Proteins and peptides: The need to improve them as promising therapeutics for ulcerative colitis

Abstract

The present review briefly describes the nature, type and pathogenesis of ulcerative colitis, and explores the potential use of peptides and proteins in the treatment of inflammatory bowel disease, especially ulcerative colitis. Intestinal absorption and the barrier mechanism of peptide and protein drugs are also discussed, with special emphasis on various strategies which make these drugs better therapeutics having high specificity, potency and molecular targeting ability. However, the limitation of such therapeutics are oral administration, poor pharmacokinetic profile and decreased bioavailability. The recent findings illustrated in this review will be helpful in designing the peptide/protein drugs as a promising treatment of choice for ulcerative colitis.

Keywords::

• inflammatory bowel disease
• therapeutic proteins
• therapeutic peptides
• ulcerative colitis

Introduction

Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of inflammatory bowel disease (IBD), which are characterized by chronic, persistent and relapsing ulceration of the bowel, resulting in weight loss, chronic diarrhea and bloody stool (Rabbi et al. 2014). Specific clinical manifestations discriminate both these diseases; however, the common symptoms include diarrhea, bloody stool, weight loss, abdominal pain, cramping, fatigue and fever (Collnot et al. 2012). In UC, the inflammatory response is limited to the colon only, involving its mucosal and submucosal layers, while in CD, the inflammation process extends to any part of the gastrointestinal tract throughout the bowel wall layers, i.e. mucosa, submucosa, muscularis and serosa (Ambrosini et al. 2007). UC is a destructive disease, mainly involving secretory and motility disorders of the large intestine (Stoyanova and Gulubova 2002, Collnot et al. 2012). The precise etiology of these diseases is not yet available now, but some studies report that immune, genetic, environmental, and mainly microbial factors, are involved in the pathogenesis of these disorders (Kanwar et al. 2008). However, one study suggests that their pathogenesis involves an imbalance in the reaction between pro-inflammatory and anti-inflammatory responses, which is mainly due to the activation of CD4 T-helper 1 and T-helper 17 cells (Figure 1) (Kayhan et al. 2013). A number of animal models for colitis have recently been recognized, which provide new insights into the pathogenesis and immune retort of this disease (Hong et al. 2002).

Figure 1. Schematic diagram showing the process of inflammation and colitis.

IBD shows variability of its incidence across different countries; it is high in the United States and United Kingdom (1 in every 250) but low in Asia, South America and New Zealand Maori (Jess et al. 2002, Gearry et al. 2006). The worldwide incidence rate of UC, which is 0.5 to 24.5 per 100,000 inhabitants, is more than that of CD, which is 0.1 to 16 per 100,000 inhabitants (Molodecky et al. 2012, Cosnes et al. 2011). In Asia, the prevalence rate of UC is more than that of CD (Ng et al. 2013). Both these diseases are due to the westernization in the diet and lifestyle of people, and are more prevalent in the developed countries as compared to the developing nations (Ng et al. 2013, Bolge et al. 2010).

The rates of prevalence and incidence of UC in India are much higher than in other Asian countries. In India, the incidence rate of UC is recorded as 6.02 per 100,000 (Sood et al. 2003), which is much higher than in the other Asian countries as a whole, where it ranges from 0.4 to 2.1 per 100,000 population (Yang et al. 2008, Lok et al. 2008, Chow et al. 2009, Morita et al. 1995). Populations of Indian ethnic origin have a higher prevalence of UC as compared to those of Chinese or Malay origin (Lee et al. 2000, Ling et al. 2002, Tan and Goh 2005). All these data provide evidence that UC is a common disease in India, distressing a large range of population.

Although a wide variety of marketed preparations are available for this disease, they provide only symptomatic relief and need further improvement (Randall et al. 2012).

Nature of the disease

This disease is predominant at a young age (< 35 years) (Table I). Increased prevalence in population is due to diet habits involving either gluten-rich food, or the high intake of commercially prepared food. Moreover, UC is more prevalent among non-smokers/ex-smokers, as compared to smokers (Gunisetty et al. 2012). One study reports that males are a little more susceptible to UC as compared to females (Li et al. 2012). An internet-based survey on UC-affected individuals revealed that more than 50% of them experienced flares at least one or more times per week or month (Bolge et al. 2010). It can be recognised that it is a familial disease, as 20% of IBD individuals have blood relatives suffering with the same or other forms of this disease. The genetic basis of this disease comes from the evidence that 36% of monozygotic twins share this disease, while the rate is only 4% in dizygotic twins (Jess et al. 2005, Halfvarson et al. 2005). More than 163 genomic loci are associated with IBD, 90 with CD, 73 with UC, and 110 for both diseases, as reported by genome-wide associated studies (GWAS) (Deuring et al. 2013). The risk of colorectal cancer (CRC) is linked with progressive inflammation in this disease, which increases by 0.5%–1%/year/, 8–10 years after diagnosis (Li et al. 2012).

Table I. Types of ulcerative colitis (Conrad et al. 2014, Travis et al. 2008, Bokemeyer et al. 2012, Bitton 2001, Limbergen et al. 2008).

		Prevalence onset
Types of UC	Parts affected	Adult	Childhood
Proctitis	Rectum	40–50%	1.4%
Left-side colitis [Most prevalent subtype]	Descending colon or Splenic flexure	30–40%	16%
Extensive colitis/Pancolitis	Hepatic flexure or entire colon	25–30%	82%

Microscopic colitis (MC) is another subtype of this disease, in which the colonic mucosa shows inflammatory disorder but the endoscopic result is near normal (Pardi and Kelly 2011). Smoking increases the risk of MC, but alcohol consumption and lifestyle factors have no effect on it (Roth et al. 2014).

Pathogenesis of ulcerative colitis

A genetically prejudiced, blown up and continual response of immunity against the body's own gut flora is an important pathogenic factor of this malady (Figure 2). Environmental factors directly and indirectly stimulate these two factors involved in the disease pathogenesis (Carbonnel et al. 2009, Gibson 1997, Danese et al. 2004, Stasikowska et al. 2012, Pearl et al. 2013, Hanai et al. 2013, Ordas et al. 2012, Farrell and Peppercorn 2002).

Figure 2. Factors involved in pathogenesis of ulcerative colitis.

Oral delivery of peptide/protein drug

The use of peptide and protein drugs has increased noticeably in the last two decades (McCrudden et al. 2013). The parenteral route is generally used for delivering peptides and proteins, but in comparison to the oral route, it has a low patient compliance, consumes time and money, and is inconvenient, sometimes also dangerous (Dorkoosh et al. 2001).

Peptides and proteins are good in many aspects, such as their high potency, good selectivity and low toxicity, but the limitation of oral administration is the enzyme degradation in the GI tract (GIT) and serum (Sobczak et al. 2014). Moreover the molecular weight and size also affect their diffusion through the epithelial layer, which decreases with increase in molecular mass beyond 700 Da (Antosova et al. 2014). Until the mid-1960s, it was assumed that the GIT digests all the orally administered peptides and proteins entirely before their absorption takes place, while little reaches the systemic circulation, having no nutritional or clinical relevance (Gitler 1964). However, later studies proposed that small amounts of such molecules enter blood circulation in the intact form (Gardner 1987). Cyclic peptides, in comparison with linear peptides, have increased metabolic stability (chemical and enzymatic) and lipophilicity, resulting in good half-life and improved penetration across biological membranes (Piekielna et al. 2013). The use of mucoadhesive microsphere preparations from polyanhydrides is another approach to target the drug to the mucous lining of the intestines, which increases its bioadhesive properties, thus resulting in increased bioavailability of the delivered proteins and peptides (Pettit and Gombotz 1998).

Cellular uptake mechanism of proteins and peptides

Detection of antibodies to food proteins in human blood circulation indicated absorption of proteins and peptides (Rundles 1987, Paganelli and Levinsky 1980). Appropriate absorption of protein and peptide is possible in the colon, as it lacks digestive enzymes and proteolytic activity; moreover, it provides longer residence time (Sinha and Kumria 2003). The major transport mechanisms involved in the colon for uptake of protein and peptide are the paracellular, transcellular, carrier-mediated, and receptor-mediated transport (Figure 3).

Figure 3. Integrated process of absorption mechanism and hindrance factor of peptide and protein in intestinal epithelial cell. [A, B, C and D stand for receptor-mediated, paracellular, transcellular and carrier-mediated transport of protein drug respectively, whereas E, F, G and H stand for luminal enzyme, mucin barrier, brush border enzyme and intracellular enzyme respectively].

Paracellular transport

Paracellular transport allows the movement of substances across an epithelium by passing through water filled pores/channels in between epithelial cells (Pappenheimer and Reiss 1987, Madara 1998). It is a passive diffusion transport regulated by a tight junction, which limits the permeation of ions and larger substances (Madara 1998). Peptide and protein molecules having a value of log P less than zero (Pappenheimer & Reiss) and a molecular radius less than 15 Å (approximately 3.5 kDa) enter via this route (Rubas et al. 1996). The tight junctional paracellular space has a dimension of 8–9 Å in the human colon (Tomita et al. 1998). Paracellular transport of polypeptides depends upon the physicochemical properties, ion concentration, pH, molecular dimension, and anomalous mole-fraction effects (Tang and Goodenough 2003, Pauletti et al. 1997, Miller and Johnston 2005), but is not much affected by the lipophilicity and hydrogen bonding capacity. It constitutes about 0.01% of the total absorptive surface area of the intestine (Pappenheimer and Reiss 1987).

Transcellular transport

Transcellular transport involves the process of transcytosis, i.e. the cell's apical membrane takes up the particle endocytically, and it is liberated at the basolateral pole (Shakweh et al. 2004). Unlike the paracellular route, which is limited to only tiny hydrophilic molecules (Morishita and Peppas 2006), transcytosis is suitable for macro-sized proteins and peptides. As peptides have a lipophilicity [log P] range of − 2.2 – + 2.8 (Conradi et al. 1991), the number of polar groups determine its permeability (Burton et al. 1996), and energy is required for such absorption to break the water– peptide hydrogen bonds (Conradi et al. 1991). It is a widely accepted transport mechanism for lipophilic substances, as the biological cell membrane has a lipid bilayer structure (Rautio et al. 2008). The intestine contains M-cells (1% of the total intestinal surface) and enterocytes as transporter cells (Giannasca et al. 1999). M-cells transport a wide range of oral proteins and peptides, as they have high endocytotic capacity (Frey and Neutra 1997, Clark et al. 2000). Transcellular transport depends upon size, charge, lipophilicity, hydrogen bond and presence of ligands. Moreover, the physiology of the GIT and the type of animal model also bear an influence (Florence 2004 & Burton et al. 1991).

Carrier-mediated transport

This process follows an active transport mechanism in which specific molecules are taken up by carriers on the cell surface. It starts after recognition of the target molecule by the carrier, and its transportation across the GI epithelium, against the concentration gradient (Bai and Amidon 1992). Small hydrophilic molecules (Barthe et al. 1999) and di/tripeptides (Bai and Amidon 1992) are actively absorbed by this process. The presence of net charge on peptides affects their affinity for intestinal carrier transport. Neutral dipeptides show the highest affinity, as compared to dipeptides containing two positive charges (Wood et al. 1992), whereas negative charges are more damaging to the affinity of the carrier system as compared to positive charges (Wootton and Hazelwood 1989).

Receptor-mediated transport

In receptor-mediated transport, protein molecules facilitate entry by acting either as receptors for ligands or ligands for surface-attached receptors (Jones 2002). In this transport system, one can increase the oral bioavailability of proteins by the modification of receptors and ligands specific for proteins and peptides. This transport system is known as endocytosis, as it involves cell invagination and vesicle formation. It comprises pinocytosis, phagocytosis, clathrin-mediated (receptor-mediated), and non-clathrin-mediated (potocytosis) endocytosis (Swaan 2002). G-protein coupled receptors [GPCR] play a significant role in exerting the action of a large number of gut peptides. GPCRs comprise a family of about 700 membrane proteins, out of which 87 are identified to have peptide ligands (Harmar 2004).

Progression obstacles: efficacy and safety considerations

Protein drugs formulated for parenteral administration are easily available, but very few formulations for oral administrations are available because of the poor bioavailability profile, as it depends upon the ability to cross the intestinal mucosa and reach the systemic blood flow (Kwan 1997). Only dimensional fractions of orally administered peptides and proteins reach the systemic circulation intact. The systemic availability of orally administered peptides [F] is calculated as

$$F=F_F\times F_G\times F_H$$

Where, F_F = dose fraction that is neither metabolised in the gut nor lost in the faeces, F_G = dose fraction that escapes degradation within GIT wall and reaches the portal vein, F_H = dose fraction that escapes liver metabolism (Langguth et al. 1997). The several factors which are hurdles in achieving good bioavailability of proteins and peptides are gastric pH, enzymatic degradation (Ikesue et al. 1993), large molecular size (Donovan et al. 1990), immunogenicity, short plasma half-life and other factors like adsorption, aggregation and denaturation (Saffran et al. 1986, Fix 1996). The surface area, mucosal thickness, residence time and nature of drug govern site-specific GIT absorption (Kompella and Lee 2001). Thus, development of an effective formulation still remains a challenge to researchers. In the pathway of effective oral delivery of therapeutic protein for IBD, metabolic liability and tissue impermeability are two important barrier factors (Hong et al. 2012).

Metabolic obstacle

In the GIT, metabolic enzymes are present everywhere including the lumen, brush border, cytosol, lysosomes and other cell organelles (Langguth et al. 1997, Reis et al. 2006). Colonic tissue contains microvilli (1 mm high projection), but it lacks villi. Microvilli are a glycoprotein coating (glycocalyx), which forms a biochemical barrier at the gut surface. The glycocalyx consists of sulfated mucopolysaccharides, and being fuzzy and fibrous in nature, it forms a weakly acidic coat over the epithelial cells (MacAdam 1993). It is associated with proteolytic activity of degrading proteins and peptides. The mucus layer present atop the glycocalyx consists of glands and goblet cells (Daugherty and Mrsny 1999). The mucus secreted by goblet cells (MacAdam 1993) is composed of mucin glycoproteins, enzymes, lipids, electrolytes [5%] and water [95%] (Phelps 1978, Ponchel et al. 1987), and restricts the physical movement of proteins and peptides (Daugherty and Mrsny 1999). The mucin glycoprotein imparts the hydrogel characteristics of mucus (Bakhru et al. 2013), and contributes to its cohesive and adhesive nature (Allen and Garner 1980, Kompella and Lee 2001). Moreover, The GIT offers a swarm of diverse metabolic enzymes such as trypsin, chymotrypsin, and elastase, which act as endopeptidases, whereas aminopeptidase and carboxypeptidase A are exopeptidases in nature (Woodley 1994). Being proteolytic in nature, these enzymes easily cleave orally administered proteins and peptides.

Tissue impermeability obstacle

Epithelial cell sheets represent tight apical junctions to most constituents, and work as a semipermeable barrier to ions, cells, solutes and water also. The epithelial cell sheet is identified and characterised as a transmembrane protein tight junction complex and includes proteins like claudins, junctional adhesion molecules (JAMs), occludin and tricellulin. These proteins contribute to the tight junctional nature of the barrier system (Chiba et al. 2008), and this is how the tight junctional structure acts as a physical barrier to peptide and protein transport (MacAdam 1993). Occludin (60 kDa) was the first protein to be identified (Furuse et al. 1993), whereas claudin (18–27 kDa) (Tsukita et al. 2001), is the major protein with barrier function, (Furuse and Tsukita 2006) which the forms the yarn [backbone] for the tight junction (Furuse et al. 1998). However, tight junctions have a role in maintaining homeostasis, and disturbance in their function leads to IBD and other disease conditions (Mankertz and Schulzke 2007). IBD-related diarrhea by a leak flux mechanism is an important characteristic of tight junction impairment (Mankertz and Schulzke 2007, Hering and Schulzke 2009). Changes in tight junctional proteins, especially increased expression of claudin-2, are noticed in UC (Schulzke et al. 2009, Mankertz and Schulzke 2007).

Strategies to improve biological activity, specificity, stability and delivery of proteins and peptides

Physiochemical properties (molecular weight, pH stability, hydrophobicity, molecular size, and ionization constant) and biological barriers (proteolysis in stomach, variable pH, poor permeation and membrane efflux) are the two important factors to be considered carefully for effective oral delivery of proteins and peptides (Mahato et al. 2003). A number of strategies in this regard have been proposed, to avoid the degradation of protein and peptide drugs (Morishita and Peppas 2006). Various approaches are summarized in Table II. They include the opening of tight junctions to increase paracellular transport (Miller and Johnston 2005), and the use of carrier systems such as microemulsions and various polymeric nanoparticles (NPs). Increasing the permeability of the cell membrane retention time in GI retention is another approach. All these techniques result in increased intestinal uptake and lymphatic absorption (Schnurch 2013, Reis et al. 2006).

Table II. Various strategies for biotherapeutics of protein and peptide.

Strategy	Method	Outcomes	References
I. Chemical modification
Modification of peptide at N- and C-terminal	Gly-Gly in methionyl enkephalin is replaced by D-Ala. Replacement of L-amino acid by D-form at C-terminal inhibits the degradation by blocking carboxypeptidases	Half-life of modified peptide is increased significantly compared to that of the parent which is due to low cleavage power of aminopeptidases on Tyr-D-Ala amide bond	(Pauletti et al. 1996)
Cyclic analog of linear one	In this method, an amide bond is formed in between various ways such in N- and C- terminal, in side chain or N- or C-termini or in two side chains	Increased half-life is noticed such as in case of α-melanotropin (α-MSH), it shows 100-fold greater half-life than its linear parent	(Al-Obeidi et al. 1989)
II. Polymeric carrier
Ionically (Fe^3+) crosslinked alginate carboxymethyl cellulose (AC) beads	AC solution is dropped into ferric chloride which contains protein, so that beads containing therapeutic protein are formed	Bioactivity and fragility of protein is preserved in such type of control release beads	(Kim et al. 2012)
New graft copolymers containing NPs using salmon calcitonin as peptide	It has hydrophilic branches and hydrophobic backbone prepared by the dispersion copolymerization method of hydrophilic polyvinyl macromonomers using polar solvent with styrene	Absorption of peptide is enhanced because of bioadhesion of nanoparticle in GI mucosa	(Sakuma et al. 1997)
N-trimethyl chitosan chloride (TMC) for peroral delivery of peptides like buserelin and octreotide	Chitosan undergoes reductive methylation to form TMC which is a partially quaternized derivative of chitosan. TMC, being an excellent absorption enhancer shows its action in neutral and basic environments	In vitro studies show that the transepithelial electrical resistance (TEER) is reduced, which opens the tight junction between the epithelial cells. This results in enhancement of paracellular absorption	(van der Merwe et al. 2004)
III. Vesicular Carrier
Archeosomes	Sulfolobus acidocaldarius extracted polar lipid fraction E (PLFE) is used for preparation which is suitable for oral insulin delivery. Fluorescent labelling of peptide is done for the absorption study	Blood glucose level is decreased significantly as compared to conventional liposome, which proves that it could be a potential carrier	(Li et al. 2010)
Nanosphere containing calcitonin-loaded PA (polyacrylamide)	Prepared according to Birrenbach and Speiser method in which inverse w/o microemulsion undergoes polymerization. Particles are obtained by washing with ethanol several times and then finally freeze dried	Nanosphere of size below 50 nm is formed with good peptide release profile, having good antiprotease activity	(Allemann et al. 1998)
Coated calcium alginate gel bead-entrapped liposome for bee-venom peptide	Solvent evaporation method is used for liposome preparation. Liposome so formed is suspended in sodium alginate solution, and then added dropwise in calcium chloride solution to form beads	In vivo study is done by γ-scintigraphy which reveals that liposomes so formed have protection of peptides from the physiological environment in the GIT	(Xing et al. 2003)
IV. Other approaches
Mini tablet and granule of Desmopressin (1-(3-mercaptopropionic acid)-8-D-arginine) vasopressin monoacetate (DDAVP)	Tablets and granules are prepared with the aid of N-trimethyl chitosan chloride (TMC) as absorption enhancer. Granules are prepared by wet granulation method	Paracellular transport of peptide increases by the action of TMC on epithelial tight junction, which is opened	(Paulsen et al. 1993)

An ideal strategy should include the following points:-

1. Increase in intestinal contact time, resulting in increased bioavailability.

2. Prevent degradation by GIT microbial flora and increase absorption of poor drug.

3. Stability and integrity of peptides and proteins should not be altered.

4. Free from GIT irritation and cost effective also.

Various strategies in this regard include:-

Chemical modification

In last few decades, various structural modifications of peptides and proteins have been done to overcome the metabolic problem (proteolysis), which also increase potency and specificity. Chemical modification includes cyclization (Gangwar et al. 1997), lipophilic derivatization (Goldberg and Orellana 2003), peptidomimetic synthesis (Allemann et al. 1998) or alteration of the carbohydrate moiety (glycoprotein) linked to the protein (Calceti et al. 2004). However, it should be noted that all these techniques should not affect the receptor- binding affinity and the intact form of the protein drugs. A study in the Caco-2 cell monolayer reveals that the conversion of a linear peptide into a cyclic form increases its intramolecular hydrogen bonding, which enhances its permeability. This is because the chances of hydrogen bond formation of peptide molecule with the aqueous solvent are reduced (Gangwar et al. 1997). Addition of functional groups is another approach in this regard, which includes acylation, PEGylation, glycosylation, and cross-linking. The hexyl-insulin monoconjugate (PEG-conjugated insulin) is such example, which shows acceptability in clinical phase for lowering the glucose level (Al-Hilal et al. 2013). PEG conjugation of proteins shows a steric effect, which is helpful in protecting them from enzymatic degradation (Rekha and Sharma 2013).

Use of polymers

Advancement in the oral delivery of protein is incomplete without a significant polymer strategy, as it is a widely applied approach concerning the intestinal absorption, enzymatic degradation and loading capacity (Kadiyala et al. 2010). Various systems of formulation include the use of polymeric NPs, liposomes, bioconjugates, hydrogels and microparticles (Di Marco et al. 2010, Yang and Frokjaer 2009). The choice of polymer should be suitable (composition and physicochemical properties), and according to the nature of peptide, so that optimised nanocomposites are achieved. Here too, PEG is the polymer best suited for the protein-polymer conjugate system, because of its good solubility, stability, biocompatibility and less toxicity (Salmaso and Caliceti 2011). The size of the protein also affects its diffusion from the polymer matrix, as it is very fast in porous gels like polyacrylamide, which leads to tissue damage (Dai et al. 2005). The size of nanocomposites also determines their efficacy, and scientists have found that 600 nm-sized NPs are the best in treating IBD because of drug accumulation and low side effect due to reduced systemic entry (Wilson et al. 2010). Biocompatible polymers (Anionic dextrans, neutral water-soluble polymers, cationic polymer, hydrophobic polymers) release the drug in the intact form and prevent protein degradation, as compared to chemical bioconjugates (Salmaso and Caliceti 2013).

Vesicular-mediated delivery

The vesicular system, being a novel method of drug delivery, provides targeted and controlled delivery of protein drugs with protection against the harsh environment in the GIT. Both hydrophilic and hydrophobic drugs can be loaded suitably in this system. Researchers have designed various carriers for such delivery, among which liposomes were designed first (Kumar and Rajeshwarrao 2011). Similarly, NPs are a part of one such carrier system, having good drug release profile and GIT stability (Rieux et al. 2006). Oral delivery of protein-loaded NPs is useful in targeting enterocytes, M-cells, L-cells and immune cells for treatment of inflammatory conditions and diseases (Rieux et al. 2013). Another novel approach in this regard includes the pH-sensitive and time-dependent vesicular system for inflammation of the colon (Pinto 2010). The most recently developed carriers in this regard include aquasomes (Umashankar et al. 2010), niosomes (Kumar and Rajeshwarrao 2011), bilosomes (Sizer 1997), and biotinylated liposomes (BLPs) (Zhang et al. 2014). Aquasomes are self-assembled three-layered structures in which the drug loading is done in the central solid core. Aquasomes provide structural and biochemical stability to a wide variety of peptides such as insulin and hemoglobin (Pandey et al. 2011), which gives an assurance for their future therapeutic use in peptide delivery (Umashankar et al. 2010). Similarly, bilosomes and biotinylated liposomes (BLPs) are conjugated with bile salt and biotin respectively, having good power of penetration and resulting in increased bioavailability (Sizer 1997, Zhang and Li 2014). The use of poly(lactic acid)-b-Pluronic-b-poly(lactic acid) (PLA-P85-PLA) vesicles is another such example for successful and effective delivery of insulin and other therapeutic peptides and proteins (Xiong et al. 2013).

Other approaches

1. Mucolytics as good permeation enhancers are used for the oral delivery of protein-containing NPs. Absorption increases upto 6-fold by using N-acetyl-L-cysteine (NAC), which is the most common mucolytic for enhancing the bioavailability of oral peptide NPs, as tested in a murine model (Carino et al. 2000).

2. The prodrug approach is another winning strategy that overcomes the metabolic problem of proteins, by altering the biopharmaceutical characteristics of the parent molecule. While designing the prodrug, covalent bonds between the carrier and parent drug play an important role in making the drug unalterable in upper part of the GIT, but allowing rapid release in the colon (Lautenschlager et al. 2014). This approach solves not only the metabolic problem, but it also includes the optimization of solubility, permeability, target delivery, elimination rate, and enzymatic and chemical stability (Gangwar et al. 1997).

3. Contact timing of the protein drug determines its absorption and bioavailability. A mucoadhesive delivery system extends contact time at the absorption site, resulting in increased concentration gradient and rapid absorption without intestinal damage [luminal dilution and degradation factor]. However, the gastrointestinal mucoadhesive patch system (GI-MAPS) is alternative form of such a delivery system gaining importance in the oral delivery of protein (Eiamtrakarn et al. 2002).

4. Nano and micro carriers of protein containing a protease inhibitor give promising results in oral delivery. The use of insulin microspheres (IMS) containing Eudragit L100 and aprotinin (AP) or the Bowman-Birk inhibitor (BBI) as a protease inhibitor, is one such example. This method shows excellent hypoglycemic effect by oral delivery of insulin in diabetic and normal murine models (Morishita et al. 1992). However, protease inhibitors and other absorption enhancers are more effective in the colon as compared to the small intestine (Yamamoto and Muranishi 1997).

5. Cell-penetrating peptides (CPPs), as the name suggests, facilitate drug translocation across the intestinal cell. CPPs may be natural or synthetic and have a short cationic sequence (Fischer et al. 2005). They were discovered in 1988, and are helpful in the oral delivery of a number of small molecules like antibodies, nucleic acids, NPs, peptides, and proteins. Some of the most commonly used CPPs are penetratin, polyarginine, TAT, and transportan (Fonseca et al. 2009). Recent findings make it clear that they have good efficacy and safety profiles for future applications of peptides and proteins (Khafagy and Morishita 2012).

6. Microfabricated patches are a novel method of oral and cutaneous delivery of protein and other drugs. They have a mucoadhesive property and increase the retention time at the intestinal epithelium. Unidirectional and controlled release are two important features of this delivery system (Sant et al. 2012).

7. Microgels and microcapsules provide a putative benefit to preserve the secondary and tertiary structure of proteins. Moreover, they also prevent the aggregation of molecules, with their enzymatic and chemical stability. Protein microgels coated with poly(ethylene terephtalate) (PET) reduce the production of pro-inflammatory cytokines (TNF-α, IL-1β, and MCP-1) in chronic inflammatory conditions (Bysell et al. 2011).

8. The use of protein and peptide drugs with a film coating is also a good treatment option for colon disorders. The film coating protects integrity and facilitates intestinal absorption of orally administered peptide and protein drugs (Maroni et al. 2013).

9. Ceramic NPs are one of the unique nano-carriers for protein and polypeptide delivery (Singh et al. 2013). They protect the structural integrity of the protein and are not affected by the pH change in intestinal delivery. Moreover, they can be rapidly prepared and have no swelling/porosity effect, which makes them better therapeutics (Yang et al. 2010). Ceramic NPs are widely used in the treatment of colitis and other diseases like diabetes, diarrhea, pain, etc. (Raghvendra 2012).

Future of biotherapeutics

Recent findings make it possible to safely and effectively administer peptide and protein drugs for colitis (Table II) and various other disease conditions. This is attributed to the excellent selectivity, potency and effectiveness of such molecules at the site of delivery (Hussain et al. 2001, Kumar et al. 2006, Sato et al. 2006). In the last decades, The FDA has approved 30 therapeutically active protein and peptide drugs, while about a hundred such molecules were under clinical investigation (Yun et al. 2013). While one study report of 2005 clearly shows that more than 40 drugs of peptide and protein origin are available in the world market, there are 270 and 400 peptides are running in the clinical and advanced preclinical phase studies respectively (Renukuntla et al. 2013). At present, about 160 protein molecules are established and licensed as therapeutics. Regulatory agencies are focused on further approval of numerous protein molecules in the coming years (Salmaso and Caliceti 2013). All these outcomes are due to the better understanding and improvement in biotechnological knowledge (genomics, cell-line culture and pharmacological study), and incessant effort by researchers. However, just as everything in this world requires continuous improvement to remain sustainable, the same is applicable in this system also, to enable non- invasive delivery. Recent outcomes assure us that in the next five years, the existing problems will be overcome, and oral drugs will be made available, which is the most challenging form of therapeutics for colitis and other disease conditions.

Conclusions

In conclusion, protein and peptide drugs have therapeutic potential in the treatment of ulcerative colitis. Moreover, peptides from dietary sources are also seen to be useful in modulating the immune function of the intestine (Shimizu 2010). Various traditional and modern strategies have been proposed towards the betterment of protein delivery systems, which are somewhat similar to other conventional non-peptide formulations. No doubt, these approaches make the molecule more effective, and when applied, significantly improve the condition of colonic inflammation. Although the mechanism of action is different for different types of peptides employed, the effect is beneficial towards disease severity. A wide variety of animal species like rats, mice, piglets and also various cell-line studies prove the effectiveness of the use of protein delivery systems. Research outcomes from the last few decades ascertain that it is a wonderful and promising therapeutic approach towards ulcerative colitis, but some improvement is still required to make it rational and applicable in commercial therapeutics.

Table III. Proteins and peptides potentially used in colitis.

Proteins/Peptides	Dosage forms	Dose	Route of administration	Comments	References
Vasoactive intestinal peptide (VIP)	Solution	20 μM	Ex vivo (Male ddY mice colon)	Dysfunction of VIP receptors in muscles and neuronal pathways are involved in colitis	(Kato et al. 2012)
Protamex hydrolysate from C. gigas	Solution	50 mg/kg	Oral (mice) and RAW264.7 cell cultures are also used	It has anti-inflammatory effect and reduces production of potent pro-inflammatory mucosal cytokines	(Hwang et al. 2012)
Trefoil factor family 1 (TFF1) peptide	Solution	100 μg/kg	Subcutaneous (Male, Sprague Dawley rats)	Systemic administration of TFF1 can reduce DSS-induced colitis and shows synergistic effect with co-administered epidermal growth factor (EGF)	(FitzGerald et al. 2004)
Polyethylene glycol (PEG)-porcine glucagon-like peptide-2 (pGLP-2)	Solution	30 μg	Intraperitoneally (male BALB/c mice)	Resistant to degradation, thus increased biological potency and reduced severity of colonic injury	(Qi et al. 2014)
Tumor necrosis factor alpha (TNF-α) and TNF receptor-binding cyclic peptide (TRBCP)	Solution	2.5 mg/kg	Intragastrically (male Wistar rats)	Combination treatment effectively reduces the inflammation of colitis by neutralization of TNF-α and blockage of TNFR1	(Yin et al. 2011)
P-317 peptide [Dmt-c(D-Lys-Phe D-Pro-Asp)NH2], a cyclic analog of morphiceptin	Solution	0.1 mg/kg, 1 mg/kg	Intraperitoneally, oral (Male BALB/c mice)	Shows anti-inflammatory effect in an opioid receptor-dependent manner, which is due to decreased expression of mRNA for proinflammatory cytokines	(Sobczak et al. 2014)
Egg white peptides (EWP)	Solution	150 mg/kg	Intra-gastric infusion through catheter (Yorkshire piglets)	Significantly decreases the expression of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IFN-γ, IL-8 and IL-17	(Lee et al. 2009)
Nuclear factor κB (NF-κB) essential modulator or NEMO Binding Domain Peptide1	Solution	10 mg/kg	Intraperitoneally (IL-10-deficient (IL-10–/–) mouse)	Histological improvement of chronic colitis with, decreased levels of inflammatory cytokines	(Dave et al. 2007)
L-cysteine	Suspension	0.144 g/kg	Intragastrically, through catheter (Yorkshire piglets)	Improvement of colon histology by reducing local chemokine expression and neutrophil influx. TNF-α, IL-6, IL-12p40, IL-1β expression is also reduced	(Kim et al. 2009)
PepT1-transportable soy tripeptide VPY	Solution	10 and 100 mg/kg	Oral (female BALB/c mice) Caco-2 cell and HT-29 cells are also used	Reduction in MPO activity and gene expression of the pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IFN-γ and IL-17 in the colon	(Nolan et al. 2012)
L-tryptophan	Solution	110 mg/kg	Oral (Yorkshire piglets)	Weight gain and reduction in gut permeability. Decreased expression of IL-6, TNF-α, IFN-γ, IL-12p40, IL-1β and IL-17 and increase in expression of apoptosis initiators caspase-8 and Bax	(Kim et al. 2010)

Acknowledgements

The authors are thankful to the Head of the Department, SLT Institute of Pharmaceutical Sciences, for providing necessary facilities and guidance. One of the authors, Mr Kantrol Kumar Sahu, is thankful to GGV for providing financial assistance in the form of a fellowship.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.