Nanoscale analysis of protein and peptide absorption: Insulin absorption using complexation and pH-sensitive hydrogels as delivery vehicles
Introduction
Major innovations in the drug delivery field have surfaced in recent years. Oral administration of therapeutic agents is the preferred means of delivering drugs because of ease of administration, low cost and high patient compliance. However, formulating a drug for oral delivery is a complicated process. Poor intrinsic protein permeability as a result of large molecular weight, degradation by proteolytic enzymes in the stomach and in the small intestine, and chemical instability are some of the major hurdles for developing effective formulations for delivery of peptides and proteins. Although there are several success stories in the development and commercialization of oral dosage forms for small molecules, very few oral delivery systems have been developed for proteins and peptides. The oral formulation of cyclosporin (Tan et al., 1995) is one of the very few examples of successful development of oral formulations for peptide drugs.
Here we evaluate critically novel methods for administration of insulin for the treatment of diabetes. Since its initial administration to humans in 1922, insulin has been the cornerstone of type 1 diabetes. Conventionally, insulin is administered by subcutaneous injections which mimic, as close as possible, secretion of insulin by healthy pancreas. However, due to compliance-related issues and other complications, more acceptable delivery systems are highly desirable. Special attention is given to oral insulin delivery which is the focus of this work.
Diabetes is characterized by the body's inability to produce or properly use insulin. Diabetes mellitus is a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. There are 18.2 million people in the United States, or 6.3% of the population, who have diabetes (McCarthy, 2004). Diabetes mellitus is occurring in epidemic proportions in many developing and newly industrialized countries (Simpson et al., 2003). Globally, it is now one of the most common non-communicable diseases and is the fourth or fifth leading cause of death in most developed countries (Park, 2004). The global burden of diabetes is estimated to rise from about 118 million in 1995 to 220 million in 2010 and 300 million in 2025 (Amos et al., 1997, King et al., 1998).
The aetiological types of diabetes are designated as type 1, type 2 diabetes and other specific types of diabetes resulting from genetic defects of β-cell function, genetic defects in insulin action, endocrinopathies (resulting from over secretion of insulin antagonizing hormones), infections and drug- or chemically induced diabetes. Further, in nearly 3–5% of all pregnancies, women develop gestational diabetes (Anon., 1999, Gabbe and Graves, 2003).
Type 1 diabetes can be classified as autoimmune diabetes mellitus or idiopathic type 1 diabetes. Autoimmune diabetes mellitus is characterized by the autoimmune-mediated destruction of the insulin secreting pancreatic β-cells. Individuals suffering from this form of type 1 diabetes typically become dependent on insulin for survival and are at a risk of ketoacidosis, a condition resulting from extremely high levels of blood glucose (over 249 mg/dl) wherein the body begins to burn fat and muscle for energy which causes release of ketone bodies in the bloodstream. Idiopathic type 1 diabetes is a diabetes of unknown origin. Some of the patients suffering from this disease have permanent insulin deficiency and are prone to ketoacidosis, but this form of diabetes shows no evidence of autoimmunity (McCarthy, 2004).
Type 2 is the most common form of diabetes and is characterized by disorders of insulin action and insulin secretion, either of which may be the predominant feature. Individuals suffering from this form of diabetes are typically resistant to the action of insulin (Lillioja et al., 1993). Nearly 16 million individuals in the United States have type 2 diabetes while about one-third of those people are not aware that they have the disease (McCarthy, 2004). This is primarily because the hyperglycemia is often not severe enough to be symptomatic. Ketoacidosis is relatively infrequent in this type of diabetes. Nevertheless, individuals suffering from this form of diabetes are at increased risk of developing macrovascular and microvascular complications. If untreated, type 2 diabetes can cause serious complications, including kidney failure, blindness, heart attack and lower-limb amputation (Anthony et al., 2004). Fortunately, in many cases, type 2 diabetes can be adequately controlled through a combination of proper nutrition and exercise. However, some people with type 2 diabetes do require oral medications or insulin injections.
To determine if a patient is normal, pre-diabetic or diabetic, health care providers conduct a fasting plasma glucose test (FPG) or an oral glucose tolerance test (OGTT) (Genuth et al., 2003). Either test can be used to diagnose pre-diabetes or diabetes. Pre-diabetes, characterized by a glucose level between that of a healthy individual and a diabetic patient, is also called impaired glucose tolerance (IGT) or impaired fasting glucose (IFG) (Benjamin et al., 2003).
The FPG test is easier, faster and less expensive to perform. With the FPG test, a fasting blood glucose level between 100 and 125 mg/dl indicates pre-diabetes. A person with a fasting blood glucose level of 126 mg/dl or higher is considered diabetic (Genuth et al., 2003).
In the OGTT test, a person's blood glucose level is measured after a fast and 2 h after drinking a glucose-rich beverage (2-h PG value). Two-hour blood glucose level of 140–199 mg/dl signals pre-diabetes, whereas the 2-h blood glucose level of >200 mg/dl, signals diabetes (Genuth et al., 2003). Although the OGTT (which consists of an FPG and 2-h PG value) is recognized as a valid way to diagnose diabetes, the use of the test for diagnostic purposes in clinical practice is limited because of its inconvenience, less reproducibility, greater cost (Genuth et al., 2003, Stern et al., 2002).
Curative therapy for diabetes mellitus mainly implies replacement of functional insulin-producing pancreatic beta cells, with pancreas or islet-cell transplants. This is the only replacement therapy that can improve metabolic control other than conventional and intensive insulin therapy (de Groot et al., 2004). Transplantation can be performed either by implantation of the pancreatic organ or by implantation of only the pancreatic islets of Langerhans. Until recently, pancreas transplantation was considered to be the only viable procedure for autonomous regulation of glucose level (de Groot et al., 2004, McChesney, 1999). However, to avoid pancreatic graft rejection, transplantation has to be coupled with lifelong immunosuppression which itself can cause severe side effect as increased susceptibility to viral, fungal and bacterial infections, and increased risk for the development of malignancies (de Groot et al., 2004, Penn, 2000, Vial and Descotes, 2003).
Islet transplants are beneficial compared to implantation of pancreatic organ since they avoid the need for major surgical procedures. A small mass of islet cells can be delivered to the liver through intraportal infusion. The procedure for islet cell transplantation was initially successful in small animal models, but was not effective in humans (Ballinger and Lacy, 1972, Street et al., 2004). However, Shapiro et al. (2000) recently developed a new immunosuppressive regimen and reported a 100% success rate in achieving insulin independence through islet transplantation in seven long-term diabetic patients. Unfortunately, like pancreatic graft transplants, islet transplantation also suffers from the necessity of lifelong immunosuppression. Another limitation of islet cell transplantation procedures is the need for adequate supply of donor islet cells. One solution to this problem that is currently an area of active research is differentiation of embryonic stem cells into the insulin secreting islet cells by manipulating the culture conditions (Hussain and Theise, 2004, Street et al., 2004). However, the precise factors and conditions that convert progenitor stem cells into the desired mature β-cells are not yet fully understood (Bouwens, 2004).
Researchers have sought to circumvent the need for immunosuppressant regimen in islet transplantation by encapsulation of the islet cells by semipermeable membrane that protects the grafts from the host immune system (Opara and Kendall, 2002, Soon-Shiong, 1999). In addition to being immunoprotective, these membranes also have to be mechanically stable, biocompatible and sufficiently permeable to insulin and should also allow oxygenation of the encapsulated cells.
For more than a decade following its discovery, insulin was used in the form of crude extracts of the pancreas of cow, pig or sheep for diabetes treatment (Dominguez and Licata, 2001). Insulin was first purified by crystallization in the presence of zinc in 1936 (Scott, 1934).
The primary structure of insulin as elucidated by Sanger (1959). Although the amino acid sequence of insulin varies among different species, certain sequences are highly conserved (Mohan, 2002) as shown in Fig. 1. The sequences porcine insulin and human insulin are almost identical, differing by only one amino acid whereas bovine insulin differs by three amino acids from the human analog. However, none of the variations in the amino acid sequence are at sites crucial for the activity and function of insulin (Mohan, 2002).
When insulin is synthesized by the beta cells of the pancreas, it is produced as a large preproharmone. This preproharmone has a molecular weight of about 11,500. It is cleaved within the cisternae of the endoplasmic reticulum of the β-cells to form proinsulin which has a molecular weight of about 9000. This molecule then splits into two pieces: insulin, with a molecular weight of 5808 in humans, and C-peptide, before being secreted outside the cells through the secretory granules. The secreted insulin has 31 amino acid long B chain and the 20 amino acid long A chain which are locked in their relative conformation by two disulfide bonds. The cleaved and secreted insulin is 51 amino acids long and has a hydrodynamic radius of about 20 Å (Oliva et al., 2000).
The plasma glucose level is maintained within a very narrow range of 3.5–7.0 mmol/l in spite of wide fluctuations introduced by food intake, exercise, other physiological and physiological disturbances (Owens et al., 2001). This glucose homeostasis is achieved by regulating release and inhibition of glucagons and insulin, both secreted by the pancreatic cells.
Increase in blood glucose triggers secretion of insulin from the pancreas. Insulin secreted from the pancreas is infused via the portal vein to the liver, where it leads to an increase in the storage of glucose with a concomitant decrease in hepatic glucose release to the circulation (Arbit, 2004). Insulin circulates in the blood with plasma half-life of about 6 min, so that it is almost entirely cleared from the circulation within 10–15 min. The freely circulating insulin then acts on several peripheral tissues including muscle, liver and fat tissue by binding to the specific insulin receptors on cells.
An insulin receptor is a hetero-tetramer with a molecular weight of about 300,000. The two alpha subunits lie entirely outside of the cell membrane and the two beta subunits penetrate through the cell membrane. Insulin binding to the alpha subunits on the outside of the cell triggers autophosphorylation of the beta subunits on several tyrosine residues protruding into the cytoplasm.
The activated beta subunits then phosphorylate and activate IRS-1. IRS-1 is an enzyme and a key mediator of insulin's biological activity. Following this, various intracellular regulators are recruited to IRS-1 and this initiates a regulatory cascade of signals with each molecule binding to IRS-1 activating its own complex cascade (Hill et al., 2004). This finally leads to increase in the permeability of cell membranes to glucose and glucose uptake increases. This increased uptake of glucose due to insulin binding occurs in the muscle cells, adipose cells and other types of cells in the body constituting about 80% of all the cells.
In addition to its role in uptake of glucose, insulin also stimulates conversion of carbohydrate or proteins to fat (lipogenesis), and increases amino acid transport into cells. It also stimulates growth, DNA synthesis and cell replication.
In the absence of curative therapy for the treatment of diabetes, insulin replacement therapy is required for all people with type 1 diabetes (McAulay and Frier, 2003). Benefits of insulin therapy in patients with type 2 diabetes are also well recognized now (Group, 1998, Malmberg, 1997, Ohkubo et al., 1995). Intensive glucose control delays the onset and retards the progression of microvascular and macrovascular diseases in patients with type 2 diabetes (Mudaliar and Edelman, 2001). Insulin therapy is, therefore, central to management of patients with diabetes. With the development and approval of recombinant technologies for insulin production, human insulin could be made biosynthetically which became the preferred method of insulin production. This also led to the development of mutant insulin analogues having improved pharmacokinetic properties for subcutaneous administration (Barnett and Owens, 1997, Bolli et al., 1999, Brange and Volund, 1999). However, for over 80 years injection or infusion of insulin into the subcutaneous (s.c.) tissue has been the only route of insulin delivery used in clinical practice. This mimics, as close as possible, secretion of insulin by healthy pancreas.
Recent developments of improved injection devices, such as insulin pens, and very sharp needles, have reduced the pain associated with the injection therapy to a considerable extent. But even with these developments, injection therapy still requires the handling of a device and is associated with pain. This often results in low patience compliance to the therapy. Clinical studies have shown that because of the non-compliance-related issues, even on insulin treatment, a significant percentage of patients fail to attain lasting glycemic control (Arbit, 2004). Furthermore, this route of insulin administration has associated side effects, such as hyperinsulinemia and localized deposition of insulin that lead to local hypertrophy and fat deposits at injection sites (Skyler, 1986).
Section snippets
Alternative routes of insulin administration
The current recommended intensive insulin therapy regimens involve multiple subcutaneous insulin injections everyday. These increases compliance constrains on patients to a great extent and reduce the overall efficacy of treatment. Hence, as discussed above, non-invasive insulin formulations that mimic physiological secretion of insulin as a means of maintaining the glucose homeostasis are highly desirable. Recent advances in alternative routes of administration as an approach to improve
Oral protein delivery
Of all the routes of delivering drugs, the oral route is the most desirable. This route of administration results in higher patient compliance than any other route of administration. If successful, this delivery system will solve the current non-compliance-related problems associated with insulin injections. The ease of this delivery system has led to several attempts to develop oral insulin formulations (Heinemann et al., 2001). Another great advantage of this route of administration is that
Conclusions
A variety of approaches have emerged in the recent past for designing oral delivery systems for therapeutic proteins and peptides, although a clinically viable solution to this long standing problem still alludes the scientific community. These approaches come from such diverse research disciplines as biomaterials, conjugation chemistry, nanotechnology, cell biology and employ different methodologies for solving the same problem. Some of these strategies have distinguishing beneficial
Acknowledgment
This work was supported by grant R01 EB000246 from the National Institutes of Health of the USA. We kindly acknowledge technical support by our colleague Dr. Jennifer Lopez who drew Fig. 1, Fig. 2, Fig. 3.
References (127)
- et al.
Resistance of metal complexes of conalbumin and transferrin to proteolysis and to thermal denaturation
J. Biol. Chem.
(1958) - et al.
Transferrin receptors in the human gastrointestinal tract. Relationship to body iron stores
Gastroenterology
(1986) - et al.
Insulin analogues
Lancet
(1997) The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins
J. Control. Rel.
(1998)- et al.
Insulin analogs with improved pharmacokinetic profiles
Adv. Drug Deliv. Rev.
(1999) - et al.
Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood–brain barrier of blood-borne transferrin and antibody against the transferrin receptor
Exp. Neurol.
(1996) - et al.
Ultraflexible vesicles, transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin
Biochim. Biophys. Acta
(1998) - et al.
Lectin-mediated mucosal delivery of drugs and microparticles
Adv. Drug Deliv. Rev.
(2000) - et al.
Oral modified insulin (HIM2) in patients with type 1 diabetes mellitus: results from a phase I/II clinical trial
Metabolism
(2004) - et al.
Causes of limited survival of microencapsulated pancreatic islet grafts
J. Surg. Res.
(2004)