WO2015011653A1

WO2015011653A1 - pH RESPONSIVE ORAL POLYMERIC PHARMACEUTICAL DOSAGE FORM

Info

Publication number: WO2015011653A1
Application number: PCT/IB2014/063336
Authority: WO
Inventors: Pierre P D KONDIAH; Viness Pillay; Yahya Essop Choonara; Girish Modi; Pradeep Kumar; Lisa Claire Du Toit; Lomas K TOMAR; Charu TYAGI
Original assignee: University Of The Witwatersrand, Johannesburg
Priority date: 2013-07-23
Filing date: 2014-07-23
Publication date: 2015-01-29
Also published as: WO2015011653A9

Abstract

The disclosure relates to a pH responsive oral polymeric pharmaceutical dosage form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, the dosage form comprising CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles. In use exposure of the dosage form to media of increasing pH facilitates swelling of the particles, and exposure of the dosage form to media of decreasing pH facilitates constriction and/or aggregation of the particles.

Description

pH RESPONSIVE ORAL POLYMERIC PHARMACEUTICAL DOSAGE FORM FIELD OF INVENTION

The field of this disclosure relates to an oral polymeric pharmaceutical dosage form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, particularly this disclosure relates to a pH responsive oral polymeric pharmaceutical dosage form for site specific delivery of a protein and/or peptide to the small intestine of a human or animal body.

BACKGROUND OF INVENTION

A vast number of medical conditions require a high volume of pharmaceutically active ingredient for systemic distribution in the body. This systemic distribution is typically achieved through administration via intramuscular, subcutaneous, inhalation, intravenous or other parenteral routes in order to ensure effective therapeutic success. In the event where the pharmaceutically active ingredient is a peptide and/or a protein the abovementioned administrative routes, even at low concentrations, may have serious toxic side effects. It is known that the use of peptides and/or proteins as therapeutic agents is complicated by their instability and side effects.

Patient compliance is another hurdle in parenteral therapy due to many factors influencing side effects and experienced during therapy.

By way of example, in the treatment of multiple sclerosis (MS), a standard therapy includes the administration of a peptide compound such as interferon beta (INF-β). Currently, INF-β has effectively been used to treat MS via subcutaneous application or via intramuscular injection. Interferons exist naturally as globular proteins comprising 5 helices. They are reported as having a Mw of 20kDa, although often run on SDS-PAGE gels with an apparent Mw closer to 25kDa due to glycosylation (Arduini et al., 1999). Their coding region encodes a predicted protein amino acid count of 197 amino acids, consisting of a signal sequence of 32 amino acids and a mature INF-β of 165 amino acids (Iwata et al., 1996). The fundamental effect of INF-β in the treatment of MS is based on reducing the immune response that is directed against central nervous system myelin, i.e. the fatty sheath that surrounds and protects nerve fibers. Damage of nerve fibers, resulting in demyelination, consequently causes nerve impulses to be slowed or halted, thus producing symptoms of MS. (Jongen et al., 2011). This subcutaneous application via intramuscular injection is commonly associated with multiple problems including pain, allergic reactions, poor patient compliance and increased chance of infection (Chiu et al., 2007).

There exists a need to develop a more effective route of administration for peptides and/or proteins in therapeutic applications.

SUMMARY OF INVENTION

In accordance with a first aspect of this disclosure there is provided a pH responsive oral polymeric pharmaceutical dosage form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, the dosage form comprising:

CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) copolymer particles, wherein exposure of the dosage form to an increase in the pH facilitates swelling of the particles, and

wherein exposure of the dosage form to a decrease in pH facilitates constriction and/or aggregation of the particles.

The pH responsive oral polymeric pharmaceutical dosage form may further include a pharmaceutically active ingredient, such that in use, an increase in the pH facilitates swelling of the particles which in turn facilitates an increase in the release rate of the pharmaceutically active ingredient from the particles, and wherein a decrease in pH facilitates constriction and/or aggregation of the particles which in turn facilitates a decrease in the release rate of the pharmaceutically active ingredient from the particle. The pH referred to herein may be the pH of an environment in which the dosage form is present, typically a biological medium such as, for example, gastric fluid and/or intestinal fluid.

The CHT (chitosan) may be functionalized. The functionalized CHT (chitosan) may be trimethyl chitosan (TMC) such that the dosage form may comprise TMC-PEGDMA-MAA (trimethyl chitosan- poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles.

The pH responsive oral polymeric pharmaceutical dosage form may be crosslinked. Typically, crosslinking may be caused by microwave radiation, UV radiation or chemical crosslinking. Crosslinking, and the degree of crosslinking, effects release rates of pharmaceutically active ingredients when the dosage form is in use. It is known that the greater the degree of crosslinking the slower the release rates will be in use. The particles may release the pharmaceutically active ingredient in a pH dependent manner wherein an increase in pH facilitates a conformational change of the CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) or the TMC-PEGDMA-MAA (trimethyl chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) causing the particle to expand, in so doing, releasing the pharmaceutically active ingredient at an increased rate of release.

The particles may release the pharmaceutically active ingredient in a pH dependent manner wherein a decrease in pH facilitates constriction and/or aggregation of the particles such that release of the pharmaceutically active ingredient is inhibited and/or curbed, and wherein an increase in the pH facilitates swelling of the particles such that release of the pharmaceutically active ingredient is facilitated. The release rate of pharmaceutically active ingredient from the dosage form is greater when the dosage form is exposed to a higher pH environment relative to when the dosage form is exposed to a lower pH environment.

The particles may release the pharmaceutically active ingredient in a pH responsive manner wherein a decrease in pH increases electrostatic attraction causing constriction and/or aggregation of the particles and inhibiting and/or curbing release of the pharmaceutically active ingredient. An increase in pH increases electrostatic repulsion causing swelling of the particles and/or dispersion of the particles into the surrounding medium and release of the pharmaceutically active ingredient. In a preferred embodiment, an acid pH increases electrostatic attraction and a basic pH increases electrostatic repulsion.

Typically, the pharmaceutical dosage form may be pH responsive such that in use the particles of the dosage form swell when exposed to a medium of basic pH, and constrict and/or aggregate when exposed to a medium of acidic pH.

The target site may be the intestinal region of the human or animal body, preferably the small intestine, further preferably the muco-epidermal layer of the small intestine.

Typically, in use, the dosage form is orally ingested and upon entry into the gastric region of the gastrointestinal tract (GIT) the particles of the dosage form constrict and/or aggregate in response to the acidic medium of the gastric region therein inhibiting and/or curbing release of the pharmaceutically active ingredient. Upon entry of the dosage form into the intestinal region of the gastrointestinal tract (GIT) the particles of the dosage form swell in response to the basic medium of the intestinal region therein facilitating release of the pharmaceutically active ingredient. Release rate of the pharmaceutically active ingredient in the intestinal region, preferably the small intestine, is faster relative to release in the gastric region.

The pharmaceutically active ingredient may be any chemical and/or biological composition having pharmaceutical properties.

The pharmaceutically active ingredient may be, but is not limited to, a protein and/or a peptide. The protein and/or peptide may be linked and/or bonded to the particle. The peptide and/or protein may be at least one selected from the following group: interferon beta, salmon calcitonin, eel calcitonin, chicken calcitonin, rat calcitonin, human calcitonin, porcine calcitonin or any gene-variant of calcitonin, parathyroid hormone, parathyroid hormone analogue PTH 1-31NH₂, parathyroid hormone analogue PTH 1-34NH₂, insulin of any gene variant, vasopressin, desmopressin, luteinizing hormone -releasing factor, erythropoietin, tissue plasminogen activators, human growth factor, adrenocorticototropin, various interleukins, enkephalin as well as all known vaccines.

The peptide and/or protein may be at least one or more selected from one or more of the following compound classes: anti-inflammatories, immunosuppressives, antibiotics, antifungals, antivirals, antimalarials, antiretovirals, antihypertensives, chemotherapeutics, diagnostic agents, probiotics and prebiotics.

The pharmaceutical dosage form may be mucoadhesive to in use adhere to mucosa of the gastrointestinal tract (GIT), particularly in the intestinal region of the gastrointestinal tract (GIT). Mucoadhesion of the pharmaceutical dosage form facilitates absorption of the pharmaceutically active ingredient into the blood stream.

There is further provided that the pharmaceutical dosage form shows, in use in vitro, a release profile of about 2.5 hours when in acidic pH and a release profile of about 8 hours when in basic pH, in which a minor amount of pharmaceutically active ingredient is released in the acidic pH, thereby inhibiting a major amount of the pharmaceutically active ingredient from release and/or degradation in the acid pH, and allowing release of the major amount of pharmaceutically active ingredient in the basic pH.

There is further provided that over about 80% of the pharmaceutically active ingredient is released in intestinal region. Where the particles comprise CHT-PEGDMA-MAA a minimum of about 21.2% of the pharmaceutically active ingredient is released in gastric region, and where the particles are TMC- PEGDMA-MAA a minimum of about 4.03% of the pharmaceutically active ingredient is released in the gastric region.

The pharmaceutical dosage form may be non-toxic in vivo. In a preferred embodiment the pharmaceutical dosage form may comprise a multitude of particles. There is further provided that the particles may be biodegradable.

The pharmaceutical dosage form may be formed into a tablet, caplet or capsule. In a preferred embodiment the dosage form is formed into a tablet by direct compression of the particles. Further preferably, the tablet may be cylindrical in shape and/or dimension prior to use. The cylindrical tablet may have a diameter of about 4 mm.

In a preferred embodiment wherein the pharmaceutical dosage form is formed into a tablet the dosage form may have a high matrix resilience and a high matrix hardness.

Constriction and/or aggregation of particles at acidic pH inhibits and/or curbs release of the pharmaceutically active ingredient in the gastric region, therein facilitating concentration of the pharmaceutically active ingredient at the target site (preferably the intestinal region, more preferably the small intestine) to ensure a steep concentration gradient of the pharmaceutically active ingredient from the muco-epidermal layer of the small intestine into the blood stream for systemic circulation. This established concentration gradient enhances permeation and paracellular transport of the pharmaceutically active ingredient into the blood stream. In embodiments where the pharmaceutically active ingredient is a protein and/or a peptide release by the particle into the small intestine causes hydration of the protein and/or peptide facilitating mucoadhesion to the muco-epidermal layer of the small intestine which facilitates the establishment of the concentration gradient of protein and/or peptide from the small intestine into the blood stream. This established gradient enhances permeation and paracellular transport of the protein and/or peptide in intact form into the bloodstream.

Where the pharmaceutically active ingredient is a protein and/or a peptide the carboxylic acid groups of the particles guard the protein and/or peptide from acid protease enzymes found in the gastric region of the gastrointestinal tract (GIT), in so doing the dosage form releases a minor amount of pharmaceutically active ingredient in the gastric region and a major amount of pharmaceutically active ingredient in the intestinal region of the gastrointestinal tract (GIT). Constriction and/or aggregation of particles at acidic pH inhibits and/or curbs release of the pharmaceutically active ingredient in the gastric region, therein inhibiting and/or curbing over hydration of the pharmaceutically active ingredient prior to reaching the target site.

The particles may be manufactured from natural and/or synthetic polymer building blocks. The chitosan (CHT) building block may have a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks may be of different or the same molecular weights, preferably in range of about 5000-9500g/mol.

In accordance with a second aspect of this disclosure there is provided a method for the manufacture of the pH responsive oral polymeric pharmaceutical dosage as described in the first aspect of this disclosure above, the method comprising the steps of:

(a) forming CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles or TMC-PEGDMA-MAA (trimethyl chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles; and

(b) adding the pharmaceutically active ingredient to the particles.

In a preferred embodiment of the second aspect of this disclosure the method comprises the steps of:

(a) forming PEGDMA from an esterification reaction between monomethoxypoly(ethylene glycol) and methacrylic acid;

(b) forming CHT-PEGDMA-MAA particles from a free radical suspension polymerization and crosslinking reaction wherein the PEGDMA of step (a), CHT and methacrylic acid are reacted together;

(c) lyophilizing the CHT-PEGDMA-MAA particles of step (b);

(d) forming the lyophilized CHT-PEGDMA-MAA particles of step (c) into a tablet via a direct compression technique; and

(e) adding a pharmaceutically active ingredient into the tablet.

The esterification reaction of step (a) may include use of a catalyst. The catalyst may be an acid, preferably sulphonic acid.

The free radical polymerization reaction of step (b) may include the addition of an initiator. The initiator may be azobisisobutyronitrile. The lyophilisation of step (c) may take place for a period of about 24 hours and may take place at a temperature of about -80D C. There is provided for the dosage form to possess no greater than 5% hydration after step (c) is complete, thereby preventing instability and breakdown of the particles.

The forming of the tablet of step (d) may take place through the use of a tablet press.

The chitosan (CHT) may have a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks may be of different or the same molecular weights, preferably in range of about 5000-9500g/mol.

The adding of the pharmaceutically active ingredient of step (e) may include the steps of:

(f) exposing the tablet to basic medium containing the pharmaceutically active ingredient, such that the tablet swells allowing for penetration of the pharmaceutically active ingredient into the tablet; and

(g) exposing the tablet to an acidic medium such that the tablet constricts and/or aggregates entrapping and/or encapsulating and/or incorporating the pharmaceutically active ingredient.

There is provided that the adding of the pharmaceutical ingredient of step (e) may occur through homogenous mixing of the pharmaceutical ingredient and the particles wherein the particles are in a lyophilized powder form that is excluded from any contact with solvent such as, but not exclusive to, water throughout the step, and wherein step (f) takes places in basic medium for 8 hours (pH7.4) to facilitate penetration of the pharmaceutically active ingredient into the particles, and step (g) takes place in an acidic medium of about pH 2.5.

In another embodiment of the second aspect of this disclosure step (b) comprises the forming of TMC- PEGDMA-MAA from a free radical polymerization and crosslinkmg reaction wherein the PEGDMA of step (a), TMC (trimethyl chitosan) and methacrylic acid are reacted together.

The is provided for a pH responsive oral polymeric pharmaceutical dosage form and method of manufacturing the same, substantially as herein described, illustrated and/or exemplified. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the disclosure will be described below by way of example only and with reference to the accompanying drawings in which:

FIGURE 1 shows a schematic illustrating how the pH sensitive, biostable particles constrict and/or aggregate in the acidic pH of the gastric region (pH 1.2), in comparison to their swelling abilities in the basic pH of intestinal region. Being mucoadhesive in nature the particles adhere to the mucus lining and release insulin (an example of a pharmaceutically active ingredient) which will further enter into the blood stream via paracellular pathways;

FIGURE 2 shows a schematic depicting an esterification reaction under standard conditions that were maintained (Kumara et al., 2006);

FIGURE 3 shows FTIR analysis of components the CHT-PEGDMA-MAA particles and their components. The order of the formulations as given from the above figure are as follows: (a) Optimized CHT-PEGDMA-MAA, (b) Optimized TMC-PEGDMA-MAA, (c) CHT, (d) MAA, (e) PEGDMA-MAA, (f) TMC;

FIGURE 4 shows a DSC thermogram of CHT-PEGDMA-MAA;

FIGURE 5 shows a DSC thermogram of PEGDM A ; FIGURE 6 shows a DSC thermogram of CHT;

FIGURE 7A shows a MH (N/mm) Grad net fd =80.016 Area Fd =0.009;

FIGURE 7B shows a MR (Kg/sec)

FIGURE 8 shows (a) a figure representing the schematics depicting the types of isotherms and (b) a figure representing the types of hysteresis loops according to the IUPAC classification system (adapted from Bawa et al., 2011);

FIGURE 9A shows a Type IV isotherm of CHT-PEGD MA-MAA in acidic medium; FIGURE 9B shows a Type IV isotherm of CHT-PEGD-MA-MAA in basic medium;

FIGURE 10 shows a 10 000X magnification of CHT-PEGD-MA-MAA in (a) basic condition and (b) acidic condition;

FIGURE 11 shows a TGA profile of (a) CHT, (b) PEGDMA and (c) CHT-PEGDMA-MAA;

FIGURE 12 shows a chromatogram of (a) commercial product and (b) drug (insulin) release of a dosage form having CHT-PEGD-MA-MAA;

FIGURE 13 shows fractional release of the pharmaceutically active ingredient from the dosage form having CHT-PEGD-MA-MAA in gastric medium: (a) formulations 1-6, (b) formulations 7-13;

FIGURE 14 shows fractional release of the pharmaceutically active ingredient from the dosage form having CHT-PEGD-MA-MAA in intestinal medium: (a) formulations 1-6, (b) formulations 7-13;

FIGURE 15 shows (a) residual plots of average particle size [(a)(i) a normal probability plot, (a)(ii) residuals vs. fitted values, (a) (iii) a histogram of residuals, (a)(iv) residuals vs. order of the data]; (b) a surface plot of average particle size; (c) residual plots of average particle size of fractional release in gastric medium at 2 hours [(c)(i) a normal probability plot, (c)(ii) residuals vs. fitted values, (c) (iii) a histogram of residuals, (c)(iv) residuals vs. order of the data]; (d) a surface plot of fractional release in gastric medium at 2 hours; (e) residual plots of fractional release in intestinal medium at 2 hours [(e)(i) a normal probability plot, (e)(ii) residuals vs. fitted values, (e) (iii) a histogram of residuals, (e)(iv) residuals vs. order of the data]; and (f) surface plots of fractional release in intestinal medium at 2 hours; all for a dosage form having CHT-PEGD-MA-MAA;

FIGURE 16 shows graphs (a)-(d) representing responses of average minimum, release maximum and release minimum properties of minimum particle size, greatest drug release in intestinal conditions and least drug release in gastric conditions for the optimized formulation; all for a dosage form having CHT-PEGD-MA-MAA; FIGURE 17 shows a DSC thermogram of (a) TMC, (b) PEGDMA and (c) TMC-PEGDMA-MAA;

FIGURE 18 shows a Type IV isotherm of TMC-PEGDMA-MAA in (a) acidic medium and (b) basic medium;

FIGURE 19 shows a 10 000X magnification of TMC-PEGDMA-MAA in (a) basic condition and (b) acidic condition;

FIGURE 20 shows a TGA profile of (a) PEGDMA, (b) TMC and (c) TMC-PEGDMA-MAA;

FIGURE 21 shows a fractional release from the dosage form having TMC-PEGDMA-MAA in gastric medium: a) formulations 1-6, b) formulations 7-13;

FIGURE 22 shows a fractional release from the dosage form having TMC-PEGDMA-MAA in intestinal medium: (a) formulations 1-6, (b) formulations 7-13;

FIGURE 23 shows (a) residual plots of average particle size [(a)(i) a normal probability plot, (a)(ii) residuals vs. fitted values, (a) (iii) a histogram of residuals, (a)(iv) residuals vs. order of the data]; (b) a surface plots of average particle size, (c) residual plots of average particle size of fractional release in gastric medium at 2 hours [(c)(i) a normal probability plot, (c)(ii) residuals vs. fitted values, (c) (iii) a histogram of residuals, (c)(iv) residuals vs. order of the data]; (d) surface plots of fractional release in gastric medium at 2 hours; (e) residual plots of fractional release in intestinal medium at 2 hours [(e)(i) a normal probability plot, (e)(ii) residuals vs. fitted values, (e) (iii) a histogram of residuals, (e)(iv) residuals vs. order of the data]; and (f) surface plots of fractional release in intestinal medium at 2 hours; all for a dosage form having TMC-PEGDMA-MAA;

FIGURE 24 shows a figure representing properties of minimum particle size, greatest drug release in intestinal conditions and least drug release in gastric conditions for the optimized formulation of a dosage form having TMC-PEGDMA-MAA. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE DISCLOSURE

The development of oral formulations for the delivery of proteins and/or peptides as pharmaceutically active ingredients has become an attractive alternative to conventional parenteral formulations (Morishita and Peppas et al., 2006). To date macromolecular peptides have been effectively used subcutaneously or as intramuscular injections. This form of administration has commonly been associated with multiple problems of pain, allergic reactions, poor patient compliance and chances of infection (Chiu et al., 2007). Many researchers have been investigating alternatives to parenteral delivery, such as nasal, pulmonary and oral routes, and of these the oral route is seen as the most beneficial delivery route, provided it is protected against all forms of degradation. Oral administration is typically deemed to be the most convenient and beneficial route of administration achieving the best patient compliance. There are various challenges associated with the oral route of protein and/or peptide delivery and include without limitation, the highly acidic pH of the stomach, presystemic enzymatic degradation of the protein and/or peptide, and its poor permeation through the intestinal membrane (Morishita and Peppas et al., 2006). Many strategies, such as the use of enteric-coated dry emulsions, microspheres, liposomes and nanoparticles for encapsulation of proteins and/or peptides have been critically evaluated (Shaji and Patole, 2008).

In accordance with a first aspect of this disclosure there is provided a pH responsive oral polymeric pharmaceutical dosage form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, the dosage form comprising CHT-PEGDMA-MAA (chitosan- poly(ethylene glycol) dimethacrylate-methacrylic acid) particles. The dosage form typically further includes a pharmaceutically active ingredient, wherein an increase in pH facilitates an increase in the release rate of the pharmaceutically active ingredient from the particle and wherein a decrease in pH facilitates a decrease in the release rate of the pharmaceutically active ingredient from the particle. The pH referred to herein is the pH of an environment in which the dosage form is present, typically a biological medium such as, for example, gastric fluid and/or intestinal fluid.

The CHT (chitosan) may be functionalized. The functionalized CHT (chitosan) may be trimethyl chitosan (TMC) such that the dosage form may comprise TMC-PEGDMA-MAA (trimethyl chitosan- poly(ethylene glycol) dimethacrylate-methacrylic acid) particles.

The dosage form may be crosslinked. Typically, crosslinking may be caused by microwave radiation, UV radiation or chemical crosslinking. Crosslinking, and the degree of crosslinking, effects release rates of pharmaceutically active ingredients when the dosage form is in use. It is known that the greater the degree of crosslinking the slower the release rates will be, in use.

The particles may release the pharmaceutically active ingredient in a pH dependent manner wherein an increase in pH facilitates a conformation change of the CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) or the TMC-PEGDMA-MAA (trimethyl chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) causing the particle to expand, in so doing, facilitating the release of the pharmaceutically active ingredient. Conversely, a decrease in pH facilitates constriction and/or aggregation of the particles, such that release of the pharmaceutical compound is inhibited and/or curbed.

A decrease in pH increases electrostatic attraction causing constriction and/or aggregation of the particles and inhibiting and/or curbing release of the pharmaceutically active ingredient. An increase in pH increases electrostatic repulsion causing swelling of the particles and/or dispersion of the particles into the medium and release of the pharmaceutically active ingredient. In a preferred embodiment, an acid pH increases electrostatic attraction and a basic pH increases electrostatic repulsion.

Typically, the pharmaceutical dosage form may be pH responsive such that in use the particles of the dosage form swell when exposed to a medium of basic pH, and constricts and/or aggregates when exposed to a medium of acidic pH.

The target site may be the intestinal region of the gastrointestinal tract (GIT) of the human or animal body, preferably the small intestine, further preferably the muco-epidermal layer of the small intestine.

Typically, in use, the dosage form is orally ingested and upon entry into the gastric region of the gastrointestinal tract (GIT) the particles of the dosage form constrict and/or aggregate in response to the acidic medium of the gastric region therein inhibiting and/or curbing release of the pharmaceutically active ingredient. Upon entry of the dosage form into the intestinal region of the digestive system the particles of the dosage form swell in response to the basic medium of the intestinal region therein facilitating release of the pharmaceutically active ingredient. Release rate of the pharmaceutically active ingredient in the small intestine is faster relative to release in the gastric region. The pharmaceutically active ingredient may be any chemical and/or biological composition having pharmaceutical properties.

There is further provided that the pharmaceutical dosage form shows, in use in vitro, a release profile of about 2.5 hours when in acidic pH and a release profile of about 8 hours when in basic pH, , in which a minor amount of pharmaceutically active ingredient is released in the acidic pH, thereby inhibiting a major amount of the pharmaceutically active ingredient from release and/or degradation in the acid pH, and allowing release of the major amount of pharmaceutically active ingredient in the basic pH.

In preferred embodiments about 80% of the pharmaceutically active ingredient is released in intestinal region. Where the particles comprise CHT-PEGDMA-MAA a minimum of about 21.2% of the pharmaceutically active ingredient is released in gastric region, and where the particles are TMC- PEGDMA-MAA a minimum of about 4.03% of the pharmaceutically active ingredient is released in the gastric region. The pharmaceutical dosage form may be non-toxic in vivo. In a preferred embodiment the pharmaceutical dosage form may comprise a multitude of particles. There is further provided that the particles may be biodegradable.

The pharmaceutical dosage form may be formed into a tablet, caplet or capsule. In a preferred embodiment the dosage form is formed into a tablet by direct compression of the particles. Further preferably, the tablet may be cylindrical in shape and/or dimension prior to use. The cylindrical tablet may have a diameter of about 4 mm. In a preferred embodiment wherein the pharmaceutical dosage form is formed into a tablet the dosage form may have a high matrix resilience and a high matrix hardness.

Constriction and/or aggregation of particles at acidic pH inhibits and/or curbs release of the pharmaceutically active ingredient in the gastric region, therein facilitating concentration of the pharmaceutically active ingredient at the target site (preferably the intestinal region of the gastrointestinal tracts (GIT), further preferably the small intestine, most preferably the muco-epidermal layer of the small intestine) to ensure a steep concentration gradient of the pharmaceutically active ingredient from the muco-epidermal layer of the small intestine into the blood stream for systemic circulation. This established concentration gradient enhances permeation and paracellular transport of the pharmaceutically active ingredient into the blood stream. In embodiments where the pharmaceutically active ingredient is a protein and/or a peptide release by the particle into the small intestine causes hydration of the protein and/or peptide facilitating mucoadhesion to the muco-epidermal layer of the small intestine which facilitates the establishment of the concentration gradient of protein and/or peptide from the small intestine into the blood stream. This established gradient enhances permeation and paracellular transport of the protein and/or peptide in intact form into the bloodstream.

Where the pharmaceutically active ingredient is a protein and/or a peptide the carboxylic acid groups of the particles guard the protein and/or peptide from acid protease enzymes found in the gastric region of the gastrointestinal tract (GIT), in so doing the dosage form releases a minor amount of pharmaceutically active ingredient in the gastric region and a major amount of pharmaceutically active ingredient in the intestinal region of the gastrointestinal tract (GIT).

Constriction and/or aggregation of particles at acidic pH inhibits and/or curbs release of the pharmaceutically active ingredient in the gastric region, therein inhibiting and/or curbing over hydration of the pharmaceutically active ingredient prior to reaching the target site. The particles may be manufactured from natural and/or synthetic polymer building blocks. The chitosan (CHT) building block may have a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks may be of different or the same molecular weights, preferably in range of about 5000-9500g/mol.

(a) forming CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) particles or TMC-PEGDMA-MAA (trimethyl chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) particles; and

(b) adding the pharmaceutically active ingredient to the particles.

(c) lyophilizing the CHT-PEGDMA-MAA particles of step (b);

(e) adding a pharmaceutically active ingredient into the tablet.

The esterification reaction of step (a) may include use of a catalyst. The catalyst may be an acid, preferably sulphonic acid. The free radical polymerization reaction of step (b) may include the addition of an initiator. The initiator may be azobisisobutyronitrile.

The lyophilisation of step (c) may take place for a period of about 24 hours and may take place at a temperature of about -80DC. There is provided for the dosage form to possess no greater than 5% hydration after step (c) is complete, thereby preventing instability and breakdown of the particles.

The forming of the tablet of step (d) may take place through the use of a tablet press. The chitosan (CHT) may have a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks may be of different or the same molecular weights, preferably in range of about 5000-9500g/mol.

In the design of a novel pH responsive polymeric pharmaceutical dosage form, the properties of its composite nature were evaluated and characterized. Co-polymeric chitosan-polyethylene glycol dimethacrylate-methacrylic acid (CHT -PEGDMA-MAA) and trimethyl chitosan polyethylene glycol dimethacrylate-methacrylic acid (TMC -PEGDMA-MAA) were prepared by free radical suspension polymerization technique with the intention of loading macromolecular peptides (the pharmaceutically active ingredient) for efficient and site targeted drug delivery.

A Box-Behnken design had been completed for acquiring the optimized formulations, with responses most desired for the polymeric systems. The manipulation of chain conformation due to its pH -responsive nature is considered the primary mechanism by which pharmaceutically active ingredient (the drug) is released by the exceptional swelling behavior in basic pH, and its contrary reaction in acidic pH, shrinking and/or constricting, therein protecting its contents in a responsive way.

Studies have shown that polymers containing carboxylic acid groups have the ability to guard peptides from the protease enzymes such as trypsin and chemotrypsin. These polymers were proposed to react by binding of divalent cations (calcium and zinc) to exhibit their enzyme inhibitory effects (Sajesh et al., 2006). The excellent mucoadhesive and antimicrobial properties of CHT and TMC provides an intact crosslinked network beneficial for absorption through the gas tro -intestinal membrane. The purpose of developing this composite molecular network is to obtain the highest degree of drug loading efficiency in the formulation, a minimum percentage of drug (or pharmaceutically active ingredient) being released in acidic medium according to its pH responsive nature and obtaining maximum release profiles in basic intestinal medium.

The free radical suspension polymerization and crosslinking reaction provides conjugation between

i. two extremely mucoadhesive polymers (CHT/TMC and poly-MAA);

ii. a synthetic (poly-MAA) and natural polymer (TMC); and

iii. two pH responsive polymers (poly-MAA and CHT/TMC),

forming a semisynthetic mucoadhesive-pH responsive conjugated oral polymeric pharmaceutical dosage form according to the invention capable of encapsulating and/or including proteins and/or peptides, facilitated by the presence of -COOH moieties. The dosage form in use protects the proteins and/or peptides from harsh gastric environment and retains dosage form in close vicinity of intestinal wall for a prolonged period.

The Applicant surprisingly found that the CHT/PEGDMA-MAA or TMC/PEGDMA-MAA polymeric architecture is characterized by three-in-one matrix types: i. a semi-interpenetrating polymer network consisting of CHT or TMC and PEGDMA crosslinked MAA wherein one polymer is crosslinked in the presence of another polymer;

ii. a polyelectrolyte complex formed between the -COOH functionalities of PEGDMA crosslinked MAA and -NH₃ ⁺ functionality of CHT or TMC; and

iii. PEGDMA crosslinked MAA conjugated to CHT or TMC forming CHT-PEGDMA-MAA.

The unique physico-chemical composition and architecture of the dosage form imparted with properties that at least ameliorate disadvantages known in the prior art. Furthermore the high resilience acrylate polymer (PEGDA crosslinked MAA) on the TMC backbone provided for a long side-chain molecular conformation capable of entrapping higher amount of protein and/or peptide when compared to known means. This entrapment is further enhanced by the use of a long chain crosslinker (PEGDA) providing an inter- and intra-chain crosslinked network. The retention of this conjugate polymer in "tethered" intestinal mucosa was mediated via two different mechanisms:

1) the entangling of PEGDA crosslinked MAA side chains into the mucus lining; and

2) the charged electrostatic interaction provided by the cationic polyquaternium chitosan backbone.

The unique physico-chemical properties of the dosage form provided by high molecular weight chitosan and PEGDA crosslinked MAA aided the prolonged retention in the intestine via a unique hard-to-soft swollen hydrogel architecture. The ability of the conjugated system to accommodate various chitosan derivatives (in terms of molecular weight) and crosslinkers and monomers with varying chain length can provide the flexibility required for the extent and rate of protein and/or peptide release from the nanoparticulate matrix.

Figure 1 shows a pH responsive oral polymeric a pH responsive oral polymeric pharmaceutical dosage 10 form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, the dosage form being a tablet and comprising CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles 12 each particle including a protein and/or peptide pharmaceutically active ingredient 14. Figure 1 shows the pH responsiveness of the dosage form 10 in the stomach (marked A) wherein exposure of the dosage form 10 to about pH 2 of the stomach (A) facilitates constriction and/or aggregation of the particles. Figure 1 further shows the pH responsiveness of the dosage form 10 when exposed to increasing pH, wherein exposure to the small intestine (marked B) of about pH 7.4 facilitates swelling of the particles 12, dissociation of particles 12 and an increase in the release rate of the protein and/or peptide 14. The proteins and/or peptides 14 cross the mucosa of the small intestine (marked C) through the mucosal epithelia (marked D) and into the blood stream (marked E). Figure 10 (a) and (b) shows a 10 000X magnification of a CHT-PEGD MA-MAA dosage form in (a) basic condition and (b) acidic condition respectively, and shows the swelling of the particles in basic pH conditions and constriction and/or aggregation in acidic pH conditions. PREPERATION OF CHT-PEGDMA-MAA and TMC-PEGDMA-MAA MATERIALS AND METHODS (CHT-PEGDMA-MAA and TMC-PEGDMA-MAA) Materials

Chitosan of medium molecular weight (CHMMW), Mw = 450kDa, Monomethoxypoly(ethylene glycol), (PEG) of different molecular weights (5000, 6000, and 9500g/mol), Na₂HP0₄, NaH₂P0₄, methacrylic acid, ammonium per sulphate, and azobisisobutyronitrile (AIBN) were obtained from Across Organics (NJ, USA). Acetonitrile (HPLC grade), water (HPLC grade) and hexane were purchased from Ranbaxy Chemicals. Humuinsulin-R, Actrpid^R (r-DNA origin) of 100 I.U./mL from Eli Lilly and Company (USA). Rebif^R interferon beta was used as a loading protein. Trimethyl chitosan was prepared by reductive methylation. Methoxypolyethylene glycol 2000, sodium iodide, methyl iodide, sodium hydroxide, sodium chloride, sodium bicarbonate, trinitro benzene sulphonic acid, hydrochloric acid, sodium deoxycholate, sodium sulphate and orthophosphoric acid were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) at reagent grade and were utilised without further purification. Acetonitrile was purchased from Sigma (St. Louis, Missouri, USA) at UPLC grade. Diether ether, ethanol and N-methyl-2-pyrrolidone were purchased from Merck (Halfway House, Gauteng, South Africa) at reagent grade and were utilised without further purification.

Synthesis of PEGdimethacrylate (PEGDMA)

The esterification reaction carried out in a 500mL round bottom flask, at a temperature of 80-90°C at 40rpm for 7 hours, was used as the basis for formulating PEGDMA. 200g of PEG, with a molecular weight of 4000g/mol was reacted in a ratio of 1:2 with methacrylic acid, using a 1.5% of monomer concentration of sulphonic acid as a catalytic stimulus in the reaction with hydroquinone, as part of a free radical inhibitor, in concentration of 0.01% of methacrylic acid. The azotropic solvent used in the reaction to remove excess water formed during the esterification reaction was toluene. The resulting polymeric reaction was then neutralized with 5% of sodium bicarbonate solution (NaHCC>3). The esterification reaction is shown in Figure 2. The final stage of the reaction involved precipitating PEGDMA by the addition of ice hexane, and drying the precipitate under vacuum oven conditions of 60 °C at 0.6kpa for a 24 ours period (Kumara et al., 2006) Synthesis of co-polymer CHT-PEGDMA-MAA

The reaction involving the free radical polymerization synthesis of CHT-PEGDMA₄₀₀₀ MAA, was initiated by reacting a ratio of 3: 1:2 of the above polymer concentration. The initiator used in the reaction was azobisisobutyronitrile, which was calculated as a 0.6% of monomer concentration. Chitosan of medium molecular weight was used in the reaction, after the acid medium was established with methacrylic acid, to allow dissolution and crosslinking with PEGDMA. The reaction was undertaken in a 500mL round bottom flask, using Millipore distilled water as the continuous medium, and purging nitrogen gas throughout the entire reaction. The optimum ratio of chitosan to be used was determined by implementing a Box-Benkhen design program, using the maximum and minimum limits for crosslinking to occur, thus determining which concentration according to the responses tested for optimum particle size and drug release in acid and basic pH, finally resulting in the desired concentration for optimum synthesis and delivery. A typical example of the reaction mixtures comprised lg PEGDMA, 2g MAA, 3g CHT, 97g deionized water, 0.018g AIBN. After 4 hours of purging nitrogen in the closed system reaction, microparticles began to form, appearing as white sprinkles of particles upon observation. After successfully completing this reaction, the crosslinked polymeric particles were washed twice with water and filtered, thereafter adjusting their pH to 7.4. The pH of the polymer was adjusted to basic condition of 7.4, creating a desirable environment for future drug loading after the polymer was freezed for 24 hours at -80°C and lyophilized to remove all excess water from polymer.

Synthesis of trimethyl chitosan (TMC)

The synthesis of TMC was carried out via a triple stage procedure. As a protocol, 80mL of N-methyl pyrrolidonone was heated at 60°C in a water bath incubating for 30min, thereafter dissolving 2g of CHT, 4.8g of sodium iodide and lOmL of 20%w/v sodium hydroxide solution. This reaction mixture was further incubated in a water bath for a further 30min at 60°C. After this period, 12mL of methyl iodide was added as soon as the reaction mixture was extracted from the water bath and inserted into a direct reflux apparatus using a Liebig condenser, under constant magnetic stirring of 300rpm, for 90min, also maintained at a temperature of 60°C. During this phase, a thick bright yellow homogenous mixture was formed, which was ready for precipitation using 250mL of diethyl ether and 250mL ethanol. The precipitate was then filtered and dried under vacuum conditions at 60±0.5°C for 48 hours. The dried polymer was then finely reduced to grains and used to undertake the next stage. The next stage is the same as the first, however, no CHT was used in the reaction, instead the polymer from the first stage was used in place of the CHT and the exact same procedure was undertaken. The next stage is the final reaction where ion exchange occurs, exchanging iodide ions for chloride ions. 80mL of 5% w/v sodium chloride was then prepared and the ground polymer from the previous stage was added to the solution, with continuous magnetic stirring for 30min. The reaction mixture was then added to 250mL of diethyl ether and 250mL of ethanol as used previously for precipitation. After filtering the mixture, the precipitate was once again dried in the vacuum oven for 48 hours at 60°C. The final TMC polymer was now ready for crosslinking and further utilization.

Synthesis of TMC-PEGDMA-MAA

The synthesis of TMC-PEGDMA-MAA was undertaken under continuous inert conditions, purging nitrogen gas throughout the reaction, reacting the polymer concentrations in a ratio of 3:1:2 respectively. This free radical polymerization synthesis was initiated using azobisisobutyronitrile, which was used as 0.6% of monomer concentration. The TMC that was synthesized as discussed, is water soluble and dissolves readily at a temperature of 70°C. Millipore water was preheated at 70°C and MAA was then added, creating an acid environment for the reaction process to occur. The reaction was carried out in a 500mL, three-neck round bottom flask under constant magnetic stirring of 50rpm. A Box-Behnken design program was used to determine the optimum concentration of TMC and % of crosslinker employed in the reaction, using an upper and lower limit for crosslinking of the polymers to occur, to determine the responses of the polymer at different pH states, thereby producing a wide variety of polymer characteristics such as particle size, drug release and drug loading efficiency to determine the optimum formulation for peptide delivery. The standard protocol implemented comprised lg PEGDMA, 2g MAA, 3g TMC, 97g deionized water and 0.018g AIBN. The entire reaction was undertaken in 6 hours, after physically seeing the formation of microparticles in the presence of purging nitrogen gas in a closed system, appearing as tiny sprinkles of particles upon observation. After successfully completing the reaction, Millipore water was used to wash the polymer, in order for any unreacted components to be removed from the solution. The pH of the polymer was adjusted to basic condition of 7.4, creating a desirable environment for future drug loading after the polymer was freezed for 24 hours at -80°C and lyophilized to remove all excess water from polymer.

ANALYSES OF CHT-PEGDMA-MAA and TMC-PEGDMA-MAA.

The following techniques were conducted on both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA. The general experimental procedures are explained hereunder, following which the results and discussions for both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA are individually discussed in detail. Determination of structural bonding elements using Attenuated Total Reflectance (ATR)

Attenuated Total Reflectance (ATR) was utilized for determination of chemical bonding between all components of both the co-polymeric systems, using a Perkin Elmer Spectrum 2000 FTIR spectrometer having a single reflectance MIRTGS detector, (PerkinElmer Spectrum 100, Llantrisant, Wales, UK), to provide a comprehensive chemical analysis of all polymers. For the CHT-PEGDMA-MAA dosage form PEGDMA, MAA, CHT and co-polymeric CHT-PEGDMA-MAA were analyzed using a diamond crystal component, and data was retrieved on a universal ATR polarization program for FTIR spectrum series, using a resolution of 4cm^"1. All samples were evaluated using a 100 scan run (SNR: 10) at 130psi, ranging from 650-4000cm^_1. Similarly, for the TMC-PEGDMA-MAA dosage form PEGDMA, MAA, TMC and co-polymeric TMC-PEGDMA-MAA were analyzed.

Differential Scanning Calorimetry (DSC) evaluation on both CHT-PEGDMA-MAA and TMC- PEGDMA-MAA dosage forms

PEGDMA, MAA, CHT, TMC and co-polymeric CHT-PEGDMA-MAA and TMC-PEGDMA-MAA were subjected to thermal degradation using a Mettler Toledo DSC-1 STAR⁶ System. Samples of a standard weight of 15mg were accurately weighed and placed in a 40μΙ_^ aluminum crucible pan. A 0.2mm hole was punctured on the lid of the aluminum crucible pan to ensure that in use the degrading contents escapes and does not cause an increase in pressure resulting in explosion of the crucible. The pan was then sealed and thermal settings were then programmed accordingly. Areas of pertinent interest focused specifically on the onset of melting point, melting peak temperature and heat of fusion. Samples were run with a heating range of 10°C/min, in order to determine sufficient detail from the resulting graphs. All polymers were run twice. The first run from 25-110°C was specifically to extract water molecules from the sample, following which the second run was increased to 25-250°C, to obtain as much thermal responses from the polymer.

Size and zeta potential analysis of CHT-PEGDMA-MAA and TMC-PEGDMA-MAA particles

The CHT-PEGDMA-MAA co-polymer, after being synthesized, was adjusted to two different pH states, using gastric medium (pH 1.2) and intestinal medium (pH 6.8), to determine its behavior at specific points of the GIT, thus acquiring specific responses by simulating physiological conditions. 5mg of polymer was added to 12mL of respective gastric and intestinal USP buffer solution, thereafter storing the particles for 30min to react in accordance to the specific pH, and shaking the contents after the incubation time to ensure a uniform distribution of particles for analysis. The polymer is completely insoluble in aqueous medium, facilitating most accurate particle size determination, since no particles can dissolve in the medium. 5mL of the sample was then drawn and put into a cuvette, which was analyzed accordingly. Zeta potential on the same sample was carried out, determining the effects different pH conditions have on the polymer charge, relating this to the behavior at this specific pH.

Matrix hardness (MH) and Matrix resilience (MR)

The mechanical properties of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA co-polymer dosage forms were determined after compressing the polymer into a cylindrical 4mm tablet with a pressure of 0.6MPa, and analyzed using a well calibrated textual analyzer (TA.XTplus, Stable Microsystems, Surrey, UK), under standard conditions (25°C, latm. Pressure), employing a cylindrical steel probe for determining MR (50mm diameter) and for determining MH, a flat tip steel probe was used (2mm diameter). Generating a force-time profile from MR analysis the area under curve (AUC) was calculated in ratios to determine the percentage of MR of the compressed polymer under specification settings in Table 1. From peak to base, AUC₂_3 (after removal of force) divided by AUC^ from the base to the peak (before removal of force), was the simple calculation applied to determine the percentage of resilience of the polymer. Force-distance profiles were attained, and the gradient of the initial and maximum force yielded MH (N/mm) as per maximum force required for deformation of the tablet.

Table 1: Parameter settings for determining MR and MH

Parameters MR^a( ) MH^b(N/mm)

Pre-test speed lmm/s lmm/s

Test speed 0.5mm/s 0.5mm/s

Post-test speed lOmm/s lOmm/s

Trigger type Auto Auto

Trigger force 0.05N 0.05N

Load cell 5Kg 5Kg

Compression strain Variable N/A

Target mode Strain (5%) Distance

'Matrix resilience; ^bMatrix hardness

Porositometric characteristics

Both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA co-polymers were stored at different pH values, pH, 1.2 and 6.8 respectively, were lyophilized to remove excess water molecules and a weight of 120mg of polymer was compressed at 0.6MPa and evaluated for surface area and porosity analysis, using a Porositometric Analyzer (Micrometritics ASAP 2020, Norcross, GA, USA), which comprises two basic steps: degassing following analysis of the composite tablet system. The tablet was degassed in a sample tube (I.D=9.53 mm), with a glass filler rod being inserted in the tube, thereby reducing the total volume space in the tube and increasing the pressure of the system for correct analysis of the sample, thereby removing any form of moisture and impurities in the sample. The entire degassing process was completed in a period of between 8-12 hours, which was also processed via stages of heating and evacuation phases. Table 2 outlines the basic parameter settings that were undertaken to carry out the porositometric analysis.

Table 2. Evacuation and heating phase parameters employed for porositometric analysis

Parameter Rate/target

Evacuation phase

Temperature ramp rate 10°C/min

Target temperature 40°C

Evacuation rate 50.0mmHg/s

Unrestricted evacuation from 30mmHg/s

Vacuum set point 500μπύ¾

Evacuation time 60min

Heating phase

Temperature ramp rate 10°C/min

Hold temperature 30°C

Hold time 900min

After the process of degassing, the sample was set up for analysis, where pore size, pore volume and surface area data were obtained using BJH and BET profiles.

Scanning Electron Microscopy (SEM) analysis

Both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA co-polymers were adjusted to specific pH, 1.2 and 6.8 respectively, were lyophilized and prepared for morphological analysis, differentiating its pH responsive nature in relation to its behavior on a morphological level, thus providing a link and confirming the properties of the polymer system. Surface area and pore structures were the main components of concern, employing a FEI ESEM Quanta 400F (FEI™, Hillsboro, OR, USA) electron microscope at an acceleration of 20.00kV, for acquiring images related to the swelling and aggregation behavior of the polymer. The samples were mounted on an aluminum spud and then coated with gold using an EPI Sputter coater (SPI Module TM sputter-coater and control unit, West Chester, Pennsylvania USA). The coated samples were then analyzed for surface morphology using a FEI Phenom™ desktop scanning electron microscope at various magnifications. Images obtained were differentiated at the two different pH states and direct comparisons were deduced.

Thermogravimetric analysis (TGA)

Components of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA and CHT-PEGDMA-MAA and TMC-PEGDMA-MAA themselves were analyzed using TGA 4000 thermogravimetric analyzer (PerkinElmer Inc, Massachusetts, USA), using a ramping of 10°Cmin^_1 from 50-500°C. Nitrogen conditions were constantly sustained throughout the run, in which 14mg of sample was loaded in ceramic pans for specialized detection to be obtained. Thermograms and their first derivates, indicating the water percentage content and the maximum temperature where degradation occurs, rate of degradation as well as onset and endset of degradation were obtained and evaluated for complete thermal degradation analysis.

Karl Fisher volumetric analysis

After lyophilization of samples of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA and compression (0.6MPa) into a tablet form, the water content of the sample was evaluated using a Karl Fischer volumetric titrator (Mettler Toledo, Columbus, USA) comprising a DN143-SC sensor. Standard conditions were maintained at 25°C, with a 596.5mV start potential and a 5.33724mg/mL titrant concentration.

Direct compression and pellet formation for evaluation of a final composite system

After synthesis and triple washing of samples of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA, the pH was adjusted to a basic medium (pH 6.8), and lyophilized to remove extra water, in order for drug to be loaded accordingly. Accurate weighing of lyophilized polymer was added to a specialized custom made punch and die system to form a pellet with diameter 4mm, employing a hydraulic pressurized system of 0.6MPa. The length of the pellet was proportional to the amount of polymer used, and thus the dose of insulin (the test example of the pharmaceutically active ingredient) used would predict the amount of polymer compressed.

Drug-loading for maximum entrapment and release kinetics

Insulin was utilized as an example of the pharmaceutically active ingredient or drug. It is to be understood that other pharmaceutically active ingredients may be utilized including other proteins and/or peptides. After synthesis and triple washing of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA samples, the H was adjusted to a basic medium (pH 7), and lyophilized, in which operating procedures of a 2 hour condensation phase at -60°C thereafter following a sublimation phase at 25mm Torr for 24 hours, undertaken on a Freezone 12 freeze drier (Lanconco, Kansas City, USA). Insulin solution (Actrapid^® HM 100) was added to the sample, and maintained at pH 7 for a period of 8 hours at 2-8°C, for maximum swelling of the polymer to occur, thereby allowing loading in this phase to be of the greatest capacity. After this duration, the pH was adjusted to an acidic medium (pH 2.5), to enable the particles to form a tight network of entrapment, thereby aggregating to form a composite system in which the drug is encapsulated into the polymeric dosage form. Filtration was then carried out, and the sample was collected for a second stage of lyophilization and ready for drug release studies.

Drug Entrapment Efficiency (DEE) determination

After the insulin (the example pharmaceutically active ingredient) was loaded in the above stage and the sample adjusted to acidic medium (pH 2.5), filtration was carried out and polymer was collected and lyophilised, collecting the filtrate and analysing it on HPLC as discussed below. This procedure was conducted for both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA dosage forms. ImL of filtrate was drawn and placed in an ANSI HPLC vial for analysis. Equation 1 was used to determine the final percentage entrapment efficiency of insulin in the dosage form.

%Entrapment Efficiency =

theoretical amount of insulin loaded- amount of insulin measured from filtrate _Λ ~ _ , ,

— :— - „. ,. ,—— X 100 (Eq.l)

Theoretical amount of insulin loaded

High Performance Liquid Chromatography (HPLC) determination of insulin concentration

The determination of insulin (the example pharmaceutically active ingredient) concentration employing CI 8 silica phase Symmetry 300 column of particle size 5um with a Waters 1525 Binary Pump with 2489 UV/visible detector and an auto-sampler attachment for multiple sample queue analysis. The wavelength absorbance was set at 214nm and a constant column temperature of 25 °C was maintained throughout the analysis. The mobile phase consisted of a mixture of ratio 24:76 solvent A: solvent B respectively. Solvent A consisted of pure acetonitrile 99.98% UPLC grade and solvent B was composed of 2.84% sodium sulphate and 0.27% orthophosphoric acid in a Millipore water mixture, with a pH consisting of 2.3.

In vitro drug release analysis

Compressed drug (insulin) loaded tablets of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA, were analyzed for a period of 8 hours. The tablet was put into a 20mL glass polytop, with a lOmL volume of simulated USP gastric and intestinal fluid respectively. To condition the system according to the physiological environment, an Orbit shaker incubator (LM-530-2, MRC Laboratory Instruments Ltd, Hahistadrut, Holon, Israel) at 37±0.5°C and 50rpm; was employed in order for sink conditions to be maintained throughout the drug release study. Each sample was analyzed in triplicate, and 0.5mL of sample was extracted at 30min time interval in simulated gastric pH and replaced with the same volume and temperature of fluid for duration of 150min. For tablets in intestinal fluid, 0.5mL of sample was drawn out every hour for 12 hours, and replaced with the same fluid at 37°C, to maintain a constant volume of distribution in the system. All extracted samples were immediately filtered using a 22um Millipore Millex filter (Billerica, MA, USA), and put into an ANSI 48 HPLC vial and stored at 4-8°C. All samples were analyzed on HPLC for no longer than a 3 hour period after acquiring said sample, to ensure no further degradation of drug occurs during storage. All samples were evaluated for its concentration using a standard curve, where the AUC for each drug peak, indicated the amount of insulin released at a specific point in time.

Mucoadhesive properties

The mucoadhesive properties of both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA co-polymers were evaluated using mucin from porcine stomach (type 2), acquired from sigma Aldrich, USA, constituted as a 0.1% solution in USP intestinal fluid (pH 6.8). 20mg of polymer was added to lOmL of the reconstituted mucin solution and was incubated for 6 hours in the orbital shacker, maintaining a temperature of 37°C and 50rpm rotations. Using UV analysis (nano photometer) at wavelength of 201nm, employing a 10 times dilution factor of pathlength 0.1mm, the concentration of the solution was determined before and after adding the dosage form in the mucin solution. The difference obtained in the concentration of mucin was analysed as a percentage of crosslinking with the polymers of the dosage forms, thereby indicating the interaction between mucin and the polymer. Equation 2 was used to calculate the percentage of mucoadhesion for each design formulation.

% mucoadhesion =

Concentration of mucin before adding polymer-concentration of mucin after adding polymer

Concentration of mucin before adding polymer X 100 (Eq.2)

Box-Behnken Design

For both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA dosage forms the determination of an optimum formulation, according to the specifications required as outlined in Table 3, a 2-factor, 2-level Box-Behnken statistical design was undertaken using Minitab^K V14 software (Minitab Inc., PA, USA), generating different concentrations of variables to ultimately provide inputs for modeling the most suitable formulation specifications required for the optimized formulation. This design generates a relative link in which alterations in variable concentrations has a significant impact on the physicochemical and in vitro responses. Upper and lower limits of chitosan concentration were 0.05-0.5g, and percentage (%) concentration of crosslinker was in the range of 3-8%^w/_w with respect to monomer concentration. These independent variables yielded responses that were evaluated with its relationship with drug release, particle size and zeta potential. Fractional release for both acidic and basic media were evaluated at a 2 hour point, for a comparison to be evaluated in insulin release. A gradual release of insulin was intended to be released from the formulation, but a small percentage of burst release was also desirable for penetration through mucus and tight junctions of the intestinal wall. Table 3 indicates the parameters of variables and the desired outcomes for each.

Table 3: The 2-factor, 2-level Box-Behnken Design variables employing MINITAB V14 software

Independent Variables Levels

Low Medium High

% Concentration of 3 5.5 8

crosslinking agent (% of

monomer concentration)

Concentration of CHT/TMC 0.05g 0.25g 0.5g

Dependent Variables Acid medium Basic medium Objective

Fractional drug release Minimum maximum Prevent release in acid

Particle size analysis Minimum maximum Polymer shrinks to

conserve loaded drug in acid

Zeta potential Minimum maximum Low in acid to prevent

dispersion among particles

Optimization of the design formulation

For both CHT-PEGDMA-MAA and TMC-PEGDMA-MAA dosage forms all response data were added to Minitab V14 software (Minitab^® V14, Minitab Inc., PA, USA), and statistical modeling was conducted to formulate a polymer blend comprising of all evaluated desired response elements, drug release, particle size and zeta potential, to deliver a formulation for optimum delivery of insulin. RESULTS AND DISCUSSION (CHT-PEGDMA-MAA)

Attenuated Transmission Resonance (ATR) analysis of all components of the polymer blend (CHT- PEGDMA-MAA)

The functional groups of the different elements or components together forming CHT-PEGDMA-MAA, were evaluated for their chemical characteristic bonds and were identified in Figure 3. Chitosan, PEGDMA, MAA and the co-polymeric micro particle CHT-PEGDMA-MAA, were analyzed using a pressure of 130psi in scanning spectrum range of 650-4000cm^~1 of 100 scans per sample, reducing the Signal to Noise Ratio (STN) to 10 for most accurate determination of peak responses. MAA was analyzed to have a peak at 1635cm ¹ for carbonyl groups, 1697cm ¹ for vinyl groups and a wide stretch of -OH bonding from carboxylic acid groups, from 3000-3450cm^_1. PEGDMA4000 revealed a peak at 1639cm^"1, for an explanation of carbonyl and vinyl group interactions. A bond representing CH stretching was also observed at 2882cm^"1, and 1466cm^"1 for CH bonding. For CHT-PEGDMA-MAA, a carbonyl group at 1695cm^"1 was clearly distinguished, and was present due to the strong interactive carboxylic acid groups of PEGDMA and MAA. This bonding is also responsible for the pH sensitive nature of the polymer. (Kumara et al., 2006). Chitosan has a characteristic peak at 1577cm^"1 (Mourya et al., 2009), which can also be seen in the polymeric blend of CHT-PEGDMA-MAA, in which the peak is shifted to 1553cm^"1 due to angular deformation of N-H bond of amino groups during the crosslinking reaction, however the intensity of peak 1553cm^"1 in the polymeric blend is reduced in comparison to peak 1577cm^"1 due to N- methylation occurring (Domard et al., 1986). The peak of 1553cm^"1 in the polymeric blend also explains the presence of N-H bending in addition to the range of peaks from 1415-1430cm^_1 which is characteristic for N-CH₃ absorption. Another characteristic peak at 1147cm^"1 redefines the bonding of chitosan to form the polymeric blend. The FTIR analysis of components of, and the CHT-PEGDMA-MAA, is shown in Figure 3. The order of the formulations shown in Figure 3 is as follows: (a) Optimized CHT-PEGDMA- MAA, (b) Optimized TMC-PEGDMA-MAA, (c) CHT, (d) MAA, (e) PEGDMA-MAA, (f) TMC.

Differential Scanning Calorimetry (DSC) of components and the co-polymeric blend (CHT- PEGDMA-MAA)

DSC was carried out on PEGDMA, CHT and CHT-PEGDMA-MAA micro particles. The starting procedure of removing water molecules from the sample was undertaken by running all samples from 25- 110°C (run 1), to prevent any false endothermic peaks arising, due to unliberated water molecules in the polymers. After this first run was undertaken, the same polymers were run from 25-350°C (run 2) to evaluate the heat flow in the sample at a temperature ramping rate of 10°C/min. This procedure was undertaken on all samples, in an endeavor to produce the most accurate thermal data possible. Figures 4, 5 and 6, represent CHT-PEGDMA-MAA, PEGDMA and CHT respectively. CHT was observed to have an exothermic melting peak of 121 °C and an endothermic crystalline peak at 306°C.

PEGDMA has significant characteristic melting points at 52.61 and 57.91°C. CHT-PEGDMA-MAA showed a melting peak at 56.28°C. At 221°C, PEGDMA also depicts a slight endothermic crystalline peak; however, this characteristic nature is not noticed on the polymeric blend, indicating that the sample has become more crystalline in nature. It can therefore be confirmed that the heat flow properties of the polymeric blend possess variations in thermal properties and can be summarized in Table 4 indicating the different peak flow properties of the polymers

Table 4: Dynamic Scanning Calorimetry (DSC) analysis of CHT-PEGDMA-MAA, PEGDMA, CHT at heating rate 10°C/min ranging 25°C to 350°C.

Polymer component Peak (°C) 1 Peak (°C) 2 Peak (°C) 3

CHT-PEGDMA-MAA 56.28

PEGDMA 52.61 57.91 221

CHT 121 306

The alteration in the crystallization peak from PEGDMA, occurring at a lower temperature in CHT- PEGDMA-MAA, indicates that the polymeric blend is more susceptible to alteration of its chemical structure than PEGDMA. This could be attributed to the bonding of the acyrlic group from PEGDMA to the methyl functional groups of methacrylic acid in the polymeric blend, changing the thermal properties to a slight degree. In addition to the exothermic melting peak of PEGDMA, the polymeric blend showed minimum area at 56.24°C, indicating a substantial increase in stability and resistance to thermal degradation due to rearrangement in bonds present in the polymeric blend, possibly due to variations in polymer chain bonds, lengths and mass.

Size of the design formulations and optimization results for size and potential analysis (CHT- PEGDMA-MAA)

The evaluation of particle size relationship with the parameters of the Box-Behnken design was determined using certain concentrations of CHT and cross-linking agent. The respective formations, as outlines in Table 5, were analyzed in different pH, comprising gastric and intestinal fluid, to elucidate the relationship between pH responsive nature and effects on their size. Formulations 2, 4, 8, 12 and 13 were all analyzed as replicates according to the design program for reliable data processing. 5mg of the lyophilized samples were then put into 12mL of respective gastric and intestinal fluid, and was allowed to remain dormant for 30min, thereafter shaken and extracted into a curvet, and allowed the rapid scattering of beams of light to determine the size of particles suspended. The hydrophobic nature of the particles made the evaluation very reliable, since none of the particles could dissolve in the medium. Particles were not filtered during the evaluation, since this would alter the size range, instead only the solvent was filtered with a 0.22um injection filter, to prevent any dust particles from giving false results.

Results from the table below, clearly indicated that an increase in CHT concentration and % crosslinking, resulted in bigger particles forming, as noticed in formulation 11. A pattern of 3% crosslinking concentration, noticeably yielded values of low particle size, indicating that this also plays a crucial role in the formation size of particles. According, to Kumara et al., 2006, carboxylic groups present in the particles, interact with ether functional groups of PEG, at a lower pH, due to the rich hydrogen bonding occurring between particles, therefore theoretically creating a smaller particle size. On evaluation of particle behavior, all formulations in acidic medium, yielded greater particles due to the ability of particles to clump together in acidic medium compared to basic medium in which particles disperse from each other. Analyzing the polydispersity index (PDI), all acidic medium particles, had much lighter PDI ranges, compared to basic medium. This reflects that particles are less uniform and stable in the acidic environment, as opposed to the basic environment, in which particles have lower PDI, and appear more uniform and stable. This also confirms the behavior of aggregation in acid pH, and dispersion in basic pH, since particles have less order of arrangement in their aggregated state, and will remain much more uniform in the dispersed behavior.

Table 5: Analysis of average particle size according to specifications of the design in gastric and intestinal fluid (CHT-PEGDMA-MAA)

% Average Average

Formulation CHT crosslinking Particle size in Particle size in

(g) of monomer acid (μιη) PDI base PDI

1 0.05 8 7.2 0.83 4.5 0.46

2 0.275 5.5 11.8 0.78 7.3 0.37

3 0.05 3 5.4 0.68 4.2 0.22

4 0.275 5.5 10.4 0.8 8.8 0.42

5 0.275 8 8.7 0.53 4.6 0.24

6 0.5 5.5 8.7 0.51 6.7 0.52

7 0.275 3 8.5 0.63 6.1 0.62 8 0.275 5.5 10.2 0.66 8.2 0.47

9 0.5 3 5.5 0.35 3.5 0.22

10 0.05 5.5 5.6 0.54 3.3 0.46

11 0.5 8 16.3 0.74 8.2 0.61

12 0.275 5.5 9.6 0.79 6.7 0.35

13 0.275 5.5 8.8 0.66 5.9 0.54

Computing these responses to the design program, formulation 8 was chosen as the optimum polymer blend, based on particle size and drug release profiles. The zeta potential of the optimized formulation revealed a value of 9.51 in acidic and -21.6 in basic mediums. These values correspond perfectly with the above explanation, since the charges present in acid medium are much lower, creating an aggregation medium, and a higher zeta potential at basic pH, explaining the dispersion of particles in this medium.

Matrix hardness (MH) and Matrix resilience (MR) of optimized formulation (CHT-PEGDMA-

MAA)

Matrix hardness and resilience tests were carried out on the optimized formulation, after it had been compressed at 0.6mPa, consisting of a 4mm diameter and a weight of 120mg. The matrix hardness was evaluated, using a 2mm diameter cylindrical stainless steel probe, where an indentation on the tablet, generated a regression curve, therefore evaluating the response generated. This test represents an indication of the intensity of the intermolecular bond forces latent in the tablet amongst the interparticulate granules or particles, thus measuring the amount of force required per millimeter of distance to induce an indentation on the tablet. This physiomechanical property of the tablet is an important parameter in maintaining the stability of the form of the tablet for appropriate drug release kinetics, since reverting back to the powder form will express a greater amount of drug released per given time.

Matrix hardness as a measure of stiffness/rigidity was determined via the area under the force- displacement curve, according to specification in Table 1, using a calibrated textual analyzer (TA.XT plus, Stable Microsystems, Surrey, UK). Attachments from this apparatus (textual analyzer) which were employing for running the following tests was a cylindrical steel probe (50mm diameter; for MR) and an attachment probe with a flat tip (2mm diameter; for MH), conducted at standard conditions of 25°C, latm pressure. MR, as depicted in Figure 7b was determined as a percentage of the ratio of the area under the curve (AUC), from peak to baseline. This ratio can be determined by the area (AUC₂-3) after removing the force initiated, over the area (AUC₁.₂) before removing force, on a force-time axis. MH as depicted in Figure 7a was calculated on the basis of the gradient generated from the curve of the initial force and maximum force derived on a force time axis (N/mm).

MH results obtained in Figure 7a indicated a gradient value close to ~0, therefore representing a tight formation of bonding in the polymer matrix, even though a small compression force of 0.6mPa was used to compress the tablet. This hardness characteristic is essential due to storage/packaging parameters, as well as more physiological parameters of force to withstand swallowing and delayed release disintegration properties for desired outcomes. Matrix resilience is an indication of the ability of a given substance to deform elastically, and revert to its original state, once the force is removed. Most compressed polymers do not share a great resilience profile, since the interparticulate granules have interfacing surfaces which have voids within the matrix structure, created during compression of the tablet process. Some hard polymer blends possess a greater number of interfacing particle surfaces (physical interaction) or a high strength of interfacing particle surfaces (chemical interaction), in which case, the compression induced will not create elastic deformation in the structure. Therefore, as the intermolecular bonds stretch in the tablet, the greater the resilience properties of the tablet. It can therefore be concluded that CHT-PEGDMA-MAA has good properties of mechanical nature, even at low pressures of compression, particles form good physical interaction, thus maintaining a good MH and MR profiles.

Porosity analysis of the optimized formulation (CHT-PEGDMA-MAA)

The optimized polymer blend was stored at different pH media, gastric (pH 1.2) and intestinal (pH 6.8) for 3 hours, and then lyophilized to remove all water particles in the polymer. The polymer in its respective medium was then compressed, as described earlier, and ready for evaluation of their porositometric nature. Brunauer-Emmett-Teller (BET) adsorption and desorption profiles were evaluated for both conditions, in an endeavor to relate drug release profiles and SEM images to the porosity of the samples. Analysis of pore characteristics determined by a technique of physical gas adsorption, which was employed over a wide range of relative pressures (P/Po) and N₂ adsorption isotherms, provided significant insight regarding pore size distributions. This polymer demonstrated type IV isotherms as depicted in Figure 8 with a type HI hysteresis loop. Figure 8 illustrates the different types of isotherms and hysteresis loops that are produced according to differences in amount of gas adsorbed onto a particular material in relation to its relative pressure.

From the hysteresis loop, a number of primary magnetic properties of a material can be determined, including : retentivity (a measure of the residual flux density corresponding to the saturation induction of a magnetic material), residual magnetism or residual flux ( the magnetic flux density that remains in a material when the magnetizing force is zero), coercive force (the amount of reverse magnetic field which must be applied to a magnetic material to make the magnetic flux return to zero), permeability ( a property of a material that describes the ease with which a magnetic flux is established in the component), and reluctance (the opposition that a ferromagnetic material shows to the establishment of a magnetic field) (Bawa et al., 2011).

The type IV adsorption isotherm explains formation of a multilayer material. This can be explained on the basis of a possibility of gases getting condensed in the tiny capillary pores of adsorbent at pressure below the saturation pressure (PS) of the gas. According to a type II and type IV isotherm, the point marked 'B' depicted on Figure 8 refers to the point at which adsorption was complete and was visible at the beginning of the almost linear mid-section of the isotherm. Table 6 indicates the differences in properties of the polymer with a change in pH.

Table 6: Porosity analysis of CHT-PEGDMA-MAA at different pH ranges of gastric and intestinal medium.

Parameter Acidic pH Basic

Surface Area

Single point surface area (m²/g) 1.0973 2.1416

BET Surface Area (i 5.0316 -359.7027

BJH Adsorption cumulative surface area 1.731 3.4400

of pores between 17.000 A and 3000.000

A diameter (m²/g)

BJH Desorption cumulative surface area 2.0094 3.4400

of pores between 17.000 A and 3000.000

A diameter (m²/g)

Pore Volume

Single point adsorption total pore volume 0.002068 0.004173

of pores less than 817.293 A diameter (cm³/g)

BJH Adsorption cumulative volume of 0.004504

pores between 17.000 A and 3000.000 A

diameter (cm³/g)

BJH Desorption cumulative volume of 0.002414 0.004565

pores between 17.000 A and 3000.000 A

diameter (cm³/g)

Pore Size

Adsorption average pore width (4V/A by 16.4411 -0.4641

BET) (A)

BJH Adsorption average pore diameter 54.975 56.834

(4V/A) (A)

BJH Desorption average pore diameter 48.052 53.087

(4V/A) (A)

It is thus evident from Figure 9 a and b that the compressed tablet exposed to basic medium possesses greater pore size and surface area properties than the tablet in acidic environment, due to its swelling nature in the basic medium. The process of lyophilization does create a free flowing powder that also creates pores in the polymer blend, however since this process is constant, all characteristics obtained were due to the changes in medium conditions only.

Scanning Electron Microscopy (SEM) analysis (CHT-PEGDMA-MAA)

The optimized polymer was left in the respective gastric and intestinal fluid for 3 hour duration, before lyophilizing the polymers. After sputter coating the sample for 60seconds, as specified earlier, samples were analyzed at a 10 000X magnifications, for acquiring images related to the specific behavior of the polymers as depicted in Figure 10 a-b. In acidic medium (Figure 10b), the structure of the polymer is very uniformly arranged, with great aggregation and minimum void spaces for leakage of drug to occur. Much to the contrary, in basic medium (Figure 10a), particles appear randomized, with no uniform distribution, arranged in a chaotic, non selective swelled state. This morphology is essential for drug release to occur in basic medium, since escape of drug due to the swelling of the polymer, is essential for maximum fractional release to occur. The greater pore sizes and surface area of the polymer in basic medium, and smaller sizes in acidic medium, corresponds significantly to the morphology of the polymer. The thickness of the walls of the pore structure in the basic medium also indicates that maximum swelling had occurred, in comparison to the acidic medium in which significant charges are present retarding the release of drug due to the strong attraction of the particles closely interacting, thereby preventing escape of the drug.

Thermogravimetric analysis (TGA) (CHT-PEGDMA-MAA)

Thermogravitational analysis (TGA) was carried out on the dry polymers CHT, PEGMDA, CHT- PEGDMA-MAA to analyze points of thermal degradation and percentage of moisture loss in the polymers. Temperatures of 50 to 550°C at a 10°C /min heating rate were undertaken. CHT showed an initial loss of moisture of 1.26% at 86.48°C and a significant step of degradation of 59.94% at 313.61°C. PEGDMA showed no initial liberation of moisture content, however at 418.12°C, 93.1% of the polymer showed thermal degradation. Evaluation of CHT-PEGDMA-MAA liberated 3.5% of water at 76°C and at 403.58°C, 44.66% of the polymer showed thermal degradation as depicted in Table 7. This degradation point can be explained as the decarboxylation reaction that occurs in the polymer blend (Tomar et al., 2011). Therefore it can be justified that crosslinking decreases the degradation point of the polymer, making it more susceptible to thermal conditions. It can also be confirmed that crosslinking occurred in the polymer, since no separate degradation points were present in the optimized formulation. Figure 11 demonstrates the changes in thermal events of the polymers.

Table 7: Thermogravitational analysis of polymers analyzed

Parameter CHT PEGDMA CHT-PEGDMA-MAA

Maximum degradation temperature (°C) 314 418.12 403.5

Water loss at maximum temperature (°C) 86.48 negligible 76

Percentage of water loss at maximal 1.25 negligible 3.5

degradation temperature (%)

Percentage of weight loss at maximal 59.95 93.08 44.66

degradation temperature (%)

Weight remaining at 500°C (%) 40.05 6.92 55.34

All graphs were analyzed according to the first derivative principle (dotted lines), which calculates the percentage water loss and percentage of degradation of the polymer, in a two stage step process, the first occurring from 50-110°C, as this range effectively removes excess water droplets in the formulation. The slight decrease of maximal temperature at which water is removed from CHT-PEGDMA-MAA in comparison to CHT, demonstrates that less water is retained after crosslinking has occurred in the polymer blend.

Karl Fisher volumetric analysis of optimized tablet of CHT-PEGDMA-MAA

The determination of water as a percentage present in the tablet was determined using Karl Fischer Volumetric analysis. This analysis was correlated with the amount of water liberated during TGA analysis of the sample. The percentage of water present in the polymer was analyzed to be 4.672%, which was the same amount of water liberated from TGA analysis. This further exemplifies the precision of the analysis process, and confirms the reliability in both instrumentation analyses. The sample was analyzed under normal conditions of 25°C under normal inert conditions, with a sample run time of 17.11min. Water concentration in the polymer is essential, since drug entrapment is dependent on the capacity of drug absorbed, so the less the water in the polymer, the greater the capacity for the polymer to absorb drug.

Drug (pharmaceutically active ingredient) loading and entrapment for design formulations (CHT- PEGDMA-MAA)

The entrapment efficiency of the design polymers were calculated as outlined in Equation 1 , and a range of values have been outlined in Table 8. It was clearly noted that the percentage of crosslinker and CHT concentration had a significant impact on the amount of drug absorbed in the polymer. The mesh sizes of the particles are essential for high loading capacity. At a basic pH of 7.4, the mesh size was substantially increased due to the swelling properties of the polymer, thereby providing a medium for easy penetration of drug into the polymeric network system. After leaving the polymer in the swelled state for 8 hours, the pH was then changed to 2.5, of which this contracts the polymer system, collapsing the network and allowing insulin (the example of a pharmaceutically active ingredient) to be tightly encapsulated in the particles. Another protective mechanism of PEG is the characteristics of the interaction between the negatively charged carboxylic groups which provides a protective medium of insulin from surrounding charges. It can also be emphasized that PEG has a high affinity for the protein, thereby allowing easy penetration and a stable environment in the particles. A correlation of particle size in basic environment, was indeed noticed, since the greater the particles swelled, the greater the entrapment observed. An understanding of lower entrapment could possibly be due to a high degree of carboxylic functional groups on the surface of the particles, still maintaining a high acid value, as noticed on FTIR analysis. Results showed that there was no definite pattern of order following the amount of crosslinker and concentration of chitosan used, however, the mean values of both concentrations of CHT and crosslinker, produced the best results for maximum drug entrapment to occur. Variations of results in the repeated formulations could be due to drug sticking to the vessel while it was being loaded, or degradation of the protein drug during various phases of the loading procedure.

Table 8: Drug entrapment efficiency of CHT-PEGDMA-MAA formulations

CHT % crosslinking % drug

Formulation

(g) of monomer entrapment

1 0.05 8 18.1

2 0.275 5.5 58.6

3 0.05 3 33.35

4 0.275 5.5 60.82

5 0.275 8 33.81

6 0.5 5.5 47.73

7 0.275 3 58.26

8 0.275 5.5 64.1

9 0.5 3 38.01

10 0.05 5.5 49.72

11 0.5 8 56.56

12 0.275 5.5 60.19

13 0.275 5.5 63.49

High Performance Liquid Chromatography (HPLC) analysis for in vitro drug release (CHT- PEGDMA-MAA)

All drug release data was analyzed using HPLC analysis for the most accurate quantification of insulin (used as an example of a pharmaceutically active ingredient). All samples were analyzed in triplicate to ensure reliable drug release profiles. Samples in acid and basic environments were evaluated and a graph of concentration vs. time was represented for all design formulations. A calibration curve as provided below exemplified the precision of calculating the concentration of insulin.

As seen in Figures 12 a-b, the formulation of insulin possessed excipients that eluted far before the peak of insulin manifested. The process of drug release occurred as soon as the tablet was inserted into its respective mediums; however for significant amount of drug to be quantified at a time, samples were taken every 30min for gastric medium and every hour for intestinal medium. In vitro drug release analysis on design formulations (CHT-PEGDMA-MAA)

Drug release data was analyzed on the basis of a fractional release vs. time profile. All design samples were studied in gastric (pH 1.2) and intestinal (pH 6.8) fluid to simulate GIT conditions. All samples were analyzed on HPLC. Samples were analyzed for 2.5 hours in gastric conditions, in which 0.5mL of sample was extracted every 30min for analysis and replaced with fluid of same volume. 0.5mL of sample in intestinal medium was extracted every hour for 12 hours, however, no further release was obtained after 8 hours, therefore, data is represented to a maximum time frame of 8 hours in basic medium. Table 9 represents the fractional release (FR) at 2 hours in both mediums.

Table 9: Fractional release (FR) at 2 hours and maximum FR in basic conditions

FR at 2 FR at 2 Maximum

Formulation CHT % crosslinking % drug hours in hours in FR in

(g) of monomer entrapment acid basic basic

1 0.05 8 18.1 0.26 0.438 0.98

2 0.275 5.5 58.6 0.385 0.499 0.82

3 0.05 3 33.35 0.177 0.520 0.86

4 0.275 5.5 60.82 0.68 0.550 0.84

5 0.275 8 33.81 0.503 0.191 0.95

6 0.5 5.5 47.73 0.268 0.142 0.57

7 0.275 3 58.26 0.298 0.058 0.86

8 0.275 5.5 64.1 0.541 0.429 0.79

9 0.5 3 38.01 0.162 0.240 0.81

10 0.05 5.5 49.72 0.391 0.455 0.53

11 0.5 8 56.56 0.292 0.132 0.64

12 0.275 5.5 60.19 0.670 0.640 0.84

13 0.275 5.5 63.49 0.588 0.633 0.86

Optimization of the formulation was intended for the least amount of drug release in acid conditions and most significant release in basic conditions. The highest drug release in basic conditions was observed in formulation 1 with the least amount of drug entrapment observed. This could be due to the polymer having less charges and interactions, thereby preventing the association with carboxyl functionality groups, which hinder the release of drug. A proportional correlation of particle size with release in acid and basic conditions was easily determined, since in most cases, as the particle size was increased (5.9- 8.8μηι), the release was also increased. Therefore, the size of the particles that were much smaller, around 3.5μπι, showed more desirable properties with greater sustained release profiles in basic conditions, and lowest release of insulin in acidic medium. In many conditions, the greater the percentage drug loading, the greater the amount of drug released in acidic medium, therefore, values in mid range, such as represented in formulation 9, also being the optimized formulation, showed the most desirable results for drug release rates. Figure 13 a-b represents formulations in gastric medium and Figure 14 a-b represents formulations in intestinal medium.

Mucoadhesive properties of the design formulations (CHT-PEGDMA-MAA)

Of significant consideration in an oral drug delivery system is the ability of the formulation to possess high mucoadhesive properties, due to enhanced abilities of penetration and absorption through the mucosal lining of the intestine. The main component responsible for a high degree of mucoadhesion in the formulation is the use of CHT, due to its highly flexible nature and ionic strength, occurring in the presence of hydrogen bonding which is significantly due to its OH and NH₂ functionalities. There also exists a great degree of strong electrostatic interactions with mucus or the charged mucosal surface, due to the cationic polyelectrolyte nature of CHT. A summary of the results obtained for mucoadession is given in Table 10, where each formulation was analyzed in triplicate, and the average values of percentage crosslinking of the formulation to mucus was obtained.

Table 10: Average percentage of crosslinking of the formulation to mucus

Formulation CHT cone % Average % crosslinking of

number (G) crosslinker formulation to mucus

1 0.05 8 20

2 0.275 5.5 21.2

3 0.05 3 6.5

4 0.275 5.5 16

5 0.275 8 18.76

6 0.5 5.5 14.5

7 0.275 3 4.69

8 0.275 5.5 18.6

9 0.5 3 17.7

10 0.05 5.5 15.6

11 0.5 8 15.3

12 0.275 5.5 16.88

13 0.275 5.5 20.15 As evaluated in the above table, the minimum amount of crosslinker used, the smaller the percentage of mucus crosslinking to the polymer occurring. This correlation is easily understood, since the strength of the particles adhering to each other is greater at higher crosslinking concentrations, as opposed to when the concentration is at a minimum. It can therefore be concluded that the polymer displays sufficient ability for successful mucoadhesion, thereby enhancing the properties of penetration and absorption through intestinal mucosa.

Box-Behnken Design and responses (CHT-PEGDMA-MAA)

A total number of 13 formulations were generated by Minitab 14^R Box-Behnken Design, in which each formulation was prepared and evaluated in triplicate, to ensure most reliable data analysis. Fractional drug release at 2 hours in gastric and intestinal conditions as well as average particle size in neutral pH, were the basis of evaluating the responses from the design. Table 11 represents the order of the formulations generated and undertaken.

Table 11: Formulations generated by Box-Behnken design according to the upper and lower limits of each variable.

Std Pt % crosslinking of

Formulation Blocks CHT (g)

Order Run Order Type monomer

1 3 1 1 1 0.05 8

2 11 2 0 1 0.275 5.5

3 1 3 1 1 0.05 3

4 9 4 0 1 0.275 5.5

5 8 5 -1 1 0.275 8

6 6 6 -1 1 0.5 5.5

7 7 7 -1 1 0.275 3

8 12 8 0 1 0.275 5.5

9 2 9 1 1 0.5 3

10 5 10 -1 1 0.05 5.5

11 4 11 1 1 0.5 8

12 10 12 0 1 0.275 5.5

13 13 13 0 1 0.275 5.5

Residual and surface plots from the responses were generated, showing greatest correlation in average particle size and fractional release in intestinal medium, with P values less than 0.3. The ideal residual histogram should be represented as a bell curve; however histograms bellow show variations in this pattern, due to different behavior of the polymer in various pH states. Responses varied in uniformity, with regard to % entrapment of drug, as well as release rates in different pH states. In some instances, insulin was released greater in acid medium at 2 hours than in basic, due to the constriction of the particles and their release of insulin while undergoing this change. However, particle size properties showed less deviation in their response. Figure 15 represents residual and surface plots of the formulations, in regard to a) average particle size, b) fractional release at 2 hours in gastric medium, c) fractional release at 2 hours in basic medium. Figure 15 shows (a) residual plots of average particle size [(a)(i) a normal probability plot, (a)(ii) residuals vs. fitted values, (a) (iii) a histogram of residuals, (a)(iv) residuals vs. order of the data]; (b) a surface plot of average particle size; (c) residual plots of average particle size of fractional release in gastric medium at 2 hours [(c)(i) a normal probability plot, (c)(ii) residuals vs. fitted values, (c) (iii) a histogram of residuals, (c)(iv) residuals vs. order of the data]; (d) a surface plot of fractional release in gastric medium at 2 hours; (e) residual plots of fractional release in intestinal medium at 2 hours [(e)(i) a normal probability plot, (e)(ii) residuals vs. fitted values, (e) (iii) a histogram of residuals, (e)(iv) residuals vs. order of the data]; and (f) surface plots of fractional release in intestinal medium at 2 hours; all for a dosage form having CHT-PEGD-MA-MAA.

Optimization of the design formulation (CHT-PEGDMA-MAA)

All inputs of average particle size, fractional release in gastric and intestinal conditions at 2 hours were put into the design program of the Box-Behnken design, generating an optimized formulation desirability of 93.01%. This optimized formulation was determined as having a concentration of 0.5g of CHT and 3% of crosslinking agent of total monomer concentration. Figure 16 indicates the program generated graphs for the optimized formulation, taking into consideration the average minimum size of the particles, greater release rates in intestinal conditions and minimum release rates in gastric conditions.

RESULTS AND DISCUSSION FOR TMC-PEGDMA-MAA

Attenuated Transmission Resonance (ATR) analysis all components of the polymer blend for TMC- PEGDMA-MAA

All components of the dosage form of TMC-PEGDMA-MAA were analyzed for their structural functional groups using a pressure of 130psi in scanning spectrum range of 650-4000cm^-1 using a 100 scan run, reducing the signal to noise ratio to 10, in order to attain most accurate peaks from each polymer. TMC, PEGDMA, MAA and TMC-PEGDMA-MAA co-polymer were evaluated separately as shown in Figure 3. The order of the formulations as given from Figure 3 are as follows: (a) Optimized CHT-PEGDMA-MAA, (b) Optimized TMC-PEGDMA-MAA, (c) CHT, (d) MAA, (e) PEGDMA-MAA, (f) TMC. MAA displayed peaks at 1635cm ¹ for carbonyl groups, 1697cm ¹ for vinyl groups and a wide stretch of -OH bonding from carboxylic acid groups, from 3000-3450cm^_1. Evaluating peaks from TMC, it is evident that the reaction to produce TMC was successful due to the peaks at 1475cm^"1 and 1559cm^"1, whereas, if this was not successful, a peak at 1577cm^"1 would indicate that the polymer still remains as chitosan (Mourya et al., 2009). Asymmetrical angular deformation with reference to methyl groups of C- H bonds, characterized at peak at 1475cm^"1, indicates that these methyl groups are covalently bonded to the amino groups on the chitosan structure, since this peak does not exist on the chitosan polymer. N- methylation occurs at 1555cm^"1, due to angular deformation of N-H bond as well as bending of the amino groups. Peaks in the range of 1415-1430cm ¹ are characteristic of N-CH₃ absorption (Tomar et al., 2011). Evaluation of PEGDMA₄₀oo_, gave characteristic broad band peak at 1639cm as an interaction between carbonyl and vinyl groups. CH stretching also gave peaks at 2882cm ¹, and 1466cm ¹, which were clearly indentified in the polymer. Evaluation of TMC-PEGDMA-MAA gave a peak at 1654cm ¹ indicating strong interactive carboxylic acid groups, which is also responsible for pH sensitive nature of the polymer. The peak at 1545cm ¹ also indicates the presence of N-H bending due to the strong interaction of methyl groups with amino groups. The characteristic TMC peak of 1475cm ¹ has shifted to 1466cm ¹ indicating the strong crosslinked nature in the polymeric blend. Figure 3 depicts all data for analysis of each component of the system.

Differential Scanning Calorimetry (DSC) of components and the co-polymeric blend (TMC- PEGDMA-MAA)

DSC profiles were undertaken on TMC, PEGDMA, TMC-PEGDMA-MAA, to determine thermal properties of the polymer, under standard constant nitrogen flow of 0.25psi. All samples were run twice, the first run was from 25-110°C, to remove water droplets from the polymer, and the second run was from 25-350°C, to determine the heat flow in the sample, ramping the temperature by 10°C/min. TMC displayed a melting peak of 214°C, and a peak of crystallization at 256°C. There is also an exothermic peak in the range of 300°C, relating to the decomposition of amine within 2-amino-2-deoxy- β-D-glucopyranose. PEGDMA gave double melting points at 52.61 and 57.91°C. In the polymeric blend, a melting peak at 54.59°C was evident, indicating that crosslinking of the polymer was evident with a modification of the melting peak from the original compound. PEGDMA also depicts a slight endothermic peak at 221°C which is not noticed on the polymeric blend, showing a higher degree of crystallinity after crosslinking. The polymeric blend displayed three distinct melting peaks at 54, 87 and 226°C. The first melting peak is attributed to the characteristics of PEGDMA with its double characteristic peak at 54°C. The second melting peak can be attributed to the shift of the second melting peak of PEGDMA from 57°C to 87°C. The peak responsible for TMC had shifted from 214C to 226°C indicating that the polymer has undergone a higher crystallinity transition to a more stable state due to crosslinking occurring, as depicted in Figure 17 a-c. It can therefore be confirmed that the heat flow properties of the polymeric blend possess variations in thermal properties and can be summarized in Table 12 indicating the different peak flow properties of the polymers.

Table 12: Dynamic Scanning Calorimetry (DSC) analysis of CHT-PEGDMA-MAA, PEGDMA, TMC at heating rate 10°C/min

Polymer constituent Peak (°C) 1 Peak (°C) 2 Peak (°C) 3

TMC-PEGDMA-MAA 54 87 226

PEGDMA 52.61 57.91 221

TMC 214 256

Zeta size of the design formulations and optimization results for size and Potential analysis (TMC- PEGDMA-MAA)

Particle size evaluation was conducted on all design formulations, with the use of varying the concentration of TMC and percentage of crosslinking agent. The formulations were evaluated in their respective pH conditions, gastric and intestinal USP buffers, to exemplify the pH responsive nature of the particles, and the transition of change the particles undergo in the respective mediums. As seen in Table 13, formulations 4,5,6,9 and 11 consisted of the same polymer composition, to determine the reproducibility of the formulations. All samples were analyzed in triplicate to ensure proper data with minimum deviations. For each sample evaluation 5mg of lyophilized polymer was put into 12mL of the respective medium and was left in a dormant state for 2 hours for full responsiveness of the polymer. The sample was then extracted into a curvet, and allowed for particle size determination to occur, with the use of rapid light scattering beams to determine particle sizes. The particles are hydrophobic in nature, which allows reliable results, since no particles can dissolve in the aqueous medium. Results from Table 13 indicate an increase in particle size as the concentration of TMC and % crosslinker are increased. Using a lower concentration of crosslinker, such as 3%, noticeably yielded lower particle sizes. At low pH values, carboxylic groups interact with ether functional groups of PEG, therefore forming strong hydrogen bonding between the particles to theoretically form smaller particles at acidic pH. After evaluation of the sizes of the particles it was distinctly clear that particles in acidic medium gave rise to larger particle sizes in comparison to basic medium. This is due to the aggregation properties of the particles in acidic medium in which particles form clumps and attract each other forming larger masses. This is the opposite effect observed with particles in basic medium due to their repulsive properties, creating a dispersed medium of particles.

Table 13: Analysis of average particle size according to specifications of the design in gastric and intestinal fluid

TMC crosslinking Av size in Av size in

Formulation PDI PDI

(g) of monomer acid basic

1 0.5 5.5 4.2 0.41 2.57 0.25

2 0.275 3 2.2 0.67 1.8 0.27

3 0.5 8 6.2 0.72 2.4 0.35

4 0.275 5.5 5.7 0.52 3.7 0.42

5 0.275 5.5 5.4 0.68 2.6 0.36

6 0.275 5.5 5.2 0.49 2.4 0.53

7 0.5 3 4.7 0.68 1.2 0.61

8 0.05 8 6.3 0.77 2.9 0.52

9 0.275 5.5 6.6 0.62 3.3 0.41

10 0.05 5.5 6.6 0.45 2.3 0.62

11 0.275 5.5 5.2 0.56 3 0.38

12 0.275 8 6.7 0.69 3.4 0.46

13 0.05 3 3.6 0.51 2.8 0.63

With respect to polydispersity index (PDI), particles in acidic medium displayed much greater values, than in basic medium. Optimal values are in ranges of 0.5 and bellow, indicating stability and uniformity of the particles. Therefore clear analysis reflects the instability of particles in acidic medium due to their clamping behavior, and greater uniformity of particles in basic conditions. This clearly confirms the behavior of aggregation in acid pH, and its dispersion properties in basic pH, since particles have less order of arrangement in their aggregated state, and will remain much more uniform in their dispersed behavior. Using the responses from the Box-Behnken design formulations for determination of the optimal polymer blend, formulation 7 was computed as the most significant optimization, based on particle size and drug release behavior. Zeta potential analysis in acid and basic conditions for the optimized formulation was 9.58 and 21.5 respectively.

Porosity analysis of the optimized formulation (TMC-PEGDMA-MAA)

Table 14: Porosity analysis of TMC-PEGDMA-MAA at different pH ranges of gastric and intestinal medium.

Parameter Acidic pH Basic pH

Surface Area

Single point surface area (m²/g) 0.1913 0.9952

BET Surface Area (i 0.2092 0.1172

I Adsorption cumulative surface area of pores between 17.000 A 3.986

and 3000.000 A diameter (m²/g)

1 Desorption cumulative surface area of pores between 17.000 A 4.4645

and 3000.000 A diameter (m²/g)

Pore Volume

*le point adsorption total pore volume of pores less than 796.892 0.004900

A diameter (cm³/g) ί Adsorption cumulative volume of pores between 17.000 A and 0.005245

3000.000 A diameter (cm³/g)

1 Desorption cumulative volume of pores between 17.000 A and 0.004725

3000.000 A diameter (cm³/g)

Pore Size

Adsorption average pore width (4V/A by BET) (A) 1672.1311 BJH Adsorption average pore diameter (4V/A) (A) 52.632

BJH Desorption average pore diameter (4V/A) (A) 42.334

It can therefore be summarized from the above data that the tablet in basic medium possesses greater pore size and surface area properties than the tablet in acidic medium as seen in Table 14. The isotherm for the polymer in acidic medium (isotherm Figure 18a) resulted in data that could not be detected, practically concluding no pores/voids in the polymeric tablet in acid medium. In isotherm Figure 18b, the swelling ability of the polymer is confirmed in basic medium clearly showing greater pore size and volume. During the process of lyophilization of the polymer, pores are created during this process, however, since this process is constant throughout, all characteristic obtained were due to the changes in pH medium conditions only.

Scanning Electron Microscopy (SEM) analysis (TMC-PEGDMA-MAA)

The samples were sputter coated for 60 seconds under standard conditions and analyzed at 10 000 times magnification for clear properties to be evaluated.

It is indeed evident from Figures 19 a-b, how the change in pH affects the surface morphology of the particles. In Figure 19b acidic environment, the structure appears uniform in arrangement, clearly identifying the aggregation of the particles and the arrangement of the microscopic interparticulate spaces so as to avoid leakage of the loaded drug, thereby protecting the contents to the maximum capacity possible. The opposite of this effect is seen in Figure 19a, in which the basic environment of the polymer completely disrupts the uniformity of the particles, resulting in a chaotic swelled state. This state of swelling of the polymer is essential for release of drug, thereby allowing maximum fractional release to occur. A good correlation with porosity analysis is also exemplified in which greater pore sizes are evident in a basic medium than in acidic conditions as seen in the morphology of the polymer.

Thermogravimetric analysis (TGA) (TMC-PEGDMA-MAA)

The evaluations of thermal characteristics of the polymers TMC, PEGDMA, TMC-PEGDMA-MAA, were analyzed for moisture percentage and thermal degradation of the polymers. The polymers were run from temperatures 50 to 550°C at 10C7min. TMC showed an initial loss of 3.7% of water at 97°C and 62% degradation at 241°C. PEGDMA showed no initial liberation of moisture content, however at 418.12°C, 93.1% of the polymer showed thermal degradation. Evaluation of TMC-PEGDMA-MAA liberated 3.7% of water at 74°C and at 409°C 52% of the polymer showed thermal degradation. At this stage of degradation, it can be explained as the decarboxylation reaction that occurs in the polymer blend, in which functional groups no longer sustain their intact structures in the polymer (Tomar et al., 2011). Therefore it can be analyzed that crosslinking of the polymers decreases the degradation point of the polymer, making it more sensitive to thermal degradation as seen in Figure 20 a-c. It can also be confirmed that crosslinking occurred in the polymer, since no separate degradation points were present in the optimized formulation. Table 15 represents thermal values for the formulations.

Table 15: Thermo gravitational analysis of polymers analyzed

Parameter TMC PEGDMA TMC-PEGDMA-MAA

Maximum degradation temperature (°C) 214 418.12 409

Water loss at maximum temperature (°C) 97 negligible 74

Percentage of water loss at maximal degradation 7 negligible 52

temperature (%)

Percentage of weight loss at maximal 62 93.08 52

degradation temperature (%)

Weight remaining at 500°C (%) 38 6.92 48

The evaluation of the graphs obtained was undertaken using first derivative principles (dotted lines), which calculates the amount of moisture loss from 50-110 °C and polymer degradation at much higher temperatures. It is evident that less water was conserved in the crosslinked polymer than the separate components, making the polymer system more stable and less prone to degradation.

Karl Fisher volumetric analysis of optimized tablet (TMC-PEGDMA-MAA)

The amount of water present in the optimized formulation was determined using Fischer volumetric analysis. This amount of water present in the polymer was compared to the amount liberated from TGA analysis. The percentage of water analyzed on Karl Fisher was 3.76% and the amount liberated from TGA was the same. This confirms the correlation between the instruments used and the precision of measurement. All samples were at standard protocol of 25 °C normal inert conditions, with a sample run time of 20.6min. It is essential for a minimum amount of water to be present in the polymer under normal conditions due to stability of the polymer, however, it is more important when loading of the drug is concerned, since greater amounts of peptide can be absorbed when less water content is present in the polymer. Drug loading and entrapment for design formulations (TMC-PEGDMA-MAA)

The design formulation was evaluated for drug efficiency as implemented in Equation 1, with a range of values as outlined in Table 16. It is clearly distinguishable that the concentration of crosslinker and TMC has a significant impact on the percentage of entrapment of insulin (the example pharmaceutically active ingredient) in the polymer. The size of the particles are also essential for loading to occur, therefore while in basic medium of pH 7.4, the mesh size of the particles are greatly increased due to the pH sensitive nature of the polymer, allowing the swelling properties of the polymer to absorb maximum drug into the polymeric network. The polymer was left in this swelled state for 8 hours, thereafter changing the pH to 2.5, which constricts the size of the particles, allowing maximum encapsulation of drug into the system. This important protective property of PEG is due to the fundamental interaction between the negatively charged carboxylic groups which provides a protective medium for insulin from surrounding charges. It is also well noted that PEG has a high affinity for protein and/or peptide drugs, creating an environment for easy penetration and encapsulation of the drug. It is easily distinguished that as the particle size increased in basic environment for loading, so too did the percentage of entrapment increase. Some formulations however displayed lower encapsulation values, possibly due to a high degree of carboxylic functional groups on the surface of the particles as noticed on FTIR analysis, maintaining its high acid value. It is evident from Table 16, that the pattern of percentage drug loading in comparison to particle size in the swelled states correlate significantly, therefore confirming the increase in particle size with the amount of drug absorbed. It is also noted that as the percentage of crosslinker and TMC increase in the formulation, the size and percentage efficiency also increase to a certain degree. Variations of results from the repeated formulations could possibly be due to drug adhering to the vessel walls during loading, or degradation of the protein during various stages of preparation.

Table 16: DEE of TMC-PEGDMA-MAA formulations

crosslinkin entrapme

Formulatio TMC Av size in Av size in

g of PDI PDI nt n (g) acid basic

monomer

1 0.5 5.5 4.2 0.41 2.57 0.25 45.5

2 0.275 3 2.2 0.67 1.8 0.27 59.4

3 0.5 8 6.2 0.72 2.4 0.35 71.18

4 0.275 5.5 5.7 0.52 3.7 0.42 60.93

5 0.275 5.5 5.4 0.68 2.6 0.36 74.69

6 0.275 5.5 5.2 0.49 2.4 0.53 72.13

7 0.5 3 4.7 0.68 1.2 0.61 58.78 8 0.05 8 6.3 0.77 2.9 0.52 51.26

9 0.275 5.5 6.6 0.62 3.3 0.41 71.97

10 0.05 5.5 6.6 0.45 2.3 0.62 16.72

11 0.275 5.5 5.2 0.56 3 0.38 63.41

12 0.275 8 6.7 0.69 3.4 0.46 99.4

13 0.05 3 3.6 0.51 2.8 0.63 34.204

In vitro drug release analysis on design formulations (TMC-PEGDMA-MAA)

In vitro drug release was carried out in gastric and intestinal USP buffer, simulating GIT conditions. All samples were detected on HPLC for insulin detection as discussed earlier. A volume of 0.5mL was drawn each time period and replaced with its respective buffer at the same temperature condition. Samples were then filtered using a 22um Millipore Millex filter (Billerica, MA, USA), and put in an ANSI HPLC vials for evaluation. Table 17 summarizes the fractional release (FR) data at a 2 hour time point in both mediums.

Table 17: Fractional release (FR) at 2 hours and maximum FR in basic conditions

% % % FR in acid % FR in Maximum

Formulatio TMC

crosslinking entrapment (2HR) basic (2 % release n (g)

of monomer HR) in basic

1 0.5 5.5 45.5 15.2 6.2 97.2

2 0.275 3 59.4 12.5 11.0 95.0

3 0.5 8 71.18 3.07 18.5 43.0

4 0.275 5.5 60.93 52.3 12.9 86.3

5 0.275 5.5 74.69 54.8 41.4 83.0

6 0.275 5.5 72.13 65.6 54.3 84.0

7 0.5 3 58.78 3.2 16.3 83.0

8 0.05 8 51.26 26.3 27.3 96.6

9 0.275 5.5 71.97 42.1 60.4 74.00

10 0.05 5.5 16.72 21.2 57.5 66.4

11 0.275 5.5 63.41 46 10.0 84.0

12 0.275 8 99.4 9 6.3 22.9

13 0.05 3 34.204 2.8 20 43.4

Each formulation was intended for minimum drug release in acidic conditions and maximum release in basic condition. The maximum drug release in the basic conditions was observed to be in the middle region of both variable limits. The accountability of greater drug release in acidic medium than basic at the fractional 2 hour duration is due to the further constriction of the polymer at a lower pH, thereby releasing drug at a faster pace, however only to a maximum degree not exceeding amounts greater than the amount released in the total duration of basic conditions in a sustained slow release manner.

Formulations having high % crosslinker, such as formulation 12, even though having the highest drug entrapment resulted in the lowest amount of drug released, due to the strong interparticulate forces preventing release from the polymer. On the other extreme, the lowest % of crosslinker in the formulation, as seen in formulation 7, showed the minimum amount of drug released in acid environments, and above 80% release in basic conditions. It can therefore be concluded that the percentage of crosslinker used has a significant impact on the amount of drug released in both acid and basic conditions. It was therefore selected by the Box-Behnken design that formulation 7 is the optimum formulation to deliver the peptide. Its protective properties in acid environment were most desirable, protecting the peptide to the maximum capacity possible and releasing drug to a substantial amount in basic environment. Figure 21 represents the release rate in gastric medium and Figure 22 represents the release rate in intestinal medium for all design formulations.

Mucoadhesive properties of the design formulations (TMC-PEGDMA-MAA)

TMC is the main polymer responsible for the greatest mucoadhesive properties of the polymeric blend due to its considerate degree of strong electrostatic interactions with mucus or the charged mucosal surface, due to the cationic polyelectrolyte nature of TMC. It has a well defined structural arrangement with improved solubility from its chitosan derivative. Table 18 summarizes the mucoadhesive results of the design formulation, analyzing each sample in triplicate, yielding average percentage crosslinking of the formulation to mucus solution.

Table 18: Average percentage of crosslinking of the formulation to mucus

Formulation % crosslink to

number mucin

1 6.46

2 6.2

3 8.2

4 2.38

5 3

6 3.1 7 23

8 21

9 2.8

10 7.61

11 2.5

12 6.6

13 6.9

Results obtained clearly demonstrate the inversely proportionate relationship between the percentage of crosslinker used and the amount of mucoadhesion to the polymer. At high concentrations of crosslinker, the bonds in the polymeric system was too strong for the polymer to expand and share charges with the mucin solution, however, at low concentrations, the polymeric system was able to share bond charges and attract mucin accordingly. It was also evident that as the concentration of TMC was increased mucoadhesion was proportionally elevated, allowing greater interaction between the polymeric system and mucin. It can therefore be concluded that the optimized formulation displays significant mucoadhesive properties, thereby enhancing the penetration and absorption of the peptide through the intestinal mucosa.

Box-Behnken Design and responses (TMC-PEGDMA-MAA)

The design formulations as depicted in Table 19 were generated by Minitab 14^R Box-Benken Design programme, synthesizing and evaluating each sample in triplicate, to obtain the most reliable data analysis, and developing the most accurate optimized formulation. The responses from the design formulations were; fractional drug release in gastric and intestinal conditions at a 2 hour duration point as well as particle size analysis. Mucoadhesive studies were also conducted on all formulations, thereby providing a comprehensive detail evaluation of all major characteristics of the formulation.

Table 19: Formulations generated by Box-Behnken design according to the upper and lower limits of each variable.

Std Run % crosslinking of

Formulation Order Order Pt Type Blocks TMC (g) monomer

1 6 1 -1 1 0.5 5.5

2 7 2 -1 1 0.275 3

3 4 3 1 1 0.5 8

4 10 4 0 1 0.275 5.5

5 9 5 0 1 0.275 5.5

6 12 6 0 1 0.275 5.5

7 2 7 1 1 0.5 3

8 3 8 1 1 0.05 8

9 11 9 0 1 0.275 5.5

10 5 10 -1 1 0.05 5.5

11 13 11 0 1 0.275 5.5

12 8 12 -1 1 0.275 8

13 1 13 1 1 0.05 3

The design program generated responses from the results of each formulation for determining the greatest correlation with response elements for the optimized formulation. The ideal histograms represent bell shaped curves for the responses, however, slight variations are evident in the responses obtained due to the unique behavior of the polymers in different pH states. Variations of the proposed behavior of the polymer can be attributed to alterations in particle sizes and % entrapment of drug as well as release rates in different pH states, in some instances, releasing greater in acid medium than in basic at the two hour duration, due to the constriction of the particles in acidic medium and their release of insulin while undergoing this change. Figure 23 represent residual and surface plots of the formulations, with regard to average particle size, fractional release at 2 hours in gastric medium and fractional release at 2 hours in basic medium. Figure 23 shows (a) residual plots of average particle size [(a)(i) a normal probability plot,

(a) (ii) residuals vs. fitted values, (a) (iii) a histogram of residuals, (a)(iv) residuals vs. order of the data];

(b) a surface plots of average particle size, (c) residual plots of average particle size of fractional release in gastric medium at 2 hours [(c)(i) a normal probability plot, (c)(ii) residuals vs. fitted values, (c) (iii) a histogram of residuals, (c)(iv) residuals vs. order of the data]; (d) surface plots of fractional release in gastric medium at 2 hours; (e) residual plots of fractional release in intestinal medium at 2 hours [(e)(i) a normal probability plot, (e)(ii) residuals vs. fitted values, (e) (iii) a histogram of residuals, (e)(iv) residuals vs. order of the data]; and (f) surface plots of fractional release in intestinal medium at 2 hours; all for a dosage form having TMC-PEGDMA-MAA.

Optimization of the design formulation (TMC-PEGDMA-MAA)

Responses from the design formulations were evaluated using the Box-Behnken program. The program then calculated the concentration of TMC and percentage of crosslinker that would be ideal for synthesizing the optimum formulation. The optimum formulation was thus calculated as having a concentration of 0.5g of TMC and 3% of crosslinker. This was indeed represented as possessing the ideal properties for delivering the peptide formulation, generating ideal properties for oral drug delivery. Figure 24 indicates the program specifications with graphs generated for the optimized formulation, taking into consideration the average minimum size of the particles, possessing greater release rates in intestinal conditions and minimum release rates in gastric conditions.

Conclusions

It is thus evident that the pH responsive oral polymeric dosage forms of CHT-PEGDMA-MAA or TMC- PEGDMA-MAA as disclosed herein above demonstrate highly efficient systems in encapsulating a protein and/or peptide, which were strategically designed to overcome gastric and intestinal conditions, which provide a poor environment for absorption of proteins and/or peptides. The polymers comprising the dosage forms were strategically crosslinked, and are capable of reaching their target site of absorption, through a pH responsive biostable mechanism of action. The excellent mucoadhesive and pH responsive properties of the pharmaceutical dosage forms described herein provide a crosslinked network beneficial for absorption through the gastro-intestinal membrane. It was indeed evident that the highest degree of loading efficiency in the formulations were demonstrated with a minimum percentage of drug being released in acidic medium according to the pH responsive nature and obtaining maximum release profiles in basic intestinal medium at its site of absorption. It can therefore be concluded that the pharmaceutical dosage forms according to this disclosure possess unique properties and are most beneficial for delivering protein and/or peptide pharmaceutically active ingredients in the most stable bioavailable form possible.

• two extremely mucoadhesive polymers (CHT/TMC and poly-MAA);

• a synthetic (poly-MAA) and natural polymer (TMC); and

• two pH responsive polymers (poly-MAA and CHT/TMC), forming a semisynthetic mucoadhesive-pH responsive conjugated oral polymeric pharmaceutical dosage form according to the invention capable of encapsulating and/or including proteins and/or peptides, facilitated by the presence of -COOH moieties. The dosage form in use protects the proteins and/or peptides from harsh gastric environment and retains dosage form in close vicinity of intestinal wall for a prolonged period.

The Applicant surprisingly found that the CHT/PEGDMA-MAA or TMC/PEGDMA-MAA polymeric architecture is characterized by three-in-one matrix types:

o a semi-interpenetrating polymer network consisting of CHT or TMC and PEGDMA crosslinked MAA wherein one polymer is crosslinked in the presence of another polymer;

o a polyelectrolyte complex formed between the -COOH functionalities of PEGDMA crosslinked MAA and -NH₃ ⁺ functionality of CHT or TMC; and

o PEGDMA crosslinked MAA conjugated to CHT or TMC forming CHT-PEGDMA- MAA.

The unique physico-chemical composition and architecture of the dosage form imparted with properties that at least ameliorate disadvantages known in the prior art.

Furthermore the high resilience acrylate polymer (PEGDA crosslinked MAA) on the TMC backbone provided for a long side-chain molecular conformation capable of entrapping higher amount of protein and/or peptide when compared to known means. This entrapment is further enhanced by the use of a long chain crosslinker (PEGDA) providing an inter- and intra-chain crosslinked network. The retention of this conjugate polymer in "tethered" intestinal mucosa was mediated via two different mechanisms:

The unique physico-chemical properties of the dosage form provided by high molecular weight chitosan and PEGDA crosslinked MAA aided the prolonged retention in the intestine via a unique hard-to-soft swollen hydrogel architecture. The ability of the conjugated system to accommodate various chitosan derivatives (in terms of molecular weight) and crosslinkers and monomers with varying chain length can provide the flexibility required for the extent and rate of protein and/or peptide release from the nanoparticulate matrix. While the disclosure has been described in detail with respect to specific embodiments and/or examples thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing may readily conceive of alterations to, variations of and equivalents to these embodiments. Accordingly, the scope of the present disclosure should be assessed as that of the claims and any equivalents thereto, which claims will be added upon completion of this provisional patent application.

References

Amidi, M.; Mastrobattista, E.; Jiskoot, W.; Hennink, W.E., 2010. Chitsan-based delivery systems for protein therapeutics and antigens. Adv Drug Deliv Rev. 62(l):59-82.

Arduini, R.M., Strauch, K.L., Runkel L.A., 1999. Characterization of a soluble ternary complex formed between human interferon-la and its receptor chains. Protein Sci. 8, 1867-1877.

Bowman, K., Leong, K.W., 2006. Chitosan nanoparticles for oral drug and gene Delivery. Int. J. Nanomedicine.l, 117-128

Bravo-Osuna, I.; Millotti, G.; Vauthier, C; Ponchel, G.,2007. In vitro evaluation of calcium binding capacity of chitosan and thiolated chitosan poly(isobutyl cyanoacrylate) core - shell nanoparticles. Int. J. Pharm. 338(l-2):284-290.

Calceti, P., Salmaso, S., Walker, G., Bernkop-Schurch, A., 2004. Development and in vivo evaluation of an oral insulin-PEG delivery system. Eur. J. Pharm. Sci. 22, 315-323.

Chiu, A.W., Ehrmantraut, M., Richert, N.D., Ikonomidou, V.N., Pellegrini, S., McFarland, H.F., Frank, J. A., Bagnato, F., 2007. A case study on the effect of neutralizing antibodies to interferon beta lb in multiple sclerosis patients followed for 3 years with monthly imaging. Clin. Exp. Immunol. 150, 61-67

Cruise, G.M., Hergre, O.D., Scharp, D.S., Hubbell, J.A., 1998. A sensitivity study of the key parameters in the interfacial photopolymerisation of poly(ethylene glycol) diacrylate upon porcine islets. Biotechnol. Bioeng. 57, 655-665.

Kumara A, Lahiri S.S, Singh H., 2006. Development of PEGDMA: MAA based hydrogel microparticles for oral insulin delivery. Int. J. Pharm. 323,117-124

Domard, A.; Rinaudo, M.; Terrassin, C, 1986. New method for the quaterinization of chitosan. Int J Biol Macromol. 8, 105-107. Eid P., Meritet J.F., Maury, C, et al., 1999. Oromucosal interferon therapy: pharmacokinetics and pharmacodynamics. J. Interferon Cytokine Res. 19, 157-169.

Fischer ,A., Heesen, C, Gold, S.M., 2011. Biological outcome measurements for behavioral interventions in multiple sclerosis. Ther. Adv. Neurol. Disord. ; 4, 217-229.

Hemant, K.S.Y. and Shibakumar, H.G.,2010. A comparative study of N-trimethyl chitosan chloride and chitosan microparticles as novel carriers for the delivery of hypertensive drug. J Pharm Res. 3(4):809- 813.

He, P., Davis, S.S., Ilium, L.,1998. In vitro evaluation of the mucoadhesive properties of chitosan microspheres. Int. J. Pharm. 166, 75-68.

Iwata, A., et al., 1996. Cloning and expression of the canine interferon-beta gene. J. Interferon Cytokine Res. 16, 765-770

Jelvehgari M, Siahi-Shadbad MR, Azarmi S, Martin GP, Nokhodchi A., 2006. The microsponge delivery system of benzoyl peroxide: Preparation, characterization and release studies. Int J Pharm. 308: 124-32.

Jongen, P.J., Hengstman, G., Hupperts, R., Schrijver, H., Gilhuis, J., et al., 2011. Drug adherence and multidisciplinary care in patients with multiple sclerosis. BMC Neurol. 11, 40

Joseph, M., Cummins, G., Krakowka, S., Thompson, C.G., 2005. Systemic effects of interferons after oral administration in animals and humans. Am. J. Vet. Res. 66, 164-176

Kittur, F.S.; Prashanth, H.; Sankar, K.U.; Tharanathan, R.N., 2002. Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry. Carbohydr Polym. 49, 185-193

Kumar, A., Lahiri, S.S., Punyani, S., Singh, H., 2008. Synthesis and Characterization of pH Sensitive Poly(PEGDMA-MAA) Copolymeric Microparticles for Oral Insulin Delivery. J. Appl. Polym. Sci. 107, 863-871

Kumari, A., Yadav, S.K., Yadav, S.C., 2010. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf. B. Biointerfaces . 75, 1-18 le Dung, P.; Milas, M.; Rinaudo, M.; Desbrieres, J., 1994. Water soluble derivatives obtained by controlled chemical modification of chitosan. Carbohydr Polym. 24(3): 209-214.

Morishita, M., Peppas, N., 2006. Is the oral route possible for peptide and protein drug delivery. Drug Discov. Today. 11, 19-20 Mourya, V.K.; Inamdar, N.N., 2009. Trimethyl chitosan and its applications in drug delivery. J Mater Sci Mater Med. 20, 1057-1079.

Bawa P, Pillay V, Choonara Y. E, du Toit L. C, Ndesendo L. V. K, and Kumar P., 2011. A Composite Polyelectrolytic Matrix for Controlled Oral Drug Delivery. AAPS PharmSciTech .12(1)

Sajeesh, S., Sharma, CP., 2006. Cyclodextrin-insulin complex encapsulated polymethacrylic acid based nanoparticles for oral insulin delivery. Int. J. Pharm. 15, 147-154

Sajeesh, S., Sharma, CP., 2006. Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery. J. Biomed. Mater. Res. B. Appl. Biomater. 76, 298-305

Sajesh, S., Sharma, CP., 2006. Interpolymer complex microparticles based on polymethacrylic acid- chitosan for oral insulin delivery. J. Appl. Polym. Sci. 99, 506-512.

Shaji, J., Patole, V., 2008. Protein and Peptide Drug Delivery: Oral Approaches. Indian J.Pharm. Sci. 70, 269-277.

Stuart, M., Bergstrom, L., 2011. Pregnancy and Multiple Sclerosis, J. Midwifery Womens Health. 56, 41— 47

Tomar, L.K., Tyagi, C, Lahiri, S.S.,Singh ,H.,2011. Poly (PEGDMA-MAA) copolymeric micro and nanoparticles for oral insulin delivery. Polym. Adv. Technol. 22, 1760-1767

Veronese, F.M., Pasut, G., 2005. Pegylation, successful approach to drug delivery. Drug Discov. Today .10, 1451-1458

Yang L, Chu IS, Fix JA., 2002. Colon-specific drug delivery: new approaches and in vitro/in vivo evaluation. Int J Pharm. 235: 1-15.

Claims

CLAIMS:

1. A pH responsive oral polymeric pharmaceutical dosage form for site specific delivery of a pharmaceutically active ingredient to a target site in a human or animal body, the dosage form comprising:

CHT-PEGDMA-MAA (chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) copolymer particles, wherein exposure of the dosage form to an increase in pH facilitates swelling of the particles, and

2. The pH responsive oral polymeric pharmaceutical dosage form according to claim 1, further comprising a pharmaceutically active ingredient, such that in use, an increase in the pH facilitates swelling of the particles which in turn facilitates an increase in the release rate of the pharmaceutically active ingredient from the particles, and wherein a decrease in pH facilitates constriction and/or aggregation of the particles which in turn facilitates a decrease in the release rate of the pharmaceutically active ingredient from the particle.

3. The pH responsive oral polymeric pharmaceutical dosage form according to claim 1 or 2, wherein the CHT (chitosan) is functionalized.

4. The pH responsive oral polymeric pharmaceutical dosage form according to claim 3, wherein the functionalized CHT (chitosan) is trimethyl chitosan (TMC) such that the dosage form comprises TMC- PEGDMA-MAA (trimethyl chitosan-poly(ethylene glycol) dimethacrylate-methacrylic acid) co-polymer particles.

5. The pH responsive oral polymeric pharmaceutical dosage form according to any one of claims 1-

4, wherein the dosage form is crosslinked.

6. The pH responsive oral polymeric pharmaceutical dosage form according to any one of claims 1-

5, wherein the target site is the intestinal region of the human or animal body, preferably the intestinal region is the small intestine, further preferably the intestinal region is the muco-epidermal layer of the small intestine.

7. The pH responsive oral polymeric pharmaceutical dosage form according to any one of claims 1- 6, wherein the pharmaceutically active ingredient is a protein and/or a peptide.

8. The pH responsive oral polymeric pharmaceutical dosage form according to claim 7, wherein the protein and/or peptide is at least one selected from the following group: interferon beta, salmon calcitonin, eel calcitonin, chicken calcitonin, rat calcitonin, human calcitonin, porcine calcitonin or any gene-variant of calcitonin, parathyroid hormone, parathyroid hormone analogue PTH 1-31NH₂, parathyroid hormone analogue PTH 1-34NH₂, insulin of any gene variant, vasopressin, desmopressin, luteinizing hormone-releasing factor, erythropoietin, tissue plasminogen activators, human growth factor, adrenocorticototropin, various interleukins, enkephalin and vaccines.

9. The pH responsive oral polymeric pharmaceutical dosage form according to claim 7, wherein the peptide and/or protein may be at least one or more selected from one or more of the following compound classes: anti-inflammatories, immunosuppressives, antibiotics, antifungals, antivirals, antimalarials, antiretovirals, antihypertensives, chemotherapeutics, diagnostic agents, probiotics and prebiotics.

10. The pH responsive oral polymeric pharmaceutical dosage form according to any one of claims 1-

9, wherein the pharmaceutical dosage form is formed into a tablet, caplet or capsule.

11. The pH responsive oral polymeric pharmaceutical dosage form according to any one of claims 1-

10, wherein the particles are manufactured from natural and/or synthetic polymer building blocks, and wherein the chitosan (CHT) building block has a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks have different or the same molecular weights, preferably in range of about 5000-9500g/mol.

12. A method for the manufacture of the pH responsive oral polymeric pharmaceutical dosage form, the method comprising the steps of:

(c) lyophilizing the CHT-PEGDMA-MAA particles of step (b); (d) forming the lyophilized CHT-PEGDMA-MAA particles of step (c) into a tablet via a direct compression technique; and

(e) adding a pharmaceutically active ingredient into the tablet.

13. The method according to claim 12, wherein the esterification reaction of step (a) includes use of a catalyst, preferably the catalyst is an acid, further preferably the acid is sulphonic acid.

14. The method according to claim 12 or 13, wherein the free radical polymerization reaction of step (b) includes the addition of an initiator, preferably the initiator is azobisisobutyronitrile.

15. The method according to any one of claims 12-14, wherein the lyophilisation of step (c) takes place for a period of about 24 hours and takes place at a temperature of about -80□ C.

16. The method according to any one of claims 12-15, wherein the chitosan (CHT) has a molecular weight (Mw) of about 450kDa, and monomethoxypoly(ethylene glycol) building blocks has different or the same molecular weights, preferably in range of about 5000-9500g/mol.

17. The method according to any one of claims 12-16, wherein the adding of the pharmaceutically active ingredient of step (e) includes the steps of:

(h) exposing the tablet to basic medium containing the pharmaceutically active ingredient, such that the tablet swells allowing for penetration of the pharmaceutically active ingredient into the tablet; and

(i) exposing the tablet to an acidic medium such that the tablet constricts and/or aggregates entrapping and/or encapsulating and/or incorporating the pharmaceutically active ingredient.

18. The method according to any one of claims 12-17, wherein the adding of the pharmaceutical ingredient of step (e) occurs through homogenous mixing of the pharmaceutical ingredient and the particles wherein the particles are in a lyophilized powder form that is excluded from any contact with solvent throughout the step, and wherein step (f) takes places in basic medium for 8 hours (pH7.4) to facilitate penetration of the pharmaceutically active ingredient into the particles, and step (g) takes place in an acidic medium of about pH 2.5.

19. A method for the manufacture of the pH responsive oral polymeric pharmaceutical dosage form, the method comprising the steps of:

(b) forming of TMC-PEGDMA-MAA from a free radical polymerization and crosslinking reaction wherein the PEGDMA of step (a), TMC (trimethyl chitosan) and methacrylic acid are reacted together;

(c) lyophilizing the CHT-PEGDMA-MAA particles of step (b);

(e) adding a pharmaceutically active ingredient into the tablet.