substances. They catalyze the myriad biochemi- cal reactions on which life depends. They cause to proceed Pancreatic Juice and Pancreatin rapidly, in aqueous medium and at physiologic temperatures, The exocrine secretion that passes from the pancreas into reactions which our greatest organic chemists can carry out the duodenum, commonly called the pancreatic juice, is only at a slow rate, employing high temperatures and organic a cocktail containing proteolytic, amylolytic, and lipolytic

4 Clinical Enzymology

By the 1940s, pancreatin enjoyed only occasional usage, as Box 1. Concocting a Distasteful “Solution” a digestive aid, generally filled into gelatin capsules by the pharmacist. A concentrated form of pancreatic amylase was “Horace Dobell, noticing that consumptives are very available as Diastase Vera. Amylase obtained from malt–– likely to have a dislike for fat, conceived the idea produced from the action of Aspergillus oryzae on sterilized that this was due to lack of pancreatic digestion, rice hulls or on wheat bran––was also marketed, under the and [he] administered an emulsion made with the brand name Taka-Diastase®, recognizing Jokichi Takamine, fat of beef stirred in milk with the pancreatic juice the Japanese biochemist who had purified this enzyme (3). of the pig.” One might therefore assume that Dobell Diastase was mainly indicated for use by people who had wanted his patients to eat fat for its caloric value overindulged in starchy food such as pasta. because tuberculosis patients undergo wasting (i.e., consumption) of body mass. Quote taken from (1). Parenteral Trypsin Early in the 1950s, The National Drug Company of Philadelphia introduced Parenzyme®, a 5 mg/mL suspen- enzymes, and contributes to the digestion of all three major sion of trypsin in sesame oil, not for its digestant properties, food components. A crude extract, generally made from hog but for reduction of local inflammation in phlebitis, ocular pancreas, and called pancreatin, has been in medicinal use inflammation, and traumatic wounds. In this case, an enzyme since before 1870. Pancreatin can be made by extracting was not being used in direct contact with its substrate but for minced, fresh hog pancreas in a variety of ways. In one action at a distance. Before further discussing this truly novel example, pancreas is extracted with 25% alcohol and the application, some background information on National Drug liquid is evaporated (1). and its Research Director, Gustav J. Martin, ScD, is in order. The proteases of the pancreatic juice are trypsin, chymotryp- One of five major Philadelphia drug sin, and carboxypeptidase, each of which attacks proteins at companies at that time, National Drug different points in the polypeptide chain. These proteases have had a pharmaceutical manufacturing been crystallized (2), and trypsin and chymotrypsin were intro- plant in Philadelphia and a biologics duced as medicinals during the mid-20th century. Yet it was plant in the Pocono Mountains in upstate pancreatic lipase that first brought medical attention to pancre- Pennsylvania. Following several mergers, atin. When pancreatin and fat from consumed food are pres- National Drug no longer exists; however, ent in a slightly alkaline environment, the lipase begins to split its biologics facility, now part of Sanofi the fat and the resultant fatty acids form Pasteur, produces a large percentage of a soap, which acts as an emulsifier. The Gustav Martin America’s supply of flu vaccine. National physician Horace Dobell (1826–1917), Drug’s research department was also located in Philadelphia believing that tuberculosis patients needed but separate from its manufacturing plant. The guiding spirit to ingest more fat, mixed pancreatin in of the research department was Gustav J. Martin. milk with beef fat and administered the emulsion to patients as a nutrient (Box 1) Martin (1910–1967) earned his doctorate at Johns Hopkins, (1). Pancreatin was also mixed with cod and served as National Drug’s Director of Research from liver oil to emulsify the latter. Jokichi Takamine 1944 to 1960. Martin thought in terms of big scientific concepts. His studies on competitive and non-competitive During the first half of the 20th century, the enzyme inhibitors led him to propound a Theory of Biological main applications of pancreatin were: 1) as a digestive aid Relativity. Of the theory’s eight main propositions, the major in dyspepsia, and 2) to predigest food. As an example of ones stated that there are no absolutes in biological systems predigestion, 300 mg of pancreatin, mixed with 1.2 g of to distinguish them from physical systems, that in both types sodium bicarbonate of systems the only absolute is time, and that all enzymes in an ounce of warm possess only relative specificity (4). Martin once met with water, was added to a Albert Einstein to brief Einstein on his theory. Biological pint of milk and kept at Relativity, however, did not fire the imagination of scientists about 43°C for 20–30 and the public as Einstein’s theory had done, and it quickly minutes. The mixture faded into obscurity. was then boiled to prevent further proteolysis, Martin had a strong practical side as well, and his research cooled, and adminis- group brought forth some novel pharmaceutical products. He tered by mouth (1). Taka-Diastase

February 2007 Volume 7, Issue 1 5 Reflections

Clinical Enzymology Box 2. Egg-White Edema The decade of the 1950s saw a flurry of laboratory and clinical research on the systemic applications of enzymes. When a solution of egg white is injected into the At a symposium in November 1956, organized by the New hind paw of a rat, it causes inflammation, resulting York Academy of Sciences, presentations were made on the in accumulation of fluid in the paw. A test sub- parenteral use of trypsin and chymotrypsin and their anti- stance, injected before or simultaneously with the inflammatory effect (9). Also reported were the use of beef egg white, will reduce the amount of fluid if it has pancreatic dornase (DNAase) aerosols to liquefy mucopu- an anti-inflammatory action. The difference in mass rulent secretions in bronchopulmonary disease (9). Today, between a treated paw and a control (egg-white Pulmozyme®––human DNA-ase prepared by recombinant alone) paw is used to assess the anti-inflammatory techniques in Chinese hamster ovary (CHO) cells––is similarly effect of the test substance. administered to improve pulmonary function in cystic fibrosis patients. There were studies on plasmin, the proteolytic-fibrino- pioneered the use of ion-exchange resins as non-absorbable lytic enzyme of blood, and on the activation of plasminogen. ® gastric antacids and antidiarrheals, as well as the use of pro- Today, tissue plasminogen activator (tPA) (e.g., Activase ) teolytic enzymes for systemic action. made by recombinant DNA (rDNA) technology, is given as an intravenous emergency treatment in myocardial infarction, Martin and others found, in animal studies, that injection of ischemic stroke, and certain pulmonary embolisms (10). trypsin inhibited egg-white edema (Box 2), a sign of anti- inflammatory action. Their experimental data showed that the These developments led Martin to proclaim that an era of enzyme acts to facilitate drainage from an inflamed area by Clinical Enzymology had arrived. He stated that until recent- increasing permeability, causing vasodilation, and reducing ly, it was deemed impossible to give a proteolytic enzyme the viscosity of edema fluid (5). parenterally, or to have it exert a systemic action. Apparently reflecting opposition he had encountered, he pictured “the As the first product in a new category, Parenzyme® quickly textbooks” turning over in their “intellectual graves.” But with gained a significant market. Nevertheless, it shared the dis- scientific caution he stated “the structure of clinical enzymol- advantages of all oily injectables: pain at the injection site, ogy is not a fairyland castle of panacea, but a solid, 2-story and formation of sterile abscesses. An aqueous vehicle was building of real but limited utility” (11). desirable, but trypsin is unstable in aqueous solution. This was solved by employing as the vehicle a 5% solution of gel- Alas, Martin’s figurative two-story structure did not endure. atin, partially denatured by autoclaving. This prevented auto- The clinical enzymology of the 1950s, comprising trypsin digestion of trypsin by providing a large concentration of and other proteases, faded into desuetude. This author, who protein (gelatin) to attach the enzyme. The product, protected worked at National Drug during that period, believes that ® by patent (6), was marketed as Parenzyme Aqueous. timing was largely responsible. The Parenzyme line and the “me-too” enzyme products were marketed before enactment of The next extension of the Parenzyme® line was a buccal tab- the Drug Amendments of 1962. Before 1962, new drug appli- let. Martin hypothesized that trypsin produces local changes cations (NDAs) only had to prove safety (and safety testing in permeability, permitting its absorption through the buccal back then did not include carcinogenicity or reproduction stud- mucosa (7). This was followed by an ointment, for local ies), so these products went through the NDA process quickly debridement, and finally an enteric, coated tablet contain- and easily. After 1962, proof of effectiveness was required, ing 20 mg trypsin for oral administration, Orenzyme®. The generally involving two controlled clinical trials. By the 1970s, enteric coating protected the trypsin from inactivation by when the FDA demanded effectiveness data to support contin- the gastric juice and allowed it to be released in the small ued marketing, the industry situation had changed. Champions intestine. Martin claimed that the intestinal tract is not totally of enzyme therapy such as Gustav Martin were gone, mergers impervious to protein molecules, pointing out that Sabin polio had occurred, and the firms involved were pursuing other new virus is active as a vaccine when administered orally (8). products or grappling with other problems. They were thus unwilling to devote resources to the needed efficacy studies. ® The commercial success of Parenzyme led to marketing As a result, we do not know whether trypsin and related prote- ® of various “me-too” products, such as Tryptar (trypsin, ases are effective medicinals. Armour); Chymar® and Chymoral® (chymotrypsin, Armour); and Ananase® (bromelain), which is a protease from the stems of the pineapple plant.

6 Clinical Enzymology

Pancreatin Survives the Table 1. Comparative Enzyme Activities Purified Enzymes Pancreatin Pancrelipase Usually, in the history of natural product medicinals, crude USP units/mg USP units/mg extracts are succeeded and replaced by their pure active Protease ≥25 ≥100 components. Foxglove leaves (digitalis) have been replaced by digoxin, thyroid powder by thyroxine, etc. Remarkably, Amylase ≥25 ≥100 this is not the case with the pancreatic enzymes. Today, while Lipase ≥2 ≥24 the trypsin and chymotrypsin dosage Based on United States Pharmacopeia 25, (2002). forms are gone, pancreatin plays a more important role in medicine than ever a much higher lipase content, as well as a higher protease before. It is used in pancreatic insufficien- and amylase content, than the old standby pancreatin (Table cy from whatever cause, and, especially, 1) (13). A high fat-splitting activity is particularly desirable in the management of cystic fibrosis (CF). for CF patients. To simplify discussion, both pancreatin and pancrelipase are referred to below as pancreatin. Cystic fibrosis, which affects about 30,000 people in the US, is the second The marketing of these products may be described as chaot- ic. As mentioned above, the use of pancreatin long predates Foxglove most common childhood-onset single-gene disorder (after sickle cell disease) in this 1938, when NDAs were first required. Thus, pancreatin country. An autosomal recessive disorder, caused by muta- products today are being marketed without NDAs and on an tions in one gene on the long arm of chromosome 7, CF is over-the-counter (OTC) basis. FDA records in 2004 showed manifested in an electrolyte transport defect resulting in thick, that twenty-three manufacturers and twenty-six private label sticky mucus. Abnormally viscous mucus affects the airways, distributors were selling a total of thirty-eight formulations resulting in pulmonary obstruction and chronic infections; it (14). These include capsules and tablets, enzymes in rapid also obstructs the flow of pancreatic juice from the pancreas release form or as enteric coated microspheres, with labeled into the small intestine, resulting in malabsorption of nutrients lipase content ranging from 1,200 to 20,000 units per dose. (12). Documented problems with these products include bioavailabil- Although no means of correcting the genetic defect has been ity issues, variability in lipase content and release rate, and discovered, and no CF-specific drugs have been developed, uneven quality of enteric coatings (14). Insufficient delivery of medicine has made enormous progress in early diagnosis by enzymes to the intestinal tract results in malabsorption, leading neonatal screening and in the treatment of CF. While in the to steatorrhea, azotorrhea, and malnutrition; enzyme overdose 1960s life expectancy for newborns with CF was less than may result in intestinal stricture and other serious problems. one year, at present the median survival is thirty-five years, FDA Attempts to Assert Control and 40% of patients are forty years or older (12). The FDA is attempting to bring the situation under control. Management of CF on a chronic basis requires a three- The Agency has decided to require NDAs for these prod- pronged approach: promoting clearance of mucus from the ucts, in order to guarantee proper quality control in terms of lungs, controlling lung infection and inflammation, and assur- product purity, potency, stability, and bioavailability. The FDA ing adequate nutrition. Clearing pulmonary secretions from also feels that proper dosage adjustment and patient monitor- the airways is done by postural drainage with chest percus- ing require the products to be prescription only, rather than sion, nebulizer therapy with sterile water or normal saline, OTC. Accordingly, on April 28, 2004, the FDA announced or oscillating positive expiratory pressure devices. Antibiotics that manufacturers of pancreatin products must submit NDAs. are used for both acute exacerbations and chronic suppres- A four-year period was provided for NDA submittal and sion of respiratory infections. Finally, the mainstays of diges- approval, during which time the products may remain on the tive aid therapy are the pancreatic enzymes which help to market because they are medically necessary (14). prevent malnutrition, once a primary cause of death among In April 2006, the FDA followed up by issuing a Guidance CF patients (12). for Industry on the required content for a pancreatin NDA. The pancreatic enzymes are administered in the form of pan- The Guidance spells out the control tests that must be in creatin, or more commonly now, as pancrelipase. The latter place for the active ingredient, the excipients, and the drug is also an extract of fresh hog pancreas, but is processed in product. No toxicology or new pharmacology studies are such a way that the enzyme activity, especially that of lipase, required. To prove bioavailability, the three separate enzyme is more completely preserved. As a result, pancrelipase has activities should be measured on aspirates from the stomach and duodenum. A dose-response relationship should be

February 2007 Volume 7, Issue 1 7 Reflections

demonstrated by clinical trials, to include pediatric as well Note: The sections of this paper dealing with Gustav Martin as adult CF patients, and the Guidance provides detailed and Parenzyme® are based in part on the author’s recollec- instructions for several types of trials, all of short duration tions of his early years in the pharmaceutical industry, as a and involving a relatively small number of patients (15). member of Martin’s research team.

Does issuance of this Guidance indicate that manufacturers have been unable to produce an acceptable NDA on their References own? The FDA may face a peculiar problem when the grace 1. Wood, H.C., LaWall, C.H. Eds. “The Dispensatory of the United States of period expires in 2008. If no firm has submitted an approv- America”, J.B. Lippincott Co., Philadelphia, 1926, pp. 813–815. able NDA, the FDA will be unable to remove pancreatin 2. Harrow, B. “Textbook of Biochemistry”, W.B. Saunders Company, Philadelphia, 1943, pp. 107–110. products from the market because they are not only medically 3. “The Merck Index, 5th Edition”, Merck & Co., Inc., Rahway, NJ, 1940, necessary, but actually life-saving. pp.191–192. 4. Martin, G.J. “Biological Antagonism”, The Blakiston Company, Inc., New A New Wave of Clinical Enzymology York, 1951, pp.476–479. The development of rDNA techniques in recent decades has 5. Martin, G.J., Brendel, R., and Beiler, J.M. Mechanism of the antiinflam- made clinical enzymology more prominent and more impor- matory action of trypsin. Am. J. Pharm. 127, 125–128 (1955). tant than ever before. The clinical uses of tissue plasminogen 6. Sullivan, M.J. and Martin, G.J. Stabilized trypsin compositions containing partially hydrolyzed gelatin. US Patent 2,930,736 (March 29, 1960; activator (tPA) and human DNAase have been mentioned assigned to National Drug Company). above. The ability to create enzymes in cell culture has obvi- 7. Martin, G.J. Ed. “Clinical Enzymology”, Little, Brown & Co., Boston, ated the need to isolate them from huge quantities of animal 1958, p. 131. tissues. As a result, it is possible to provide replacement thera- 8. Martin, G.J. Ed. Ibid. p. 228. py for genetic diseases caused by the inability of the patient’s 9. St. Whitelock, O.V. Ed. “Proteolytic Enzymes and Their Applications”, New York Academy of Sciences, New York, 1957. body to make an essential enzyme. The following few exam- 10. Physicians’ Desk Reference, 60th edition, Thomson PDR, Montvale, NJ, ples illustrate what this field of technology is accomplishing. 2006, pp.1225-1229. 11. Martin, G.J. Ed. “Clinical Enzymology”, p.1. The best known example of rDNA replacement therapy is 12. Hendeles, L., Kuhn, R.J., and Milavetz, G. The role of pancreatic enzyme Cerezyme® (imiglucerase), an analog of human β-glucocer- therapy in the treatment of cystic fibrosis. Pharmacy Today 12(9), 40–51 ebrosidase, used for long term treatment of Type I Gaucher (2006). disease (16). Recently, news of a merger between two small 13. The United States Pharmacopeia (USP 25)/The National Formulary (NF 20), U.S. Pharmacopeial Convention, Inc., Rockville, MD, 2002, pp. companies revealed that one of them is conducting Phase 1303–1305. III clinical trials on a potential competitive enzyme product 14. Exocrine pancreatic insufficiency drug products. Federal Register 69, (17). Among ten biopharmaceuticals approved in 2005, 23409–23414 (2004). there were two enzymes: Hylenex® (human recombinant hyal- 15. Guidance for industry: Exocrine pancreatic insufficiency drug products–– submitting NDAs. Food and Drug Administration, April 2006. uronidase) and Nagluzyme® (human recombinant N-acetyl- 16. Physicians Desk Reference, 60th edition, op. cit., p.1263. galactosamine-4-sulfatase), an enzyme replacement therapy 17. Anonymous. Protalix merges with Orthodontics. Chem. Eng. News, for mucopolysaccharadosis VI (18). Approved earlier was August 28, 2006, p.18. Aldurazyme® (laronidase), a polymorph of human alpha- 18. Walsh, G. Biopharmaceuticals: Approval trends in 2005. BioPharm L-iduronidase, used for treating mucopolysaccharadosis I International, August 2006, pp. 58–67. (19). Myozyme®, for the rare α-glucosidase deficiency called 19. Physicians Desk Reference, 60th edition, op. cit., p.1261. 20. Bridges, A. 1st treatment for rare disease gets OK. Phila. Inquirer, April Pompe Disease, was approved in April, 2006 (20). 29, 2006. 21. Kuehn, B.M. Enzymes might cut celiac symptoms. JAMA 296, 382 Inability to digest gluten, a protein constituent of wheat and (2006). other grains, produces celiac disease. Currently the only treatment is avoidance of foods which contain gluten, such Stanley Scheindlin, DSc, as bread and pastas. Research has now been reported RPh, holds graduate degrees that might eventually lead to an enzyme mixture capable of in pharmaceutical chemistry breaking down gluten for celiac sufferers (21). and worked in drug product development and regulatory It is impossible to know whether Gustav Martin, that early affairs. Now retired, he is a proponent of clinical enzymology, might have foreseen a part-time consultant and writes time when vital enzymes could be made to order, so to freelance articles for pharmacy- speak. Were he alive today, however, he would feel vindicat- related specialty publications. ed. His modest two-story building is growing rapidly toward skyscraper heights. doi:10.1124/mi.7.1.1