Past Present Future Salivary Glands Drug Delivery System Biology Essay

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Tissue engineering is a highly exciting field of research that aims to repair damaged tissues as well creates replacement organs. Recently there has been a growing public awareness of a relatively new field of applied biological research called tissue engineering. This field builds on the interface between materials science and biocompatibility, integrates cells, natural or synthetic scaffolds, and specific signals to create new tissues (Mooney DJ, Rowley et al; 1997). This field is increasingly being viewed as having enormous potential (Ferber D, Laufrenburger D A; 1999)

Some of the earliest attempts at the tissue replacement dating back thousands of years, involved teeth (Craig RG; 1996). In modern times, dentistry has continued to place considerable emphasis on, and be a leader in the study and use of biocompatible materials.

Tissue Engineering Takes Different Forms:

Clinical problems relating to the loss or failure of tissues extend beyond dentistry to all field of medicine. Currently the replacement of lost or deficient tissues involves prosthetic materials, drug therapies, and tissue and organ transplantation. But these prosthetic materials can not replace simple structures like tissues. Such problems have motivated the development of tissue engineering, which can be defined as a combination of the principle and methods of the life sciences with those of engineering to develop materials and methods to repair damage or diseased tissues and to create entire tissue replacements.

Many strategies have been used to engineer new tissues and organs but in almost all we have to combine a material with either bioactive molecules that induce tissue formation or cells grown in the laboratory. The bioactive molecules are frequently growth factors proteins that are involved in natural tissue formation and remodeling, the basic hypothesis underlying this approach is that the local delivery of an appropriate factor at a correct dose for a defined period of time can lead to recruitment, proliferation and differentiation of a patient's cells from the adjacent sites. These cells can then participate in tissue repair and regeneration at the required anatomical locale.

The second general strategy uses cells grown in the lab and placed in a matrix at the site where new tissue or organ formation is desired. These transplanted cells usually are derived from a small tissue biopsy specimen and have been expanded in the laboratory to allow a large organ or tissue mass to be engineered. Typically, the new tissue will be formed in part from these transplanted cells.

In both approaches specific materials deliver the molecules or cells to the appropriate anatomical site and provide mechanical support to the forming tissue by acting as a scaffold to guide new tissue formation (Shullman L B et al; 1997).

Drug Delivery Technology Being Used:

The number of products based on new drug delivery systems has significantly increased in the past few years, and this growth is expected to continue in the near future. The latest Biopharmaceuticals present a challenge to drug delivery scientists because of their unique nature and difficulty in delivery through conventional routes. Therefore nowadays the stress in on the delivery of these complex molecules through different routes, including oral, nasal, pulmonary etc

Gene Therapy:

Generally gene therapy is not considered to be an example of tissue engineering. However, gene transfer to well differentiated cells arguably can be viewed as a way to engineer a tissue. Gene transfer has been used for about 10 years and began with the treatment of two children suffering from a severe combined immunodeficiency resulting from an inherited reduction in the enzyme adenosine deaminase, or ADA. These patients were treated with the gene therapy and ADA gene was transferred into their own lymphocytes in the lab, followed by the return of these cells to the patients. To date both the patients have survived.

Re-engineering is used in restoring or altering of a tissue's natural functions. It includes the development of any application of modern biology leading to a health-related beneficial phenotypic change in the tissue (salivary glands) of interest. Two general methodologies are currently used to accomplish such a result, termed gene

Therapy and tissue engineering. Gene therapy refers to any technical approach used for transferring a gene to a patient for clinical benefit. Gene transfer can occur by direct delivery to a patient in vivo, or can result from the transfer of a gene to cells in the laboratory. The latter can be the patient's own cells or cells from another source, and gene transfer is followed by the delivery of the cells to the patient, i.e., ex vivo gene transfer (Verma and Somia, 1997; Anderson, 1998)

Tissue engineering uses biological and engineering concepts and methods to develop materials to repair damaged or diseased tissues, and to create entire tissue replacements (Langer and Vacanti, 1993;Nerem and Sambanis, 1995)

Re-engineering The Salivary Gland:

The primary role of salivary glands in human biology is to produce an exocrine secretion, saliva, which provides much of the innate host defense for the upper gastrointestinal tract (Mandel, 1998).Many salivary gland proteins are antimicrobial that can control the bacterial fungal and viral population with in the oral cavity. The other main functions include the remineralization of enamel, forming the acquired pellicle, and role in maintenance and repair of mucosal structure. Saliva also acts as a solvent which dissolves tastants for the presentation of the taste buds. It also protects the host from consuming spoiled and potentially harmful food. Additionally, saliva is critical for the food bolus formation, translocation, and initial processing.

Thus it can be concluded that any condition causing a decrease in the saliva production is highly undesirable and will have a negative impact on the patient. Clinically there are 3 major conditions for the reduced saliva output.

Iatrogenic or Pharmaceutically Induced Hypo secretion (Sreebny & Schwartz,1986; Atkinson & Fox , 1992)

Therapeutic Radiation for Head & neck Cancer.

Sjogren's syndrome.

The first one is reversible and is usually corrected by change in dose or medication type, or is accepted as an unfortunate but necessary side effect for treatment of a more threatening condition. While Sjogren's syndrome is an auto immune exocrinopathy. This condition effects the female population as compared to the males. Both these conditions can cause irreversible changes in the parenchyma, loss of fluid and proteins secreting acinar cells. At present, there is no adequate treatment for the patients with irreversible gland damage, and major reason for reengineering salivary glands is to offer these patients better treatment options.

There are also at least 2 other reasons to re engineer salivary glands. First, Salivary glands are secretory tissues with the ability to produce and deliver considerable quantities of proteins intended to function at distant sites. While the bulk of this activity is exocrine in nature, salivary glands can secrete in an endocrine manner and lead to systemic effects (Kaqami et al; 1996, Goldfine et al;, 1997, He at el, 1998; Baum et al, 1999)

Thus salivary glands are a worthy target tissue for engineering to take advantage of these protein secretory properties. This has the application for improving the treatment of either a systemic protein deficiency disorder or an upper GI tract condition. Second, salivary glands are a tissue with a relatively easy and virtually non invasive access (Mastrangeli et al, 1994; Delporte et al, 1997; Baum & O Connell, 1999). By cannulation of the main excretory ducts exiting in the mouth, direct contact with the luminal membranes of almost every epithelial cell present is achieved (Cook et el; 1994). Because of this circumstance, salivary glands provide a convenient experimental target for investigators in the field of re engineering of the epithelial tissue. Indeed, the oral cavity generally provides excellent models with easy access for many experiments in gene transfer and tissue engineering (Baum and Mooney, 2000)

Practically the re engineering of the salivary glands can have 4 goals.




Regenerate Gland Tissue

Gene Transfer To Salivary Glands:

Gene transfer can be accomplished by either viral or non viral methods (Verma & Somia; 1997). Viral methods have the advantage of providing much greater gene transfer efficiency but with a potential safety risk. Non viral methods, although currently much less efficient in accomplishing successful gene transfer, are considered to have less safety risk. It is not difficult to transfer genes to the salivary glands by means of intraductal cannulation methods for vector delivery (Mastrangeli et al, 1994). A wide variety of different genes have been successfully transferred to rodents in vivo (Baum et al; 1998). At present recombinant adenoviruses are the most effective vectors for transferring genes to the salivary glands (Baum & O Connell, 1999). With adenoviral vectors, a substantial proportion of glandular parenchyma cells, both acinar and ductal, can be infected after delivery through cannulated ducts. For example, (Delporte et al; 1997) reported that 15 to 30% of the epithelial cells in rat submandibular glands expressed the transgene product following adenoviral infection.

Two other recombinant viral vectors have been used for gene transfer to salivary glands. Barka and van der Neon (1996, 1997) used a retroviral vector to infect rat salivary glands. Gene transfer occurred, but transgene product expression was extremely low. Retroviral vectors require cell division for successful infection, and salivary gland cell divide very slowly in vivo. Braddon et al.1998 reported that a recombinant adeno- associated viral vector was able to mediate gene transfer to mouse salivary gland cells in vivo. AAV offers the potential for relatively stable gene transfer to both dividing and non dividing cells (Kotin, 1994; Flotte and Carter, 1995)

There have been only 2 attempts on gene transfer into a salivary gland to restore the function of the salivary glands from irreversible salivary gland damage. In both the cases the same experimental strategy was used with the same recombinant adenovirus and in both the cases glands had been damaged by ionizing radiation (Delporte et al; 1997) .The method which had been chosen actually requires some of the epithelial cells to survive in the gland along with reasonable maintenance of access through Stensen's duct. The irradiation leads to loss of fluid secreting acinar cells in the gland (Delporte et al; 1997) constructed the recombinant adenovirus AdhAQP 1 to transfer to aquaporin 1 to the irradiated rat submandibular glands.

GLP-1 -Glucagon Like Peptide 1:

Type 2 diabetes mellitus is one of the most prevalent metabolic disorders presently affecting most of the population worldwide. Some of the people are aware of their situation while some are still unaware. It results from the failure of the Beta cells to produce sufficient insulin to meet the demands of the body. Medications currently in use have limited success in controlling blood glucose levels, hence, the complications of the disease. At the same time there is a failure in the beta cell function as well is another aspect of the disease and we are currently unable to reverse the process. According to the latest research done the GLP 1 or the glucagon like peptide 1 is an effective method to lower the blood glucose levels in the diabetic patients.

Glucagon Peptide 1 :


GLP-1 is secreted by intestinal L cells in response to nutrients ingestion (Mojsov et al.; 1986). Exogenous administration of GLP-1 reduces blood glucose levels in both normal and diabetic patients (Nathan et al.; 1992) by in suppressing glucagon secretion (Komatsu et al; 1989)

Glucagon-like peptide 1 (GLP-1) is a hormone that is encoded in the proglucagon gene. It is mainly produced in enterendocrine L cells of the gut and is secreted into the blood stream when food containing fat, protein hydrolysate, and/or glucose enters the duodenum).

GLP-1 action in the p-cell is mediated by binding of the peptide to a specific, seven-membered transmembrane receptor (Thorens et al., 1993). Activation of this G-coupled receptor causes an increase in intracellular CAMP concentration (Drucker et al., 1987) and activation of protein kinase A (PKA). GLP-1 acts directly through the cAMP/PKA pathway to enhance and sensitize b-cells to glucose-stimulated insulin secretion (Figure 1). The metabolism of glucose in the p-cell causes an increase in the amount of adenosine triphosphate (ATP) and a rise in the cytoplasmic ATP:adenosine diphosphate (ADP) ratio, leading to closure of the KATP channels. GLP-1 may close the KATP channels via a PKA-mediated phosphorylation. These channels possess sites that are phosphorylated by G-protein stimulated PKA (Beguin et al., 1999). The ability of the specific PKA inhibitor Rp-CAMP to reverse the GLP-I action on the KATP channels confirms the involvement of PKA in this step (Holz et al., 1993).

Subsequent membrane depolarization results in the activation of voltage-de- pendent L-type Ca+ channels, an influx of Ca+, and exocytosis of the insulin-containing granules. GLP- 1 is known to cause a rise in intracellular Ca+ concentration [Ca+] (Yada et al., 1993). This effect is attributable both to greater activation of the L-channel, which could be due to its phosphorylation by PKA, and also to a mobilization of the intercellular stores of Ca+, which may or may not be PKA dependent (Bode et al., 1999). GLP-1 also increases insulin secretion in a calcium-independent manner by mobilizing secretory vesicles to enter the readily releasable pool. Again, this is by both PKA-dependent and -independent means (Gromada et al., 1998).

Glucagon like Peptide 1 Genetic Aspect:

Glucagon-like peptide (GLP)-1 is a peptide hormone that is expressed as part of the prohormone, proglucagon. In the intestinal endocrine L cells, proglucagon is specifically processed to generate GLP-1 (1-37) by prohormone convertase 1/3 (Dhanvantari S, Seidah NG, Brubaker PL; 1996), which is further cleaved to GLP-1 (7-37) by removal of its amino terminus. The carboxyl terminus can also be amidated (Orskov C et al; 1989); however, this posttranslational modification is not required for biological activity (3-5). Thus, GLP-1 (7-37) or GLP-1(7-36-amide) are the bioactive forms of GLP-1 released into circulation after a meal. GLP-1 acts as an incretin and functions to slow gastric emptying and enhance insulin function by increasing insulin secretion from pancreatic-cells and increasing cell mass. The biological half life of GLP-1 is about 2-3 min (Kieffer TJ et al; 1995). It is deactivated in the blood by protease and DDP IV.

GLP-1 & Diabetic Patients:

Because of the usefulness of GLP in diabetic patients specifically type 2, many approaches are being used to increase its effectiveness by generating noncleavable GLP-1 analogs, DDP IV inhibitors, GLP-1 receptors agonists and sustainable and ultimately regulatable levels of GLP-1 by gene therapy.

However for effective use large amount of GLP has to be given with frequent injection so as to compensate for the small biological half life. Where as for the gene therapy it has to be made sure that it passes through safely through plasmid so as to produce a constant supply bioactive GLP-1. Unfortunately these approaches have only been found successful in animals because in Human testing the repeated injections can cause severe pain while the gene therapy can cause transduction in several organs leaving few options if adverse effects develop.

Use of Gene Therapy in Salivary Glands:

Salivary glands are exocrine glands that have both the secretory pathways that allow the release of secreted proteins through the CSP into the bloodstream (endocrine) and similarly through the RSP into the Saliva (exocrine). Thus if there is a problem or deficiency in one of the system or pathway the other is by default present. Thus the salivary glands are an excellent target site for the gene transfer with the potential application for systemic treatment of single protein deficiency syndromes such as those involving GH, IGF-I and Leptin.

Use of the salivary gland for the gene therapy has several uses.

It has the capacity to synthesize a large amount of proteins.

The vector is not diluted after the retroductal delivery so the lower number of vector particles can be administered to obtain sufficient levels of the proteins required.

These glands are encapsulated so the vector administrated is well confined with in the limits.

Salivary glands diseases are not life threatening so if something or some adverse reaction does take place, a gland can be removed.

The vector administration can be accomplished as an outpatient procedure similar to the contrast x ray during sialography.

Gene Transfer and Testing of Insulin Level:

The tests performed by Votetakis et al were done on mice in which he transferred the genes in to the salivary glands of the mice and then tested the GLP-1 peptide and respective insulin levels as well. Both the submandibular glands of male mice were transduced by retroductal infusion with GLP-1. After 24 hours the mice were subjected to an overnight fast. This time only the serum was analyzed for glucose insulin level, GLP_1 level. Two groups of mice were transduced and subjected to and overnight fast. Serum was obtained and analyzed for GLP-1, Insulin and Glucose. The remaining mice per group were subjected to an IP injection of glucose and at 30 min after injection and sera were collected and analyzed for the same molecules. The results of the test have been plotted in the table below and have been represented in the figures.

Alloxan Treatment:

The submandibular glands of the two groups of mice were transduced with Ad GLP-1. One day later and after an overnight fast the mice were injected with alloxan to induce diabetes and blood glucose levels were monitored for 3 days. After the experiment, the submandibular glands were analyzed by hematoxylin and eosin staining. The changes were normal but were returning to normal as soon as the experiments were finished. The table given below shows the comparison between the groups in the glucose tolerance tests. The mice serum was checked for insulin, glucose and GLP-1 levels and compared. The difference between the Ad luc and the GLP-1 is given and shows the difference. The Test were performed Ad Glp-1 mediates the bioactive form of GLP-1 that is secreted consecutively from cells in culture and is resistant to degradation by DDP IV. Thus Ad-GLP-1 delivery to salivary gland would result in secretion of bioactive form of GLP-1 that would be released into the circulation and will have a longer biological half life.

Ref: Voutetakis A et al. Endocrinology. 2010 Sep; 151 Epub 2010 Jul 7

According to the tests the serum glucose and insulin level had no major difference in the groups under investigation. But when we measured 30 min after the glucose tolerance test the level if glucose and insulin increased but were significantly less compared with the control group. This showed that the Ad GLP-1 treated mice cleared the glucose more efficiently as compared to the control group thus having for insulin sensitivity.

Then the findings were extended to the mouse model having diabetes. Mice were transduced with Ad GLP-1 or Ad Luc vectors. After the overnight fast, the mice were given 1 injection of alloxan to cause diabetes. Glucose level was monitored for 3 days. With elevated levels of GLP-1 the mice developed diabetes but at a very increased level as compared to the Ad-Luc Mice. The expression of the transgenic GLP-1 construct in the submandibular provided the protection against the diabetic phenotype induced by alloxan.

GLP-1 and Salivary Glands Future:

The incretin function of GLP-1 has led to broaden its use in the treatment of diabetic type 2 patients. Since a lot of study has been done on the GLP-1 so a lot of new therapeutic approaches have been developed and have been based on increasing the efficacy of GLP-1 by either preventing it from being inactivated or providing more to the system.

A lot of work has been done on the tissue engineering but still a lot of work has be done till we reach the critical level of success. The neural and vascular supply has to restored as well for the proper functioning of the gland. All this engineering will be quite significant for the future development in dentistry especially in field concerned with mineralization of enamel and restoration of the function of lost structures. May be in future it might be possible that further work may be done to help work the salivary gland as a drug delivery system.