Abstract

Enzymes play a central role in maintaining the metabolic balance within the human body. Acting as catalyst enzyme activity can either slow down or speed up metabolic reactions. Therefore, a reduction or total lack of enzyme creates an imbalance leading to disorders. In modern medicine, enzymes are being formulated in the laboratory and adopted in the treatment of a variety of diseases and disorders. The digestive enzymes supplements; lactase and pancreatic, are used in the treatments of lactose intolerance and pancreatic ulcers and pancreatitis respectively. Cystic Fibrosis and celiac disease, serious conditions affecting children, are also treated using enzyme therapies of rhDNase and prolyl endopeptidase respectively. Lastly, L-asparaginase is by far the most successful treatment therapy for Acute Lymphoblastic Leukemia. This paper will look at the molecular make up of these therapeutic enzymes that facilitate their operation, the clinical efficacy and dosages and sensitivity.

Keywords: Therapeutic, Treatment, Enzyme, Asparaginase, Cancer, Leukemia, Clinical

List of Abbreviations

CF- Cystic Fibrosis

CFTR – Cystic Fibrosis Transmembrane Conductance Regulator

RhDNase – Recombinant human deoxyribonuclease 1

Introduction

Therapeutic enzymes are enzymes which are used medically or adjunct together with other enzymes for the treatment of diseases (Hwang et al.2013). Therapeutic enzymes have been used for the last four decades. All the cells in the human body require enzymes so that they can perform efficiently and effectively. When these enzymes lack, the body becomes prone to attack by diseases. Enzymes are responsible for converting the food eaten to energy; they regulate the growth and development of the body and break up various substances in the cells. Medically important enzymes are used singly or combined with some therapies to cure diseases (Vellard, 2003). These enzymes have two characteristics which are crucial for their task. The first one is that they act on their specific targets with specificity and secondly, they can convert several target molecules and form the wanted products. Some major therapeutic enzymes include chitinase, nattokinase, asparaginase, lipase, Serratiopeptidase, Thrombolytic drugs, L-amino acid –lipase and Glutaminase among others.

This paper is going to look into three enzyme therapies (digestive, asparaginase, and CF enzyme therapy). It will analyze how these enzymes are used and the various diseases that are treated using each specific enzyme. Asparaginase is used in the treatment of acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma and acute myeloid leukemia (AML). Recombinant human DNase (rhDNase) is used as a CF enzyme therapy (Patel et al.2000) RhDNase is an enzyme which has the ability of breaking down DNA strands found in air path secretions. Specific digestive enzyme therapy used in metabolic pathologies particularly in neonates and infants will be addressed as well.

1. Therapeutic Enzymes.

Therapeutic enzymes are briefly described as enzymes artificially synthesized or biologically derived that are used either in isolation or in combination with other therapies to treat diseases and disorders. However, the effectiveness of these treatments is inhibited by some factors (Chang, 2013).

1. Factors Affecting use of Therapeutic Enzymes

• The relative sizes of these enzymes make it difficult to distribute around the body. Therefore, new approaches are required to ensure that enzyme therapies are applicable in the treatment of several human genetic diseases. One such method is the deliberate targeting of enzymes. For instance, enzymes that exhibit covalent coupling are used to target particular monoclonal antibodies thus avoiding side effects
• Therapeutic enzymes are known to have relatively short circulation spans within the body. In comparison to the immunological challenges of disguising using covalent modification, this is tolerable. However, although such methods are known to be effective at increasing the circulation spans, they are known to result in increases in immunological responses and further lead to blood clots and coagulation,
• Therapeutic enzymes are foreign proteins in the body. Naturally, the body reacts to such foreign bodies by producing antibodies (Bellanti, 2013). The outcome of this antigenic reactions is a severe, life-threatening allergic reaction that increases in severity with continuous use. However, by creating a disguise for this enzyme as it enters the body, often as a non-protein molecule, can lower the risk of such reactions. A good example of such coating is in asparaginase. The enzyme is coated with a covalent attachment of polyethylene glycol (Harris, 2013), which has so far proven effective with little immunogenicity. Thus, such reaction elicits the use of animal obtained enzymes. Although at a higher cost to purchase and prepare in comparison to those of microbial origin.

It is important that the sources of therapeutic enzymes be evaluated to lower the possibility of contamination. Once prepared, therapeutic enzymes are made available for sale as lyophilised, biocompatible buffering salts with little diluent added (Gurung, Ray, Bose, & Rai, 2013). However, therapeutic enzyme preparations are expensive to buy in comparison to other therapeutic treatments. Thus these treatments may not be available to every patient that needs them

Currently, therapeutic enzymes are being adopted in the treatment of cancer (Chen, Huang, & Chen, 2013). Asparaginase has so far had higher degrees of success, especially during the treatment of acute lymphatic leukemia; pharmacological indications of asparaginase are based on the fact that the tumor cells associated with leukemia are deficient of aspartate ammonia ligase activity. This deficiency means that these cells cannot synthesize L-asparagine, therefore, needing to extract it from bodily fluids (Vrooman, Stevenson, Supko, O’Brien, Dahlberg, Asselin&Laverdière, 2013). They key advantage of asparaginase is that it has a minimal effect on the functioning of healthy cells. However, its actions result in lower concentrations of free exogenous thus starving the tumor cells (Timmerman, Holton, Yuneva, Louie, Padró, Daemen, &Polyak, 2013). With a 60% complete remission, the intravenously administered treatment is a viable option in cancer treatment.

2. Digestive Enzyme Therapy

An enzyme therapy is, in simple terms, a medical plan of diet supplements mostly consisting of enzymes obtained from plants and animals. The primary objective of an enzyme therapy is to smoothen the digestive process and bring about a general improvement in the body’s metabolic balance (Anwar, Nanda, Bhatia, Akhtar, & Mahmood, 2013). Often, enzyme therapies are recommended for patients suffering from disorders of digestive systems. These disorders include cystic Fibrosis, Gaucher’s disease, celiac disease, and (Sosnay, Siklosi, Van Goor, Kaniecki, Sharma, &Masica, 2013). Furthermore, enzyme therapy is believed to be useful in the treatment of cancer. Although research is still in progress, scientists explain that these enzymes digest the protective membranes of the cancerous cells, enabling the body defenses to attack and eliminate these cells (Timmerman, Holton, Yuneva, Louie, Padró, Daemen, &Polyak, 2013). Apart from gastrointestinal disorders, this therapy is also applicable in the treatment of a variety of conditions such as food allergies, hypoglycemia, and nutritional disorders.

The digestive system is a complex yet well-orchestrated system. Enzymes play a vital role in the digestive system, beginning from the mouth with salivary amylase breaking down starches, to lactase for dairy products, lipase for fats and protease for proteins. These enzymes ensure that they bodies utilize fully all the nutrients available in the food that we eat (Bischoff, Barbara, Buurman, Ockhuizen, Schulzke, Serino, & Wells, 2014). The consumption of large amounts of plant enzymes will allow the body to use fewer enzymes, this, in turn, will enable these enzymes to be more useful in maintaining the body’s metabolism at peak.

The enzymes that are used in enzyme therapy are often extracted from plants such as papayas and pineapple and animals such as cows and pigs. Typically, enzyme supplements are either administered as tablets or pills, over a period of dosage as the physician or nutritionist would recommend. For instance, if the condition being addressed is not digestive, then doctors recommend that the supplements be taken at least an hour before any meal. The reason behind this is that when taken early, the supplements are absorbed quickly into the blood to start their intended functions (Anguiano,Pohlenz, Buentello,& Gatlin, 2013). When treating digestion disorders, then most often doctors recommend that these enzymes be taken immediately before a meal, and with a good quantity of fluids. Vitamin A also forms an excellent accompaniment.

In the treatment of gastrointestinal diseases and other digestive disorders, pancreatic enzyme supplementation, and lactase supplementation are often used. Pancreatic enzyme supplementation is recommended for patients with some pancreatic diseases as well as those that indirectly affect the pancreas. These conditions include acute pancreatitis, Schwachman syndrome, cystic fibrosis, and pancreatic cancer (Bilton et al., 2013). Lactase supplementation is usedmainly in the treatment of lactose intolerance and hypolactasia. Therefore, the supplements play a crucial role in mitigating the effects of lactase deficiency in both adults and neonates. The two enzyme supplements are discussed further in the subsequent sections.

1. Asparaginase

Asparaginase is hydrolase capable of converting asparagine, an amino acid that facilitates the function of neoplastic cells (Salzer, Asselin, Supko, Devidas, Kaiser, Plourde& Hunger, 2013). In human cells, asparagine deficiency is compensated by alternative pathways for synthesis achieved through artificial synthesis from aspartic acid and glutamine. The outcome of asparagine deficiency is the inhibition of both DNA and RNA and eventually, plastic cell apoptosis. In recent times, asparaginase has been indicated in the treatment of cancer, especially in Lymphoblastic Leukemia (Davila, Riviere, Wang, Bartido, Park, Curran, & Qu, 2014). It has been introduced as part of a multi-drug chemotherapy in both adults and children. So far, the chemotherapy approach has had significant outcomes in chemotherapy sessions as well as in remission, with up to 90% in results. However, asparaginase has a few negatives, notably the risk of thrombosis, protein synthesis inhibition, impaired liver functions, kidneys failure and complications of the central nervous system (Raja, Schmiegelow, Albertsen, Prunsild, Zeller, Vaitkeviciene, Kanerva, 2014). The explanation for the high risk of thrombosis is the inhibition of the synthesis of anti-coagulant proteins such as antithrombin.

Figure 1: Asparagine aspartate

1.    Asparaginase as Treatment of Acute Lymphoblastic Leukemia

The treatment of lymphoblastic leukemia has gone to a new and better level of the adoption of asparaginase. Asparaginase treatment protocols are also adopted in pediatric treatment regimens and some adult protocols. As mentioned earlier, the adoption of newer treatment methods has ensured in improvements in patients with lymphoblastic leukemia (Schultz, Carroll, Heerema, Bowman, Aledo, Slayton, &Gaynon, 2014). Recent estimates place the survival rate in children at approximately 80% whereas that of overall survival at 90%. However, in comparison to children outcome, about 38 to 50 percent of all cases have long term effects. The variation between the two results is attributed to favorable genetics, better compliance to dosages, effective treatment regimens and poorer tolerance to some chemotherapy treatments.

Asparaginases, are bacterially derived enzymes that are used in the management of acute lymphoblastic leukemia. Asparaginase is available in three preparations; a pegylated form of asparaginase also known by its abbreviated form PEF-asparaginase, Native asparaginase, and Erwiniaasparaginase. Although in certain countries these preparations are not available. Currently, clinical trials and evaluations are being conducted on a fourth asparaginase preparation, E. Coli-asparaginase (Vrooman, Stevenson, Supko, O’Brien, Dahlberg, Asselin&Laverdière, 2013). This new development is designed to contain a sequence of amino acid similar to that present in asparaginase medal. Furthermore, scientists are proposing a new approach to enzyme activity stability and maintenance, and achievement of a reduction information of anti-asparaginase antibodies by introducing an encapsulated asparaginase into red blood cells. A pegylated recombinant Erwiniaasparaginase is undergoing clinical studies.

Indeed, asparaginase preparations are essential in treating acute lymphoblastic leukemia. However, optimal formulations and dosages remain a matter of concern. This matter is brought about by rapid deamination of amino acids present in asparagine. This deamination results in depletion of asparagine mostly found in blood plasma and minimally, in cerebrospinal fluid (CSF). The dosages varied significantly during clinical trials, an indicator of the difference in potency of the products.

Action of L-Asparaginase on Cancer Cells (Acute Lymphoblastic Leukemia)

The main aim of the chemotherapy process in cancer treatment, is to identify isolate and destroy cancerous cells in the body. In order to do this, doctors need to identify weaknesses in the cells where treatments are directed. The rapid cell division associated with cancerous cells, has proved problematic in previous years. However, current treatments are designed to target the main process of cell division (David et al,2014). L-asparaginase, is effective in cancer treatment mostly because it is capable of targeting the protein synthesis action of cancerous cells, inhibiting their action.

Amino acids are an essential component of cell division. This importance lies in the fact that amino acids are building block of proteins. In most cells, the process of asparagine synthesis is controlled by the enzyme, asparagine synthetase. Asparagine formation occurs when the enzyme, asparagine synthetase, combine as aspartate with an anime compound to form an asparagine amide group. This process of asparagine formation, ensures that the cells have sufficient supply and do not thus need external supply. Notably, some cells still depend on blood nutrients to obtain asparagine (Vrooman et al, 2013). These cells are therefore weakened this supply is diminished.

Another path for asparagine formation is through the use of glutamine. In this instance, the glutamine is the primary source of amine whereas in bacterial asparaginase amine is derived from ammonia. L-Asparagine, a more purified from bacterial cells, is the main element used in chemotherapy treatment of blood cancer. The L-Asparagine works by removing the amine group from asparagine, resulting in the release of aspartate ammonia. Under normal circumstances, the asparaginase enzyme is involved in protein synthesis. However, when introduced into the blood in large quantities, the enzymes will breakdown all asparagine present starving the target cells. This targeted starvation, is by far an effective strategy in managing rapidly dividing cells, like those of cancer.

In Acute Lymphoblastic Leukemia, the blood cells become cancerous and begin to target other cells within the blood. However, these cells depend on blood to obtain necessary nutrients for functionality and essential synthesis. The L-asparaginase enzyme utilizes this weakness to eliminate these cancerous cells (Schultz et al, 2014). The enzyme cuts off the supply of asparagine to these cells by break down blood asparagine. Unable to synthesize proteins, the cancer cells die and are eliminated. However, the key demerit of this therapy lies in the body’s immunology. L-asparaginase being a protein, will elicit antigen antibody reactions and may result severe reactions such as anaphylaxis. There is a solution to this challenge. The L-asparagine enzyme can receive a coat of polyethylene glycol that acts as a mask, hiding it from the body’s immune system.

2.    Hypersensitivity to Asparaginase.

Like any other drug, there are associated side effects. Asparaginase use has been associated with anti-asparaginase antibody production. This has been observed in nearly sixty percent of patients at a point during therapy with asparaginase. Most often, the antibodies are produced in response to the native strain E. coli-asparaginase. In comparison, the PEG-enzyme has a significantly lower occurrence of hypersensitivity (Shinnick, Browning, & Koontz, 2013).Hypersensitivity to asparaginase is associated with symptoms which include; rash, pain, edema, erythema and anaphylaxis (August, Miller, Dalton, &Shinnick, 2013). Administration through intramuscular means produces more skin reactions compared to intravenous administration. Notably, hypersensitivity to asparaginase mostly occurs in post inductions treatments, particularly when re-induction and intensification have not taken place for weeks or months. Scientists and biomedical researchers have come up with several explanations as to why reactions are limited during remission induction. Due to the delay in immune response, from the time of full activation to the production of antibodies, the responses of the body to asparaginase are inhibited during this stage (Adkinson, Bochner, Burks, Busse, Holgate, Lemanske&O’Hehir, 2013). However, if symptoms are to occur, they can be masked corticosteroids used during induction. Studies have shown that hypersensitivity to asparaginase has similarities in different age groups. In contrast, other studies have shown younger patients, as well as infants, are less likely to develop hypersensitivity reactions or produce antibodies as compared to adults and teenage patients.

Clinical hypersensitivity is not always as a result of antibody production. Instead, rapid inactivation of enzyme asparaginase results in lower than optimal depletion of asparagine. A feature is referred to as ‘silent hypersensitivity’ commonly occurring in at least thirty percent of patients. As a result of silent activation of asparaginase, anti-asparagine antibodies produced may increase the body’s resistance to asparaginase leading to higher levels of asparaginase in the blood plasma, as well as a reduction in its efficacy (Emadi, Zokaee, &Sausville, 2014).

In response to clinical hypersensitivity, it is recommended that for those patients exhibiting such symptoms as a result of the use of one product, may only be switched to another to reduce the possibility of further reactions. However, this treatment may not be effective in patients with silent inactivation since antibody monitoring is not carried out routinely (Regan et al., 1977). The other form of asparaginase, PEG-asparaginase, is less reactive with the body’s immune system. Its component, monomethoxy polyethylene glycol is known to be responsible for this low immunogenicity. It is usually the replacement drug of choice on patients who develop sensitivity to asparaginase (Kim, Yang, Kim, Kwak, Eom, Hong, & Nam, 2014). However, in some cases, antibodies formed against asparaginase may react with PEG-asparaginase often inducing silent inactivation. These antibodies may also cause a fast decline in asparaginase activity. Under such circumstances, PEG-asparaginase is not a viable option for patients with asparaginase hypersensitivity, and alternatives need be sought.

Figure 2: Effect of intensification using asparaginase on event-free survival (Emadi  & Sausville, 2014)

Asparaginase type	Dose	Concomitant steroid medications	Antibody-positive patients	Citation
E. Coli	10 000 IU/m2 IM three times weekly for nine doses during induction and nine during re-induction	Prednisolone	35.5%	Woo et al. 2000 (46)
	6000 IU/m2 IM three times weekly for nine (induction) and six (intensification) doses	Prednisolone/dexamethasone	26–42%	Avramis et al. 2002 (28)
	6000 IU/m2 SC two-times weekly for 14 doses (induction/intensification)	Prednisolone	20%	Larson et al. 1998 (47)
PEG	2500 IU/m2 IM for a total of four doses (induction) and one dose (intensification)	Dexamethasone	11%	Hawkins et al. 2004 (48)
	2500 IU/m2 IM for one dose (induction) and one dose (delayed intensification)	Prednisolone/dexamethasone	2–11%	Avramis et al. 2002 (28)
Erwinia	10 000 IU/m2 IM three times weekly for a total of nine doses (induction/re-induction)		33%	Wang et al. 2003 (49)
	30 000 IU/m2 IV or IM daily for a total of 10 doses (induction), twice-weekly for a total of four doses (re-induction)	Prednisolone	21%	Albertsen et al. 2002 (50)
	30 000 IU/m2 IV or IM daily for a total of 10 doses (induction), twice-weekly for a total of four doses (re-induction)	Prednisolone/dexamethasone	8–10%	Albertsen et al. 2002 (53)

E. Coli, Escherichia coli; IM, intramuscular; SC, subcutaneous; PEG, polyethylene glycol; IV, intravenous.

There are several uses of the asparaginases enzymes. They are used in industrial activities and, in pharmaceutical purposes as well.

Medical purposes of asparaginases enzymes: E-coli strains constitute the major source of medical asparaginase. Some of them include Asparaginase Medac, Ciderolase, and Oncaspar. There is a new recombinant E. coli asparaginase called Spectrila which has been developed (Deirde & Claire, 2007). Asparaginase enzymes can be given through injections or as subcutaneous and intramuscular injections. One major advantage is that they do not cause any tissue irritation.

Food manufacturing: Asparaginases are used as processing aids in the manufacture and processing of food. They are used so that they can prevent the formation of acrylamide. This is a carcinogen that is present in most starchy food products especially snack (Barterlink et al. 2006). Acrylamide which is a carcinogen is produced when starchy foods are being prepared. Starchy foods naturally have amino acid asparagine, when they are heated; the asparagine goes through a process referred to as the Maillard reaction. This process is responsible for the brown color in fried foods. It is responsible for the toasted flavor and their crust (Amena et al. 2010). When the Maillard process is taking place, there are some carcinogens which are produced (such as acrylamide and some heterocyclic amines).

When asparaginase is added prior to cooking any starchy foods, asparagine is transformed into ammonium and aspartic acid, which is also another amino acid. Consequently, this ensures that asparagine does not part in the Maillard reaction. When asparagine takes part in the Maillard reaction, it results in the production of acrylamide; therefore, the absence of asparagine plays a role in hindering the formation of acrylamide (Fusseti et al. 2002). It is not possible to entirely eliminate acrylamide because of the pathway formations of some asparagine-independent components. The use of asparaginases reduces and lowers the level of acrylamide present in starchy foods. It does not alter with the appearance or the taste or flavor of the food.

2. Lactase Supplementation.

The enzyme lactase controls the digestion and absorption of lactose in the body. A deficiency of this enzyme leads to lactose malabsorption, creating a gap in the nutritional requirement of the human body. The enzyme lactase is secreted by the intestinal villi located in the small intestines. Its primary role is the hydrolysis of lactose into galactose and glucose.

In neonates, the concentration of lactase is considerably high. However, the levels decrease after weaning, although this decline is controlled by an individual’s genes and therefore varies from person to person (Carter &Attel, 2013). Furthermore, research links this decrease to primary lactose malabsorption, in individuals whose lactase levels fall below a set threshold. Secondary hypolactasia occurs when the mucosal brush that lines the small intestine or when the gastrointestinal transit time increases exponentially. It is important to note that lactose intolerance only happens when the malabsorption of lactose results in gastrointestinal symptoms (Johansson, Sjövall, & Hansson, 2013).

1.    Features of Lactase Enzyme Supplementation.

Lactase enzyme supplementation is composed of exogenous enzymes. These enzymes are typically derived from yeast and fungi. Microbial exogenous lactase is considered by doctors to be the most reliable therapeutic option in the treatment of lactase related gastrointestinal diseases and disorders. The most common of administration of exogenous Lactase, is through capsules and tablets, often before the consumption of dairy products (Misselwitz, Pohl, Frühauf, Fried, Vavricka, & Fox, 2013). However, in some situations, these supplements may be administered with milk. In the current market, supplement formulations are in abundance and research into their efficacy has so far had positive results.

Lactase is regarded as part of the β-galactosidase group of enzymes. Lactase is also referred to as a glycoside hydrolase responsible for hydrolysis of lactose into galactose and other glucose monomers. The optimum operating temperature for this enzyme is estimated at 37 degrees with the optimum ph. of 6. During the metabolism of lactose, the β-glycosidic bond present in D-lactose, is broken down into D-galactose and D-glucose. These two components are subsequently absorbed into the intestinal wall and finally into the bloodstream.

Studies have revealed that substrate configuration in most products is retained even after the catalytic action of D-Lactose. This type of retention is possible through a system of double displacement. Further studies have pointed to glutamate nucleophile as the main substance responsible for initiating the process of hydrolysis. The glutamate nucleophile attacks the enzyme from the side containing galactosyl carbon within the β-glycosidic bond. After D-glucose is formed, Mg-dependent acid catalysis enables its release from the system. Moreover, the enzyme is removed when a nucleophilic attack by water takes place, releasing D-galactose.

Preprolactase is the main product involved in the translation process of lactase processing and synthesis. Preprolactase contains a primary polypeptide with around 1927 amino acids. These is further divide into three domains; a mature lactase segment, a 19-amino acid signal sequence, a prosequence absent in the mature lactase, a membrane spanning hydrophobic anchor and a hydrophilic carboxyl terminus.

The signaling sequence is located within the endoplasmic reticulum. It produces a biological signal that is sent to the Golgi apparatus. At the Golgi apparatus this signal is glycosylated and proteolytically transformed into a mature form. The prodomain, in these process, acts as a conduit in the endoplasmic reticulum, assisting in the prevention of trypsin cleavage and enabling the bio-signal to adopt a three dimensional structure for transportation into the Golgi apparatus. In the human body, the lactase contains a single polypeptide chain capable of localization in the membranes of intestinal epithelial cells. This polypeptide is oriented with an N-terminus, located outside the cell and a C-terminus in the cytosol. The LPH is composed of two catalytic glutamic acid sites, Glu-1749 and Glu-1273. Glu-1749 is connected to the lactase enzyme activity whereas Glu-1273 provides a site for phlorizin hydrolase function.

Figure 3: Schematic of processing and localization of lactase translation (Anguiano et al. 2013)

2.    Enzyme Activity and Dosage.

Naturally, there are slight differences in the efficiency of the variety of enzymes in hydrolyzing lactose. For instance, K.Latics derived lactase have a higher efficacy in comparison to A.niger derived lactase. Furthermore, the enzymatic activity of the formulations is highly dependent on the composition itself which varies from company to company (Rodriguez-Colinas, Fernandez-Arrojo, Ballesteros, &Plou, 2014). Therefore, it is important to understand and appreciate the variety to give a prescription that best suits the patient.

In a recent case study to determine the efficacy of some of the products, it was discovered that all the products contributed to a significant reduction in the symptoms of lactose intolerant individuals. The products (Dairy ease, Lactacid, and lactogest) were administered to lactose intolerant patient at set dosages. Four capsules of Lactogest, two tablets of Lactacid or two tablets of Dairyease amounting to approximately 6000IU and 3000IU equivalent to two capsules of Lactogest. From the observations, all the enzyme supplements were able to significantly reduce the symptoms when 20g of lactose were administered to the patients. However, when a new product, a beta gel was introduced in the study, in both 3000UI and 6000UI, no positive results were observed in patients who took 50g of lactose. This study goes ahead to prove the differences in performance of these drugs and their sustainable thresholds of lactose in lactose-intolerant patients.

3. Pancreatic Enzyme Supplementation

EPI, often associated with many pancreatic diseases and disease directly related to the pancreas, is a serious, life-threatening condition affecting millions of people annually. These conditions include; Cystic fibrosis, pancreatic cancer, chronic and acute pancreatitis and Schwachman. In some circumstances, EPI symptoms are as a result of gastrointestinal and pancreatic surgery. The most visible of EPI is a sudden and significant loss of weight (Perano, Couper, Horowitz, Martin, Kritas, Sullivan, & Rayner, 2014). With the inclusion of patients with high daily fecal fat excretion content and other steatorrhea-related symptoms, patients require enzyme therapy.

Pancreatic enzyme supplements are also indicated for abdominal pain relief in pancreatitis. These exogenous enzymes are intended to provide negative feedback and regulations on the secretion of endogenous enzymes (D’Haese, Ceyhan, Demir, Layer, Löhr, &Foerster, 2014). However, this remains a matter of investigations, in particular with the noticeable reduction in duct pressure in the pancreas. Clinical trials and experimentations are ongoing to establish a criterion for predicting the clinical response in affected patients.

1.    Features of Pancreatic Enzyme Supplementation

There are three distinct groups of pancreatic enzymes, divided according to their functions. These are proteolytic enzymes, amylolytic enzymes, and Lipolytic enzymes. Proteolytic enzymes are mainly composed of trypsinogen and chymotrypsinogen and their active forms trypsin and chymotrypsin. The amylolytic enzymes include the pancreatic amylase responsible for the further breakdown of starch. The lipolytic enzymes are composed of principal lipase in charge of the breakdown for further digestion of lipids (Perano, Couper, Horowitz, Martin, Kritas, Sullivan, & Rayner, 2014). The exogenous pancreatic enzymes are extracted from bovine or porcine sources. Pancreatic enzymes are also synthesized from microbial sources such as Rhizopus arrives and Aspergillus.

As indicated in various studies, microbe-derived enzymes have far much better advantages compared to animal-derived enzymes. For instance, microbe-derived enzymes require far much lower dosages to be effective and operate within a broader pH range. Pancreatic enzyme supplementation is available commercially. In commercial formulations, the supplements are available either as ‘enteric coated’ or ‘nonenteric coated’. The primary purpose for these enzymes being enteric coated is to enable them to pass safely through the acidic milieu and linings of the stomach and duodenum. It is important to note in this context, which the efficiency by which these enzymes work, decreases significantly with a reduction in ph. The enzyme lipase, for instance, is completely denatured when the ph. falls below 4.

The FDA, the agency in charge of safety and standardization of medical drugs in the United States, initially had no restrictions and did not place any notable emphasis on the safety and efficacy data. However, new FDA regulations were put in place that required the submission, to the FDA, of new drug investigation application and approved clinical trials to endorse the use of pancreatic enzyme formulations. As a result of these new guidelines, several pancreatic enzyme formulations were removed from the market. However, six products: Pancreaze, Ultresa, Pertzye, Viokace, Creon, and, Zenpep managed to get the FDA approval and are currently available in the market.

Advance in scientific research and technology has led to new forms of pancreatic enzyme supplements. Liprotamase is one such outcome of these advancements. A biotechnology-derived enzyme, liprotamase is a non-porcine supplement that has three stable enzymes. Crosslinked crystalline lipase, amorphous amylase, and crystalline protease. Each of these enzymes is resistant to proteolysis and is stable in low ph.; therefore, no coating is needed. In clinical trials of this drug, patients diagnosed with cystic fibrosis were given a dosage of one capsule per day. The drugs were well tolerated in the patients. Furthermore, there was increased fat and protein absorption vis a vis reduction in stool weight in these patients.

2.    Dosages of Pancreatic Enzyme Supplementation

Before placing a patient on pancreatic enzyme supplement, the doctor or physician should assess the etiology as well as the severity of the pancreatic disorder and symptoms of the patient. Other clinical features such as body weight, age, genotype and other factors need to be taken into a lot of consideration. As indicated earlier, pancreatic enzyme dosages contain lipase enzyme. For patients diagnosed with steatorrhea, a minimum dose of about 25000 to 50000 U is required, mostly before meals. In cystic fibrosis patients, a dosage of between 500 to 3000 U of lipase for everyone is suitable. For children, a dosage of less than 6000 and 10000 U of lipase per kilogram per day is recommended. The reason for this low dosage in children is that children under the age of five years consume less fat per kilogram and therefore require fewer enzyme dosages.

3.    Enzymatic Activity

The enzymatic activity of pancreatic enzyme supplements is dependent on a number of factors. These include the species of the animal from which it was derived, its age, sex, and husbandry. For instance, research indicates that the human pancreatic physiology is much similar to that of pigs than to any other animal species (Timmerman, Holton, Yuneva, Louie, Padró, Daemen&Polyak, 2013). With enzymatic activity approximately fifty percent, these enzymes are preferred over those derived from beef. However, it is important to note that the commercial formulations differ regarding enzyme content, that is to say, the either contain lipase, amylase or protease.

4. Enzyme Supplementation in Celiac Disease.

Celiac disease is associated with acute inflammation of the small intestine, brought about by ingestion of gluten (Rubio-Tapia, Hill, Kelly, Calderwood, & Murray, 2013). The peptides of this gluten are usually rich in glutamine and proline and thus cause the body’s immune system to react, especially in those patients that are genetically intolerant to gluten. This intolerance is often found in variants rye, wheat, and barley. Notably, while there are medical approaches to treating celiac disease, most doctors and physician recommend a gluten-free diet as the best remedy for the management of celiac disease (Ivarsson, Myléus, Norström, van der Pals, Rosén,  Högberg, &Karlsson, 2013).

Recent research and clinical trials have identified prolyl endopeptidase as a possible therapy for celiac disease (DiGiacomo, Tennyson, Green, &Demmer, 2013). Prolyl endopeptidase, or PEP, are proteases capable of breaking down proline found in peptides. The main reason for their effectiveness is their capacity and ability to enhance the breakdown of gluten peptides. However, commercial preparations and formulations are currently not in the market. More intensive and conclusive research need to be conducted before it is cleared for use in human beings.

5. Erwinia Asparaginase in Treatment of Acute Lymphomatic Leukemia

With the absence of guidelines for the use of asparaginases, the relative efficacy of these drug has become a bone of contention (Plourde, Jeha, Hijiya, Keller, Silverman, Rheingold, & Corn, 2014). Studies conducted using Erwinia asparaginase, and other native strains of asparaginase preparations on dosages and treatment schedules have so far had an inconsistent result (Tong, Pieters, Kaspers, te Loo, Bearings, van den Bos, & Missing, 2014). Therefore, it is recommended that more studies are conducted to demonstrate the efficacy of Erwiniaasparaginase and provide the necessary guidelines on its optimal use (Tong, Pieters, Kaspers, te Loo, Bearings, van den Bos& Missing, 2014).

		North America, UK, Australia and New Zealand	Europe (BFM zone)	Rest of World
Children	Naïve patients	First-line: PEG-asparaginase	First-line: E. coli-asparaginase	First-line: E. coli-asparaginase
		Second-line: Erwiniaasparaginase	Second-line: Erwiniaasparaginase or PEG-asparaginase	Second-line: Erwiniaasparaginase or PEG-asparaginase
	Relapsed	First-line: PEG-asparaginase	First-line: PEG-asparaginase	First-line: E. coli-asparaginase
		Second-line: Erwiniaasparaginase	Second-line: Erwiniaasparaginase	Second-line: Erwiniaasparaginase or PEG-asparaginase
Adults	Naïve patients	First-line: E. coli-asparaginase or PEG-asparaginase	First-line: E. coli-asparaginase or PEG-asparaginase	First-line: E. coli-asparaginase
		Second-line: Erwiniaasparaginase or PEG-asparaginase	Second-line: Erwiniaasparaginase	Second-line: Erwiniaasparaginase or PEG-asparaginase

BFM, Berlin-Frankfurt-Münster; PEG, polyethylene glycol; E. Coli, Escherichia coli

Table 2: Current regional use of asparaginase (Vrooman et al. 2013)

6. Human DNase in the Management of Cystic Fibrosis

Cystic fibrosis at its acute stages is associated with progressive lung destruction, infectious exacerbations and other respiratory symptoms (Li & Somerset, 2014). These symptoms are associated with the presence of a persistent form of bacteria and an accumulation of viscous purulent in the bodily airways within the lungs (Lazarus & Wagener, 2013). These purulent secretions are known to contain relatively higher concentrations of extracellular DNA, which is released by leukocytes (Sly, Gangell, Chen, Ware, Ranganathan, Mott, & Stick, 2013).

Recombinant human DNase (rhDNase)

Purulent sputum produced by people suffering from cystic fibrosis and other related respiratory diseases usually has high concentrations of DNA which are produced by degenerating polymorphonuclear leucocytes. Higher DNA content in CF mucus is what leads to an increase in the viscosity of the mucus and mucus elastic modulus (Patel et al. 2008). The addition of exogenous DNA to sputum increases both viscosity and elasticity.

Purulent sputum has both DNA and a large volume of broad-spectrum protease; these two are products of neutrophils. DNA ensures that the protease does not destroy the mucin (Slattery et al. 2000). The vulnerability of mucin is increased upon the removal of DNA and as such, it becomes prone to attack by protease.

There are over 1500 mutations of the CFTR gene that are currently known. Of all these mutations, the most common is a three base-pair deletion which leads to deletion of the phenylalanine residue at amino acid position 508 (this is known as the delta F508) (Bartelink et al. 2006). All these mutations can be divided into five main groups. Their categorization is based on their predicted and possible functional repercussions. Class I-111 is considered as severe mutations and they do not produce any functioning CFTR protection. The second category is class IV and V mutations which lead to mild pancreatic diseases. There are a few functioning CFTR proteins but they are in very small quantities. The first category of Class 1-111 is associated with severe clinical phenotype but genotype-phenotype relationship is variable with crucial heterogeneity in severity of lung disease (Ramjeesingh et al. 2003). The most lethal manifestation of cystic fibrosis is chronic progressive lung cancer. Though there are antibiotic therapies and chest physiotherapies which try to deal with the problem, it is one of the causes of disability and death. In every 2500 births, the occurrence of CF is one (Riordan, 2008).

It is however important to note that this ration accounts for the defective genes which are present in homozygous people. Most individuals are born with a defective CFTR gene and, they do not know (Hwang et al. 2013). A lot of research has been done on CFTR and CF and, this has resulted in several treatment procedures and possible cures. Airway secretions play a significant role in the respiratory dysfunction in cystic fibrosis. The thick tenacious secretions are viscous and it is not easy to expectorate them. Consequently, they block the airways and thus reduce the volume of air in the lungs while cutting down the expiratory flow rates (Randell, 2006). CFTR genes have very high concentrations of DNA (more than fifteen milligrams per milliliter) and this makes them very viscous and tenacious. These DNA are derived from disintegrated inflammatory cells in the body. Naturally, DNA has a tendency of forming viscous, thick, purulent secretions in cystic fibrosis which have same features as those present in conc. solutions of DNA. Even through mucus obstruction is believed to the main cause of CF lung disease, the chronic infection of the respiratory tract is a more destructive and dangerous process. The excess mucus becomes a breeding ground for the growth of pathogenic organisms such as Pseudomonas aeruginosa, Staphylococcus aureus and Haemophilus influenza and, once established, these organisms become impossible to get rid of. To prevent the damaging of the lungs, it is imperative to eliminate as much sputum as possible from the lungs every day (Lukacs & Verkman, 2012).

The clinical trials of recombinant human DNAse (rhDNase) were evaluated by cloning sequencing and expressing the human enzyme (Chia, Menzies & McKeon, 2013). The chosen test methods were in vitro ad showed the potential viscoelasticity of the rhDNase. Furthermore, this particular enzyme was known to reduce the adhesiveness as well as improve the mucociliary transportability in cystic fibrosis produced sputum. Further studies have shown the relative tolerance of this enzyme in individuals with symptoms of dyspnea, FVC, and FEV.

A new study, this time under placebo controls, was conducted in some adults and children with symptoms of cystic fibrosis (Altenburg, de Graaff, Stienstra, Sloos, van Haren, Koppers, &Boersma, 2013). The main aim of this study was to determine the direct effect that rDNase had in the risk posed by respiratory exacerbations, thus requiring parenteral antibiotics. In comparison to patients treated with placebo, human rhDNase treated patients, who received a daily dosage once or twice, experienced a reduced risk of respiratory exacerbations. Furthermore, patients under treatment with human rhDNase reported fewer days under treatment with parenteral antibiotics and subsequently, fewer days in the hospital over the duration of the study (Subbarao, Stanojevic, Brown, Jensen, Rosenfeld, Davis, &Ratjen, 2013). Notably, the inhalation of rhDNase did not result in a risk of anaphylaxis; however, upper airway symptoms such as pharyngitis, hoarseness and voice alterations were noted in some patients. However, these symptoms were mild and transient (Subbarao et al, 2013). Therefore, it was conclusive from this study that administration of rhDNase by aerosol means was safe and tolerable and led to a reduced risk of exacerbations due to infections, hence reducing the need for parenteral antibiotics, improving pulmonary action and generally, the well-being of the patient.

2.6.1  Clinical Efficacy of rhDNase enzyme therapy

In the clinical trials of rhDNase enzyme, scientist sought to confirm whether an increase in Polymerized DNA concentrations in purulent sputum, result in complications in patients with cystic fibrosis. The study further sought to establish whether the application of an aerosolized form of rhDNase therapy would result in a decrease in complications (Subbarao et al, 2013). The efficacy of rhDNase was further tested using well designed placebo-controlled and dose ranged treatment in patients with cystic fibrosis.

The dose-ranging phase of the study involved patients that were considered clinically stable. The studies were conducted over a period of 10 days displaying relatively positive results. The forced expiratory volume (FEV) test results showed significant improvements. For instance, on administration of 0.6 mg or 2.5 mg dosages of rhDNase, once a day, over a period of 10 days, FEV results improved by up to 15%. In another study, twice daily dosages of 2.5mg of rhDNase improved FEV outcomes by up to 13 percent. All these results are in comparison to placebo controlled results (Altenburg et al, 2014).

A four-year study with rhDNase was conducted to assess the long term effects of the therapy. The study subjects, were two groups, comprising of patients with cystic fibrosis, that had received rhDNase. The other group had a control group of patients who had not received this therapy. Notably, in patients who did not receive rhDNase experienced significant exacerbations compared to those who received rhDNase (Chia et al, 2014). These studies show that the rhDNase had a reliable effect on the long term.

2.6.2.              Controversies in rhDNase use.

rhDNase has shown tremendous results in both short-term and long-term studies. Reductions in exacerbations were reduced by up to 13% during the course of study. However, not all cystic fibrosis patients undergoing treatment with this therapy showed significant improvement. In fact, there is distinct variation in patient response to the therapy (Altenburg et al, 2013). Though more trials are currently underway, possible treatment regimens and strategies to reduce the failure of rhDNase in select cases are already in place.

Among such proposals, are the nebulizers used in aerosol administration of rhDNase. State rules and regulations specify the guidelines on the nebulizer that is appropriate for use. In addition to that, in comparison using FEV results, alternate day administration of rhDNase, achieved similar results to those of daily dosing. However, the study was conducted over a short duration, thus the outcome is not very conclusive.

Anti-inflammatory therapy for Cystic Fibrosis

Intense, neutrophils-dominated inflammation is characteristic of the cystic fibrosis airway phenotype and it is one of the therapeutic means which has not been fully utilized. There were trials which were conducted using alternate-day systemic corticosteroids and the results indicated that there were some benefits incurred through the use of prednisone. It improved the functioning of the lung. Several studies have also been conducted on inhaled steroids and the results have shown that they improve and enhance the air path reactivity (Tannenbaum et al. 2007). In addition to that, nonsteroidal anti-inflammatory drugs are also beneficial when it comes to therapeutic treatment of cystic fibrosis. A high dose of ibuprofen increases the pulmonary function especially for children who are aged between five to thirteen years. The use of N-acetylcysteine as treatment of cystic fibrosis is also being done. It has the ability of restoring the levels of glutathione which reduce because of the presence of neutrophils and elastase activity present in the sputum.

Pancreatic enzymes preparations (PEP)

Pancreatic enzymes preparations are mostly used as enzyme replacement therapy. They have played a crucial role in enhancing the nutritious status of patients suffering from cystic fibrosis. These pancreatic enzymes contain amylase, lipases and proteases mixed in different quantities (Wong et al. 2012). PEPs come in form of capsules which have a enteric coated microencapsulated enzymes which can be microspheres or minitablets. They have a film which is resistant to acid and it ensures that the enzymes are not inactivated by gastric and upper intestinal acidity.

Bio-repair

This is also referred to as gene therapy. It has stood out as one of the possible cures for cystic fibrosis. The transfer of CFTR gene to the epithelium of the cystic fibrosis air paths is possible (Derichs, 2013). Gene therapy has the ability of correcting the CFTR defect in the bile duct cells. The high mitotic rates of the labile cells which are in the lung airways and in the gastrointestinal tract have to be dealt with to make sure that that there long-term expression of the transferred gene.

Tob ramycin

Tob ramycin is one of most successful anti-pseudomonal treatment for CF since it enhances and improve s the functioning of the lungs through the control of infections caused by bacteria and eliminating viscous secretions. It provides greater access to the lungs (Wong et al. 2005). Acute lymphocytic leukemia is a type of cancer where the bone marrow produces lymphocytes which are not mature. It affects the cells in the body severely.

Conclusion

The application of enzyme therapy in treatment and disorders, especially in chronic terminal diseases has increased in the last decade. Asparaginase for instance is slowly being adopted as the drug of choice in the treatment of acute lymphoblastic Leukemia. Although cases of hypersensitivity to asparaginase are reported, they are mild and manageable, indicating the safety of these drugs. The rhDNase enzyme is also showing significant improvements. For patients with Cystic Fibrosis, rhDNase is known to reduce severe exacerbations by up to 15% in both short term and long term trials. Although there are known side effects, the prevalence is still low, making the therapy a much safer enzyme to use. For lactose intolerant patients, lactase enzyme has proven effective so far. The clinical trials with a variety of dosage levels are positive in clinical trials. Pancreatic enzyme supplements in all three forms; proteolytic enzymes, amylolytic enzymes, and Lipolytic enzymes show varied results in clinical trials. However, recent FDA standards and requirements have led to the absence of certain pancreatic enzyme supplements from the market. Although with proper clinical trials and assessment they can resume their presence in the American market. Therefore, it is possible to see that therapeutic enzymes indeed open a new frontier in the world of medicine. More studies and clinical trials are required to assess the efficacy and safety of these drugs. However, current results are proving promising. Thus the medical should begin to adopt the usefulness of this drug to their treatment regimen to improve the outcomes and treatment of their patients.

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