Local delivery of macromolecules to treat diseases associated with

the colon

A. Bak a, M. Ashford b, & D. J. Brayden c*

a Advanced Drug Delivery, Pharmaceutical Sciences, 'IMED Biotech Unit, AstraZeneca,

Gothenburg, Sweden, b Advanced Drug Delivery, Pharmaceutical Sciences, 'IMED Biotech

Unit', AstraZeneca, Macclesfield, UK, c UCD School of Veterinary Medicine and UCD

Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

*Corresponding author: E-mail: david.brayden@ucd.ie

Tel: +353 1 7166013, Fax: +353 1 7166104.

Abstract

Current treatments for intestinal diseases including inflammatory bowel diseases, irritable

bowel syndrome, and colonic bacterial infections are typically small molecule oral dosage

forms designed for systemic delivery. The intestinal permeability hurdle to achieve systemic

delivery from oral formulations of macromolecules is challenging, but this drawback can be

advantageous if an intestinal region is associated with the disease. There are some promising

formulation approaches to release peptides, proteins, antibodies, antisense oligonucleotides,

RNA, and probiotics in the colon to enable local delivery and efficacy. We briefly review

colonic physiology in relation to the main colon-associated diseases (inflammatory bowel

disease, irritable bowel syndrome, infection, and colorectal cancer), along with the impact of

colon physiology on dosage form design of macromolecules. We then assess formulation

strategies designed to achieve colonic delivery of small molecules and concluded that they

can also be applied some extent to macromolecules. We describe examples of formulation

strategies in preclinical research aimed at colonic delivery of macromolecules to achieve high

local concentration in the lumen, epithelial-, or sub-epithelial tissue, depending on the target,

but with the benefit of reduced systemic exposure and toxicity. Finally, the industrial

challenges in developing macromolecule formulations for colon-associated diseases are

presented, along with a framework for selecting appropriate delivery technologies.

Key words: colonic drug delivery, oral macromolecule delivery, large intestine,

inflammatory bowel disease, colorectal cancer, , enteric infections.

Contents

Glossary…………………………………………………………………………………..

1. Introduction..................................................................................................................

2. Colon physiology: normal and diseased states.............................................................

2.1 Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS)

2.2 Diarrhoea arising from GI infection

2.3 Colorectal cancer

3. Colonic delivery approaches for small molecules: learnings for macromolecules….

3.1 Time- and pH-based based triggers

3.2 Bacterial enzyme-based triggers

3.3 Mucoadhesive approaches

3.4 Nanoparticles and hydrogels

4. Colonic delivery systems for classes of macromolecules............................................

4.1 Peptides and proteins…………………………………………………………...

4.2 Antibodies.............................................................................................................

4.3 RNA therapeutics..................................................................................................

4.4 Cell-based systems.............................................................................................

5. Development challenges for colonic macromolecule delivery................................

6. Conclusions and future perspectives............................................................................

Glossary

ASO: antisense oligonucleotides

5-ASA: 5-amino salicylic acid

C. diff.: Chlostridium difficile

CD: Crohn’s Disease

CsA: cyclosporine

DNBS: dinitrobenzene sulfonic acid

DSS: dextran-sulphate sodium

HA: hyaluronic acid

IBD: inflammatory bowel disease

IBS: Irritable bowel syndrome

IBS-C: constipation-associated IBS

IBS-D: diarrhea-associated IBS

IBS-C, D: mixed type IBS

I. P.: intra-peritoneal

I.V.: intravenous

S.C.: sub-cutaneous

NP: nanoparticle

MP: microparticle

PLA: poly-lactic acid

PLGA: poly (lactic-co-glycolic acid)

P. O.: per oral

ROS: reactive oxygen species

SCF: simulated colonic fluid

SGF: simulated gastric fluid

SIF: simulated (small) intestinal fluid

siRNA: small interfering RNA

TNBS: trinitrobenzene sulfonic acid

TNF-α: tumor necrosis factor

UC: Ulcerative colitis

1. Introduction

Oral dosage formulation designed for controlled release in the lower part of the small

intestine and the colon to treat region-associated diseases has been achieved for a range of

small molecules, but not yet for macromolecules [1]. The main colonic disease targets for

both small- and macromolecules are inflammatory bowel diseases (IBD), encompassing

Crohn’s disease (CD) and ulcerative colitis (UC), while to a lesser extent there are

formulations aimed at diverticulosis, colonic amoebiasis, and irritable bowel syndrome (IBS)

[2]. Unlike UC, CD and IBS are not restricted to the colonic region, but they typically

involve colonic pathology as a part of their overall signature, as do enteric infectious disease

and colon cancer for which there are no oral colonic release products to date. One aim of this

review is to determine whether macromolecules can also be targeted to the colon for local

delivery using the same technologies that have been designed for small molecules, or whether

there is a need for alternative technologies designed to protect these labile payloads and to

deliver them to the region more accurately. Our macromolecule definition covers amino-acid

based therapeutics (e.g., peptides, proteins, antibodies), and nucleic acid-based structures

(e.g. antisense oligonucleotides, siRNA, mRNA, CRISPR-Cas-9). Though not

macromolecules per se, probiotics and bacteria are included for their potential as cell-based

vectors to release macromolecules in the colon.

Our starting point is a list of oral macromolecules for local gastrointestinal tract (GI) therapy

which, according to public domain resources, were in clinical development in 2016 [3]. We

have updated it here and have restricted inclusion to colonic targeting (Table 1). The group

includes: stable cyclic peptides of < 2000 Da in molecular weight (MW) to treat constipation

associated-IBS (IBS-C), as well as macrocycle antibiotics to treat chlostridium difficile (C.

diff.) and staphylococcal enteric infections. Also included in Table 1 are a range of

approaches to treat IBD: an antibody against tumor necrosis factor (TNF)-α, bacteria that

secrete interleukin (IL)-10, as well as several modified stable antisense oligonucleotides. Of

these, selected macrocycle peptides predicated on the structure of linaclotide (to treat IBS-C)

and on the structure of vancomycin (to treat enteric infections) have been approved by the

FDA, whereas the outcomes for oral antibodies and oligonucleotides in pivotal clinical trials

remains to be seen. Local delivery of macromolecules through oral formulation might be

regarded as being more achievable than systemic delivery since it is not constrained by

having to include formulation components to address low permeability, where there are

perceived concerns over possible toxicity and immunogenicity of both the payload and

permeation enhancement components of the dosage form [3]. Irrespective, the labile nature of

the macromolecule must also be addressed by both systemic and local delivery approaches. In

addition, local delivery of macromolecules to the colon has its own formulation challenges,

including variability in targeting the colon due to intra-subject GI physiology differences,

which can be accentuated by disease, gender, age, and diet. Secondly, an assumption is often

made that targeting of macromolecules will automatically be achieved if regional delivery to

the colon can be accomplished by a formulation: this might be true for antibiotics with

actions in the lumen. For macromolecules acting at the epithelium, however, there is still the

barrier of the overlying mucus layerto overcome [4]. . For other macromolecules, the target

is more likely to be within the epithelial monolayer or the submucosa, so there is a

requirement for epithelial cell apical membrane permeability followed by intracellular drug

release to bind the intended receptor, but with limited permeability across the basolateral

membrane. Here, we assess several of the approaches and technologies for delivering a

range of macromolecule classes to the colonic region for several diseases, but with a focus on

IBD. Fig. 1. presents the molecules that are approved for the different grades of UC or CD,

and it is apparent that macromolecules comprise the majority assigned to moderate-to-severe

cases. It is important to also note that most formulation strategies for colonic delivery of

macromolecules in clinical development outlined are typically limited to simple enteric-

coated capsules, granules or tablets, likely due to their track record in delivering small

molecules to the colonic region.

2. Colon physiology: normal and diseased states

The human colon is divided into four sections: ascending (or proximal), transverse,

descending (or distal), and sigmoid, with a total length of up to 1.5 m, compared to the 6.0 m

length of the small intestine. While the small intestine is designed to absorb essential

nutrients using emulsification, a high epithelial surface area for absorption, and receptor

systems, the colon has a dominant role in maintaining water balance, supporting the inter-

dependent microbiome, and the production of faeces. Its milieu is complex: a semi-solid

mass of faeces, volatile short chain fatty acids, methane, and carbon dioxide, minerals, salts,

water, as well as very high concentrations of up to 1800 microbial species, with

predominantly anaerobic bacteria at densities of up to 10 12 CFU/g in the caecum and colon

[5]. Microvilli are present on transverse furrows of the colon and they comprise 1.2 m2

surface area of epithelia designed for electrogenic chloride secretion and/or sodium

absorption, closely associated with the passive diffusion of ions and water to maintain

osmolarity. The luminal fluid of the normal colon has a wide pH range of 5.5-8.0 across its

four parts [6], values than can overlap with that of the latter part of the small intestine; this

variability has implications for achieving reproducible colonic release from dosage forms

with pH-dependent coatings. Within the colonic regions, pH tends to rise from 6.4 (ascending

colon, due to contribution by short chain fatty acids), to 6.6 (transverse colon), and to 7.0

(descending colon) [7], but there is large intra- and inter-subject variability across these

values.

Colonic transit is slow and variable, ranging from 24-42 h, depending on dietary composition

(e.g. meat-eaters versus vegetarian), with possible differences between male and female,

thereby reducing the accuracy of delivery systems based on time-based release for a

population. This compares to the somewhat more predictable 3-4 h mean range for small

intestinal transit, although there is also variability in that figure. As the composition of the

colon lumen comprises a high percentage of gas and semi-solid materials, fluid volume is

estimated at just 13 mL in the healthy colon [8] and this reduces capacity to reproducibly

dissolve colonic dosage forms. In studies of how colonic physiology changes with age,

compromised levels of colonic neurotransmission and low production of nitric oxide reduce

peristalsis, extend colonic transit times, and promote water retention [9]. With age therefore,

the longer retention times and lower fluid volumes observed in the colon might balance out

somewhat in terms of dosage form dissolution. In terms of inter-species comparisons,

differences in colon physiology with respect to length, the presence or absence of furrows,

transit times, and luminal pH values between rat, dog, pig, and human have been summarized

[10]. Fig. 2. is a summary of the main variables that can contribute to dosage form targeting

and performance in the normal and diseased human colon, respectively. These factors need

to be considered both when designing colonic delivery systems and in using preclinical

models where comparative physiological differences in the colon will have an impact on

performance.

2.1 IBD and IBS

The disease processes in IBD pathologies alter colonic physiology. In active UC, the colonic

luminal pH is considered somewhat more acidic in patients compared to normal subjects,

however the original data is based on limited numbers of subjects and is not a consistent

finding: pH values of 2.3, 2.9, and 3.4 were seen in just 3/6 UC patients, the other three UC

patients having pH values no different from normal subjects [11]. A previous study in 11 UC

patients was nuanced in its conclusion, with more acidic pH values detected only in the

ascending colon compared to the same region in normal subjects [12]. Data from CD patients

is inconclusive, but one study detected acidic pH values of approximately 5.3 in the

ascending and descending colons of four patients compared to controls [13]. The rationale for

the more acidic colonic pH in IBD may relate to an altered microbiome leading to increased

production of lactic acid. Still, recent reviews [14, 15] assume that the colonic pH

differences measured in some IBD patients are representative of the entire cohort. Moreover,

the evidence was from studies where the accuracy of the measurements in the limited fluid

volume of the colonic lumen was not determined and, moreover, it is likely that values will

likely fluctuate with disease progression and severity. This is important because pH

differences may need to be less variable if they are going to be relied upon as an approach for

accurate colonic-targeting of macromolecules, and moreover, more acid pH values have

implications for retaining macromolecule stability

Other physiological differences between the human GI tract of IBD patients and that of

normal subjects appear to be more consistent across cohorts. Patients display up to 20%

longer small intestinal transit times [16], an increase in colonic epithelial permeability (due in

part to inflammatory cytokine action on tight junctions) [17], reduced surface mucus (due to

low numbers of goblet cells) [18], build-up of cationic charge across the epithelial surface

(due to over-expression of transferrin) [19], and an altered microbiome [20]. Hua et al. [14]

have highlighted that many IBD sufferers undergo resection of intestinal regions and that

some of the consequences of a shortened bowel are altered luminal pH and transit times,

impaired regulation of the ileal brake that controls food transit, and reduced small chain fatty

acid digestion, each of which with associated implications for colonic dosage form design.

While retention of the dosage form in the colon may be more challenging in IBD due to

increased rates of bowel movements, delivery into the epithelium or to a downstream target

may on the other hand occur more readily due to increased epithelial permeability. In

addition, there are opportunities to exploit epithelial differences in surface charge to target

macromolecule-entrapped particulates and hydrogels to promote retention in the IBD colon

wall. Furthermore, apart from surface charge differences, there are also likely to be changes

in receptor and transporter expression between normal colon and colon from IBD patients, as

ascertained by immunohistochemistry for the decreased expression of P-glycoprotein and

Breast Cancer Resistant Protein in UC [21] In the pathophysiology of IBS-C, diarrhea-

associated (IBS-D) or mixed type IBS (IBS-C, D), low grade colonic inflammation seems to

occur in a subset of patients, and there is evidence of an overall reduction in numbers of

intestinal endocrine cells and an increase in macrophages, however evidence for microbiome

alterations in the colons of IBS patients is conflicting [22]. IBS is also associated with

reduced GI transit times in women, linked perhaps to prostaglandin production during

menstruation [23].

2.2 Diarrhoea arising from GI infections

Diarrhoea is the ninth leading diagnosis in the ambulatory setting in the USA with over 2.5

million annual encounters with the healthcare system [24]. The problem is even more

prevalent world-wide and diarrhoea is the second leading cause of illness in children under

the age of five [25]. Disease-causing organisms span a range of bacteria, viruses, and

parasites including enterohemorrhagic E. coli, Salmonella, Shigella, Cyclospora,

Cryptosporidium, Giardia, Campylobacter Jejuni, C. diff.), caliciviruses, and enteric viruses.

Pathogenesis can impact colon physiology and hence affect the performance of locally-acting

dosage forms. Toxins can trigger signaling molecules including intracellular cyclic

AMP/GMP and Ca2+ to drive colonic electrogenic epithelial ion and water transport [26].

Cyclic AMP activates the cystic fibrosis transmembrane regulator (CFTR) on the apical

membrane, and Ca2+ similarly activates a Cl- channel [27]. Pathogens including

enteropathogenic E. coli (EPEC) can also prevent Cl- reabsorption into the enterocyte via a

toxin-independent process resulting in internalization of the Cl-/OH- apical exchanger [28].

There is also evidence that decreased Na+ reabsorption into enterocytes also occurs in

diarrhoea and is coupled to increased Cl- secretion. For example, cholera toxin mediates an

increase in intracellular cAMP, which inhibits Na+ uptake via the Na+-/H+ exchanger and

increases electrogenic Cl- secretion across the colon via CFTR [29]. Some of the main ion

and water transport features of the colon are shown (Fig. 3).

Internalization of aquaporins occur at the peak of liquid diarrhoea in a rodent model and is a

non-osmotic mechanism that contributes to the increased water content of colonic matter

[30]. In addition, toxins secreted by intestinal pathogens may increase epithelial permeability

[31], which could lead to an altered concentration of the active pharmaceutical ingredient

(API) in the colonic mucosa. For example, C. Diff. infections are a common cause of

diarrhoea and can be a consequence of broad spectrum antibiotic therapy. In these infections,

the colonic mucosa is exposed to two potent toxins, which bind to surface receptors, are

internalized, and bind covalently to proteins of the RHO-subfamily involved in regulating of

the F-actin cytoskeleton. The proteins are inactivated leading to cell-rounding, perturbed

epithelial tight junctions, and eventually cell death [32, 33]. EPEC infections induce

phosphorylation of myosin light chain, which leads to cytoskeleton contraction and increased

tight junction permeability. The same study also showed a redistribution of occludin from the

epithelial tight junctions to the cytosol [34]. Lastly, there is an increase in colonic motility in

diarrhoea, which can lead to reduced retention of locally-acting dosage forms and hence

incomplete drug release in an appropriate time-frame. Engaged pressure sensors will produce

a bowel movement and propel matter forward, and there will be more bowel movements

during an infection due to the larger intestinal matter volume [35, 36]. Like IBS, it is possible

that bacterial overgrowth can increase colon motility in diarrhoea [37]. In terms of

treatments, controlled- or triggered release of poorly-absorbed macrocycle antibiotics in the

colon to treat C. diff. infection would confer an advantage over current untargeted

formulations. For example, the non-absorbable macrocycle antibiotic, rifamycin, is

formulated as a controlled release system delivered to the colon and was recently filed in the

EU to treat a range of bacterial infections (Table 2).

2.3 Colorectal cancer

Colorectal cancer is the third most common cancer in the world [38, 39] and is characterized

by the development of malignant cells in the colonic epithelium. The interplay between the

gut bacteria, immunogenicity of the cancer cells and pre-existing immunological conditions

have a large influence on the tumour microenvironment [39]. Colon cancer often develops

over 10 years with dysplastic adenomas arising from the colonic mucosa being the most

common precursors [40]. In general, colorectal cancer is divided into the microsatellite

stable/chromosomal unstable pathway and the microsatellite unstable pathway, the latter

being primarily located in the ascending colon [41]. From a molecular pathway perspective,

the activation of WNT signaling pathway and the de-activation of Transforming Growth

Factor (TGF)-β signaling pathway results in increased activity of the MYC regulator gene

which codes for a transcription factor and is associated with most colon and rectal cancers.

Sporadic cancers make up ~50-60% of colon cancers in the US and, of these ~70% follow the

chromosomal unstable pathway in which large segments or whole chromosomes may be lost

or duplicated, while ̴15% follow a pathway in which small changes in DNA at microsatellites

result in cancer-causing genetic mutations [40]. About two-thirds of sporadic cancers arise

distal to the splenic flexure, with about 40% arising in the rectum. The liver is the most

common site of colon cancer metastases, present in ∼20–25% patient at initial diagnosis,

with a further ∼25% developing them thereafter. Treatments for colon cancer include

combinations of surgery, therapy and chemotherapy, the regimes dependent on stage and

localization. Surgery is the main treatment for patients affected by local disease, often

followed by adjuvant therapy for those where the cancer has spread beyond the mucosa. If

there is more advanced disease, a colectomy is required which will alter the colonic

physiology and local microenvironment, with an impact on future local therapies. Despite the

advances in surgical procedures with less invasive procedures, local relapse occurs in ∼5–

10% patients usually within first 2 years of surgery [42].

The majority of chemotherapeutics for colon cancer are combinations of small molecule

cytotoxic molecules administered via the intravenous (IV) route. Common regimens are

FOLFOX: fluorouracil (5FU), folinic acid (also known as leucovorin), and oxaliplatin, and

FOLFIRI: 5FU, leucovorin and irinotecan. Capcetabine (Xeloda®), an oral small molecule

prodrug of 5FU, is also used. It was developed to improve the efficacy and tolerability of

5FU through its tumour-specific conversion to the active drug by thymidylate phosphatase,

an enzyme with elevated levels in many cancers. In practice, capecitabine has improved

tolerability but has similar efficacy to 5FU. In relation to molecular-targeted macromolecules,

the anti-angiogenic, vascular endothelial growth factor (VEGF) inhibitors, bevacizumab

(Avastin®), ramucirumab (Cyramza®) and Ziv-aflibercept (Zaltrap®), or the epidermal

growth factor receptor (EGFR) inhibitors, cetuximab (Erbitux®) and panitumumab

(Vectibix®) are being added to treatments as injectables. More recently, injectable

immunotherapies (tremelimumab, pembrolizumab, nivolumab) have been explored in clinical

trials with mixed results, however when segmented, disease of the ascending colon responds

better, which has been attributed to more active immune cells in that region [39]. Harsh

cytotoxic regimes have several side-effects including diarrhoea, which in turn impact release

of locally-administered drugs. Early detection through colonoscopy and surveillance of

genetic and IBD high-risk groups aims to identify early disease, which opens possibilities of

earlier therapies and local delivery opportunities. Indeed, some companies are focused on

colonic delivery of diagnostics for colon cancer detection: Cosmo Pharmaceuticals [43] are

delivering methylene blue staining along the colon length using its MMX® colonic delivery

technology to make it easier to identify polyps, small adenomas, and cancers via endoscopy.

The role of neoadjuvant chemotherapy prior to surgery in locally-advanced resectable colon

cancer is also under investigation [44]. Adoption of neoadjuvant therapies creates

opportunities for local delivery and more specific therapies as disease understanding

progresses, so avoiding non-specific debilitating chemotherapy prior to surgery. In addition,

it may be possible to use local delivery post-surgery in some patients with limited spread

rather than chemotherapyOverall, oral site-specific delivery of drugs should result increased

tumour concentrations in colorectal cancers and fewer side effects, and thus enable more

efficient delivery of both small- and macromolecules for new and existing targets, and hence

more efficacious therapy

3. Colonic delivery of small molecules: learnings for macromolecules

Marketed small molecules using controlled release approaches for colonic delivery are

primarily for IBD and include mesalamine, sulfasalazine, prednisolone, and budesonide (Fig.

1). The rationale for regional targeting of 5-amino salicylate (5-ASA) moieties (e.g.

mesalamine and sulfasalazine) were to prevent premature absorption of in the stomach and

small intestine and to concentrate 5-ASA in the inflamed colonic region in either free or

prodrug formats [45]. For corticosteroids, the rationale had an additional feature: local

targeting to treat inflammation with the intention to reduce systemic exposure and associated

side-effects [46]. In these examples the principles of dosage form design were based on

identifying features of colonic physiology that are differentiated from the small intestine, as

well as an understanding of GI transit times and inter-subject variability. Yet, even with the

currently marketed small molecule colonic release products for IBD, there is still large inter-

subject variability in the accuracy of delivery to the colon, as is described below in relation to

specific formulation approaches.

Polymer-based drug delivery originated to encapsulate small molecules for release in regions

of the GI tract, and there is an opportunity to leverage some of these technologies to protect

labile macromolecules. Polymers can be designed from a range of monomers or co-

monomers to confer a variety of structures and architectures that give rise to properties useful

for colonic delivery including the capacity to be triggered by physiological features of the

normal- or diseased colon. Film coatings and coated pellet systems for small molecules have

been recently reviewed [47, 48]. Systems targeting the colon typically are differentiated in

the basis of GI transit times, inter-regional pH changes, bacterial enzymes, mucoadhesion,

nanotechnologies and disease-associated triggers. Expanding methodologies to produce both

micro- and nanoparticles as well as hydrogels have allowed elegant approaches for colonic

targeting to be explored; Table 2 compares the polymers used for each of these trigger types

for small molecules. From the following, it is apparent that colonic delivery approaches for

small molecules is highly relevant for macromolecules.

3.1 Time-and pH-based triggers

Time-delayed systems are generally based on ethylcellulose polymers and several of these

older systems are used to deliver small molecules to the colon. The inter- and intra-subject

variability of gastric emptying limits the selectivity of this method of targeting, although a

sustained release of 5-amino salicylic acid (5-ASA, Pentasa®) is marketed for the treatment

of UC. This dosage form consists of micro-granules of drug coated with a semi-permeable

ethylcellulose membrane. 5-ASA is released slowly and at a consistent rate from the stomach

onwards, but significant release in the colon is still low. The most commonly used pH-

dependent polymers are poly(methacrylate)-based, i.e. Eudragit®. The Eudragit® coatings

yield delivery system properties based on acid or basic groups in their structure and are

designed to give different pH solubility profiles in intestinal regions. Both Eudragit S® and

Eudragit L® are designed for colon targeting since they are respectively soluble at pH > 7

and pH > 6. However, as the pH drops in the ascending colon the molecule may not release

if protected only by Eudragit S® and therefore combinations of Eudragit® S and L are often

used [49], although this can lead the converse of premature release in the small intestine.

For the treatment of UC, 5-ASA products containing Eudragit® coatings include Asacol®

(Eudragit S®), Claversal ® and Salofalk ® (both Eudragit L®). A systematic study

however, indicated large variability in release rate of tablets coated with Eudragit® S when

subjected to the range of pH values present in the GI tract, highlighting a relative lack of

specificity [50]. Furthermore, Schellekens et al (2007) [51] also tested Salofalk®, Asacol®,

and Pentasa® timed-release tablets and granules in a GI simulation system and showed that

the percentage of the dose released in simulated colonic fluid was quite low for each.

Variability in the in vivo release in humans of core tablets coated with Eudragit S® was also

suggested from a gamma scintigraphy study in human volunteers [52]. Another study

compared GI concentrations of 5-ASA from three colonic formulations of in healthy human

volunteers: Pentasa® capsules (microspheres coated with ethylcellulose); Apriso® capsules

(Eudragit L® coated granules) and Lialda® (multi-matrix delayed-release tablet coated with

Eudragit S®). While the three formulations differed in their GI luminal concentrations of 5-

ASA (and hence plasma profiles), similar concentrations of the primary 5-ASA metabolite

were found in the faeces, thereby indirectly suggesting similar release and dissolution profiles

from each formulation in the colonic region [53].

To improve localisation to the ileo-caecal junction, a double coating method was designed,

comprising an inner layer of Eudragit S® neutralised to pH 8.0, a buffer, and an outer layer

of Eudragit S®. These tablets disintegrated more consistently in vivo compared to single-

coated tablets, where disintegration was erratic, and mainly in the terminal ileum and at the

ileo-caecal junction [54]. The accelerated in vivo disintegration of this double-coating

Eudragit S® construct may overcome the limitations of conventional enteric coatings

targeting the colon and can help avoid the pass-through of intact tablets. On the other hand,

since the colonic luminal pH in IBD might be more acidic in some but not all IBD patients,

such variability adds complication to the use of pH alone as a trigger for release.

3.2 Bacterial enzyme-based triggers

Polymers can release small molecules in response to the bacterial enzymes produced in the

colon. Two classes dominate: the azo-reductases and polysaccharidases. Polysaccharide-

based polymers including pectin, chitosan, chondroitin sulphate, galactomannose, and

amylose have generally regarded as safe (GRAS) status and are degraded by colonic bacteria

[55]. Pectin, chitosan and glassy amylose have had the most success for small molecule

delivery to the colon. Nevertheless, in their unmodified form, they do not have ideal

properties of carriers, with neither the capacity to both fully protect macromolecules nor to

form robust coatings for solid dosage forms in aqueous solutions. They have therefore been

combined with film-coating materials or used in double coated formats to achieve

improvements for small molecule and macromolecule colonic delivery.

Pectins are a family of heterogeneous polysaccharides composed mainly of D-galacturonic

acid with its methyl ester linked via α (1-4) glycosidic bonds. The linear backbone of

galacturonic acid units are interrupted by occasional neutral sugar residues such as rhamnose,

galactose, arabinose, glucose, mannose and xylose. Pectin is sourced from citrus fruits or

apple pomace and is isolated to produce pectins of varying degree of methoxylation, which

determines its solubility and gelation properties. It is available in a range of molecular

weights from 50-150kDa, corresponding to several hundred to ̴1000 saccharide units. Pectin

is not degraded by gastric or intestinal enzymes; however, it is readily broken down by

pectinolytic enzymes produced by colonic bacteria. It has been explored comprehensively as

a coating for tablets [56]. One of its main disadvantages is its high solubility where premature

release of its cargo can occur, this can be overcome by using a high methoxy pectin

potentially applied as a compression coat or low methoxy pectin in the presence of calcium

ions which through cross-linking reduces the solubility of pectin and makes it more

susceptible to enzymatic attack by pectinases in the colon [56]. One of its main disadvantages

is its high solubility where premature release of its cargo can occur. Other strategies to

reduce its solubility and to improve its coating capacity are to combine it with Eudragit® and

hydrophobic film-forming polymers (ethyl cellulose or cellulose acetate). Examples include

the entrapment of 5FU in pectin microspheres coated with Eudragit® [57] and of 5-ASA in

pellets coated with pectin and ethyl cellulose [58]. In addition, small beads coated with a

precise ratio of pectin to ethyl cellulose film and with a determined thickness suggested site-

specific delivery to the colon in both dog and human as measured by scintigraphy [59].

Tablets of several small molecule drugs intended for IBD treatment were coated with a mixed

pectin and cellulose acetate coat along with a secondary coat of Eudragit® L100. The

mechanism of release was due initially to dissolution of the Eudragit® coat, followed by

degradation of the pectin creating micropores to enable near zero order drug release [60].

Further studies used chitosan to form a polyelectrolyte complex with pectin combined with

hydroxymethylcellulose (HPMC) and localised delivery to the colon was evident from

scintigraphy [61].

Chitosan is obtained from the partial N-deacetylation of chitin. It is biodegradable and

positively-charged at acidic pH. Like pectin, it needs to be combined with other materials to

improve its properties to provide a robust coating and reduce its solubility in the upper GI

tract. It has therefore been combined with Eudragit®, cellulose, and polyvinyl acetates.

Chitosan/polyvinyl alcohol (PVA) (Kollicoat® SR 30D) mixtures were used to coat

theophylline- and 5-ASA pellets [62, 63] and provided colonic release in rat studies.

Chitosan was also mixed with cellulose acetate resulting in a semi-permeable membrane and

tuneable osmotic release of budesonide [64]. Other approaches to prevent premature release

have include the formation of polyelectrolyte complexes with other bacterial enzyme-

susceptible polysaccharides including chondroitin sulphate [65]. In addition, chitosan-based

pellets of rutin, (an aglycone prodrug of quercetin) have been in combined with sodium

alginate, and following coating, demonstrated efficacy in a rat model of UC [66]. Recently, a

double layer-coated colon-specific delivery system has been developed, consisting of a

chitosan-based polymeric sub- coating of a core tablet containing citric acid for microclimate

acidification, followed by an enteric coating of Eudragit E100® and ethyl cellulose [67].

Amylose, the linear polymer of starch, has been exploited for colon targeting because its

glassy amorphous form resists pancreatic α-amylase in the small intestine, while being

fermented by the colonic microbiota. Glassy amylose in combination with ethyl cellulose was

sprayed onto pellets containing 5-ASA with 13C-labelled glucose [68]. Following dosing to

human volunteers, 13CO2 breath testing correlated with arrival of the pellets in the colon, and

this was confirmed by scintigraphy. A similar amylose ethyl cellulose coating was used to

coat prednisolone-entrapped pellets (COLAL-PRED ®) and completed a Phase III trial for

the treatment of UC [69] (NCT00299013). A high amylose starch (Hylon VII) and ethyl

cellulose (Surelease®) mixture was used to coat 5-ASA pellets for delivery to rabbit colon,

yielding a delayed Cmax and Tmax that indicated that the coating had performed [70].

Recognizing the differences in colonic bacterial populations and related enzyme activities in

difference disease states, studies have identified polymeric film coatings that allow for drug

targeting to the colon under pathophysiological conditions. Nutriose FB06® (a branched

wheat dextrin with non-digestible glycoside linkages) and Pea Starch N-735 were combined

with ethyl cellulose for their sensitivity to degradation by fecal samples from CD and UC

patients and to media simulating the contents of the stomach, small intestine and colon [71].

These mixed films were then used to coat 5-ASA pellets and were investigated for their

capacity to target the colon and treat rats with chemically-induced UC. The films revealed

good colonic targeting and a better pharmacodynamic response than Pentasa® pellets [72].

Synthetic azo-polymers have also been explored for colonic drug delivery using the abundant

azo reductase enzymes as release triggers [7]. Straight chain azopolymers were used to coat

budesonide-layered pellets that were in turn over-coated with Eudragit® L. When

administered to rats with 2, 4, 6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in rats,

the pellets displayed higher efficacy than an oral solution or a rectal enema of budesonide

[73]. Concerns over possible safety issues due to the chemistry involved and the need for

organic solvents are the main limitations for use of azo-polymer approaches. Bacterial- and

pH enzyme-triggered approaches have also been compared in human subjects when

theophylline pellets were coated with either Eudragit S® or an amylose /ethyl cellulose

combination [74]. The authors concluded that the bacterial enzyme concept provided more

accurate colon-specific delivery than the pH approach. Finally, there is a combined approach

in trials to achieve colon targeting from the same tablet as measured by scintigraphy in

humans: an outermost Eudragit® S 100 coat overlying a bacterial-sensitive polysaccharide

coat (Phloral™ [75]). The latter offers insurance against failure of the pH-dependent

polymer to completely dissolve in the event of the pH threshold not being met in some

individuals. This approach was used in a successful Phase III trial for mesalazine [76] and

the formulation is due for product launch in 2019. Furthermore, the double coated approach

has flexibility to be used for both tablets and capsules.

3.3 Mucoadhesive approaches

Mucoadhesive systems are intended to keep the delivery system in intimate contact with the

intestinal epithelial membrane, however the mucous layer turns over within 24-48 hours in

humans and may be altered in the diseased state. Mucoadhesion in the colon might be more

successful than in the upper GI tract due to comparably lower smooth muscle motility and

higher mucus residence time, although this might be offset by the slower transit of the dosage

form. Several key properties of polymers for mucoadhesion have been identified. These

include high molecular weight to promote adhesion between the polymer and the mucus, as

well as optimum polymer chain length and flexibility to promote the interpenetration of

mucus network (but still with a molecular weight range to allow drug diffusion). Additional

factors include high viscosity, cross linking density, availability of adhesive bonds,

appropriate polymer concentration, charge, degree of ionisation, degree of hydration and

optimal pH for adhesion. Mucoadhesive concepts for drug delivery have been recently

reviewed [77].

Polysaccharides have also been widely investigated as mucoadhesives with potential for

colonic delivery and these include alginates and chitosans [77]. Chitosan forms hydrogen

and ionic bonds between its positively-charged amino groups and the negatively-charged

sialic acid residues of mucin glycoproteins. Alginate is a linear and anionic polysaccharide

derived from brown seaweed. It is composed of alternating blocks of α-1, 4-l-guluronic acid

(G) and β-1,4-d-manurunic acid (M) units and is available in various grades. Its

biodegradability, low toxicity, chemical versatility, pH-dependent solubility, capacity to form

gels in aqueous media and its mucoadhesive nature are key properties that have led to its

investigation for colonic drug delivery. The mucoadhesive properties of alginate are thought

to originate from the initial wetting and swelling of the polymer providing intimate contact

with the mucosa, followed by interpenetration and entanglement between mucin and the

polymer chains, promoting hydrogen bond formation. The use of alginate in microparticles

for oral delivery to the colon alone and in combination with other polysaccharides and/or pH

sensitive polymers has recently been analyzed [78].

Synthetic polymers including the Carbopols®, a copolymer of acrylic acid cross-linked with

polyalkenyl ethers or divinyl glycol, Carbomer® (acrylic acid-based polymers) and thiolated

polymers (thiomers) have also been investigated. Thiomers are modified versions of

polymers such as chitosan, acrylic polymers, and alginates designed with thiol-bearing side

chains, which are capable of superior physical interaction with mucus due to covalent bond

formation between the thiol groups and cysteine-rich subdomains of mucus glycoproteins

compared to the parent non-thiolated polymer [79]. ThioMatrix (Vienna, Austria) is

exploiting the potential of thiomers for the non-invasive delivery of a variety of drugs using a

proprietary technology, although the clinical experience so far seems to be in ocular- and not

intestinal delivery. Adhesive pellets of 5-ASA have also been prepared with Carbomer® 940

and HPMC as the core materials and coated with Surelease® and Eudragit® S100 as the

outer layer. These pellets were mucoadhesive, targeted to the colon, and efficacious in a rat

UC model [80]. In a similar approach, thiolated chitosan/alginate composite microparticles

coated by Eudragit S-100® were formulated for colon-specific delivery of 5-ASA and

curcumin and were used to successfully treat a rat UC model, demonstrating superior

mucoadhesive properties of the thiolated chitosan/alginate composites over non-thiolated

ones [81]. It is important to emphasize that no oral mucoadhesive tablet- or capsule-based

approach for the colonic delivery of any molecule has succeeded in clinical trials despite over

30 years of effort. In analyzing the possible reasons, it may be related to a combination of

rapid mucus turnover, lack of predictive power from current animal models, and intra-subject

variability in GI and colonic physiology.

3.4 Nanoparticles and hydrogels

Polymeric nano- based delivery approaches offer advantages over more traditional colonic

dosage forms in terms of the potential for better targeting to and/ or accumulation in inflamed

tissues, either passively (biophysical targeting) or via addition of an active ligand, giving the

possibility of improved localisation in diseased tissues. Oral nanoparticle delivery

approaches for IBD have been reviewed recently [14, 82] in respect of the design,

formulation, and their evaluation in preclinical studies. An optimal particle size may allow

preferential interaction with diseased tissue and uptake by macrophages and other immune

cells. The effect of size on fluorescent styrene particles (0.1µm -10µm) was investigated [83]

and the smallest diameter particles bound more avidly to the inflamed colon of a rat UC

model compared to healthy colonic tissue. Nano- formats have enabled the effect of surface

charge to be investigated in respect of electrostatic interactions, with a trend suggesting that

cationic nanoparticles adhere preferentially to negatively-charged mucus, whereas anionic

nanoparticles seem to favour positively-charged proteins in inflamed colonic epithelia,

however conflicting results have been reported. For example, it is well-known that there is

heightened attraction of anionic surfaces of particle formulations to the cationic surface of the

inflamed colon. Work by the Rubinstein group demonstrated that twice as many anionic

liposomes adhered to the inflamed colonic epithelium as neutral or cationic liposomes in the

dinitrobenzene sulfonic acid (DNBS) rat model of UC [84]. The concept was advanced

further when they showed superior efficacy of a group of anti-oxidants entrapped in anionic

liposomes compared to unentrapped molecules in the same rat model [85]. This is discussed

in detail with respect to peptides in Section 4.1.

More recently, biodegradable polymers are being investigated for colonic delivery. Poly-

lactic acid (PLA) and poly (lactic-co-glycolic acid) (PLGA) have been mainly investigated.

PLGA nanoparticles (100 nm) encapsulating the immunosuppressant, tacrolimus, penetrated

three times more into inflamed colonic tissue compared to healthy tissue in a rat colitis model

following rectal administration [86]. However, Schmidt et al [87] demonstrated preferential

accumulation of 3µm- but not 250 nm diameter PLGA particles in inflamed lesions of the

colon from patients suffering from IBD when the particles were administered via enema. The

PLGA nanoparticles appeared to be transported across inflamed mucosal layers rapidly,

whereas microparticles were retained at the mucosal surface. This is contrary to data in

previous animal studies where smaller size appeared to lead to better accumulation, however

the types and surface properties of those particles were different and there were species

differences in intestinal surface area, lumen conditions, and mucosal physiology. The total

fraction of PLGA particles retained in the Schmidt study was also much lower than seen in

previous animal studies where, for example, up to 15% of injected dose was retained [83].

Studies on both how particles interact with the colonic epithelium in the normal and diseased

state, as well as the experience to date with particle-entrapped small molecules have informed

the design of modified particles for macromolecular delivery to the colon.

Challenges for all nanoparticle constructs designed for colonic delivery are to achieve

acceptable drug loading, to avoid premature release, and to minimise burst-release profiles.

To address these three hurdles, PLGA nanoparticles entrapped with budesonide [88] or

curcumin [89] have been coated with Eudragit® S100 and afforded better colonic

localisation and efficacy in a rodent UC model than the drug either in solution or suspension.

That the surface coating of a nanoparticle is as least as important as its diameter and charge

was demonstrated in a study where ethyl cellulose nanoparticles were coated with

polysorbate-20 and the result was accumulation in the inflamed colon of the TNBS mouse

model of UC [90]. Naeem et al. [91] designed a dual pH- and bacterial enzyme-release

trigger by preparing polymeric nanoparticles with a mixture of azo-polyurethane and

Eudragit S®: these particles were selectively distributed in the inflamed colon of rats,

yielding 5.5 fold higher accumulation than those made solely with Eudragit S ®.

Another approach to generate a disease-triggered nanoparticle delivery system in the colon is

to take advantage of high levels of reactive oxygen species (ROS) produced at sites of

intestinal inflammation and in cancerous tissue. Biopsies taken from patients with UC have a

10-100-fold increase in mucosal ROS concentrations in inflamed lesions and these correlate

with disease progression [92]. In cancer, the situation is further complicated since

therapeutics (e.g. doxorubicin) may contribute to elevated ROS concentrations during

induction of apoptosis. Two types of ROS-scavenging nanoparticles have been developed by

Nagasaki’s group: pH-dependent and independent. These “redox” nanoparticles were

prepared via self-assembly of an amphiphilic block copolymer comprising nitric oxide

radicals at the side chains of the hydrophobic segment. They are ~40nm in diameter with low

poly dispersity and have high colloidal stability owing to the PEG shell. Orally-administered

pH-independent nanoparticles accumulated in the inflamed colonic mucosa of mice,

effectively scavenged ROS, thereby thus reducing inflammation in a mouse model of UC.

They also inhibited the development of colitis-associated colon cancer in a mouse model in

the absence of systemic absorption [93, 94]. The group also compared the efficacy of redox

nanoparticles in combination with doxorubicin in a colitis-associated colon cancer model in

mice and showed that the pH-dependent and pH -independent nanoparticles could improve

drug uptake into cancer cells but not healthy cells, reduce adverse effects, and improve

efficacy [95]. The results indicate that the combination of anti-oxidative nanoparticulate

approaches with conventional chemotherapy could be a promising strategy for well-tolerated

anti-colon cancer therapy. Macromolecules can potentially be added as components of these

type of constructs.

Studies have also been carried out to ascertain the potential of lectins as ligands to improve

colon targeting of nanoparticles. Wheat germ agglutinin and peanut agglutinin were

covalently attached to the surface of PLGA nanoparticles entrapping budesonide and yielded

enhanced bioadhesion to inflamed tissue and improved efficacy versus untargeted

counterparts in a mouse UC model [96]. Coco et al (2013) [97] synthesized mannose-

functionalized PLGA nanoparticles, which had a ̴ 3-fold higher uptake in inflamed mouse

colon tissue compared to controls. In addition, ligands for targeting enterocytes, M cells, and

goblets cells can also be used to improve colon targeting of nanoparticles, however

prevention of premature ligand exposure would be required to ensure site- specificity for the

colon if the receptors are present throughout the GI tract. Despite the elegance of the actively-

targeted nanoparticle approaches to achieve colonic localization, to date none have

progressed beyond in vitro cell culture and rat studies.

Colonic delivery of small molecules via triggered hydrogels has also been explored.

Targeting to inflammatory tissue can be achieved by taking advantage of the depletion of the

mucus layer and in situ accumulation of cationic proteins including transferrin, anti-microbial

peptides, as well as by exploiting increased epithelial permeability. The inflammatory

process is accompanied by release of cytokines and extracellular matrix metalloproteinases.

These features can be used for the electrostatic attraction of negatively-charged delivery

systems incorporating a stimulus for release. For example, Zhang et al. [98] developed a

hydrogel based on micro-fibers of the amphiphile, ascorbyl palmitate, which is capable of

spontaneous self-assembly into a hydrogel. Dexamethasone was entrapped in the hydrogel

and the resulting construct displayed an anionic surface, which was attracted to the cationic

inflamed epithelial surfaces in vitro, to rodent colitis models, and to ex vivo inflamed colonic

mucosae samples from IBD patients. Dexamethasone was released by enzymatic degradation

and the system was more efficacious in a mouse UC model and less toxic than free

dexamethasone administered via enema.

4. Colonic delivery systems for classes of macromolecules

4.1 Peptides and proteins

The challenges for oral delivery of peptides and proteins in the context of achieving systemic

delivery have been covered recently in terms of approaches in clinical development [99].

Here, we address the potential of targeted colonic delivery of peptides and proteins where the

approach is designed for treating the diseased colonic epithelium and/or sub-epithelial tissue.

It is unclear whether leveraging the technologies used to date to deliver small molecules to

the colon will suffice to deliver challenging labile macromolecules, but perhaps they can be

adapted. The undecapeptide, cyclosporine (CsA, Mw 1203 Da), is in development for

moderate-to-severe IBD using oral dosage formulations designed to reach the colon. It is a

stable cyclic hydrophobic immunosuppressive and the marketed oral microemulsion

(Neoral®, Novartis, Switzerland) has an oral bioavailability of 30-40% [100]. Unusually for

a peptide, absorption of CsA is limited by solubility rather than permeability and it is also

subject to its bioavailability being impeded by interplay between intestinal P-glycoprotein

efflux and the cytochrome P450 3A family [101]. Oral CsA formulations that can deliver

locally to the colon are being investigated as a treatment for severe UC. Intravenous (IV)

administration of the calcineurin inhibitor is currently reserved for severe UC sufferers, who

are non-responsive to corticosteroids and are potentially facing colon removal [102]. A

Cochrane Library Review of the safety and efficacy of randomized clinical trials of injectable

CsA concluded however, that while short term use could be beneficial in assisting steroid-

refractory UC patients achieve temporary remission, chronic use was questionable due to

limited evidence of efficacy and the increased risks of nephrotoxicity, hypertension,

infection, and neurological side effects [103]. On the basis of more recent clinical data

however, CsA is nowadays considered a valid alternative to infliximab in steroid-refractory

UC patients [104] and the use of CsA for this condition has therefore become more common.

A Phase IV clinical trial (NCT00542152) in steroid-refractory UC patients compared efficacy

of periodic i.v.-administered infliximab (Remicade®, Janssen, Belgium) with an initial i.v.

dose of CsA, followed by daily oral Neoral® for three months, however the results have not

yet been reported. The toxicological argument around the potential for nephrotoxicity to be

caused by high blood levels of CsA is spurring efforts to formulate a colonic-delivered format

with restricted distribution beyond the GI tract. Such locally-targeted formulations may also

allow conversion of CsA from i.v. - to oral formulation for patients with lymphoma or

myeloma who develop life-threatening autoimmune intestinal graft-versus-host disease

following an allogeneic bone marrow transplant [105]. In such patients, the optimum route of

delivery and the most appropriate GI region for presentation of CsA remains uncertain [106],

but nevertheless this might be a second disease scenario where delivery of CsA to the

inflamed colon could be beneficial.

In devising approaches for local delivery of CsA to the colon, preclinical research has

focused firstly on creating polymeric micro-and nanoparticulates to deliver CsA across

mucus and into the epithelium. One of the first studies to stimulate this effort was when

CsA-entrapped PLA microparticles of diameters 1-5 µm at an oral dose of 0.2 mg/kg/day

were administered by daily gavage for a week to the dextran-sulphate sodium (DSS)-induced

murine UC model [107]. The particle formulation of CsA was more efficacious than free CsA

with respect to histology scores and myeloperoxidase activity, but importantly, serum levels

of CsA were much lower for the microparticle group. This study was built upon when

efficacy of CsA-entrapped PLGA particles of both 160 nm and 2.7 µm average diameters was

also demonstrated in DSS-treated mice following oral dosing [108]. In that study, plasma

CsA concentrations were measurable for only the larger particles, thus presenting a

toxicological argument in favour of using CsA-entrapped PLGA nanoparticles. A more

complex nanocarrier construct comprised PLGA as a core polymer surrounding CsA, over-

coated by Eudragit FS30D® [109]. These ̴250 nm diameter nanoparticles released the bulk

of their CsA at pH 7.4 and were efficacious in the DSS mouse model of UC following oral

delivery. Overall, these data were consistent with human rectal administration studies of UC

patients to the effect that PLGA nano- and microparticles preferentially attach to the

ulcerated colon per se [87].

CsA is also suitable for entrapment in lipid-based nanoparticle systems. Guada et al. [110]

describe efforts to entrap CsA in a group of lipid nanoparticle prototypes based on ratios of

lipid: aqueous phase components. Following oral administration to the DSS mouse model, it

was disappointing that neither these constructs nor Neoral ®were effective in reducing acute

colonic inflammation in the model although studies on efficacy in chronic inflammation

models have not been tested yet. Toxicity studies in normal mice following repeated oral

gavage suggested some potential for reduced nephrotoxicity and lower blood levels from the

CsA delivered in lipid systems compared to Neoral® [110]. It is worth noting that these lipid

formulations were not designed for colonic targeting and could be re-visited in that regard.

Using other nanocarrier concepts to deliver CsA to the colon, Courthion et al [111] recently

synthesized a CsA-entrapped self-assembled micelle nanocarrier composed of methoxy-PEG

and hexyl- substituted PLA for rectal delivery to a mouse model of UC. With a diameter of

30-50nm and a slightly negative zeta potential, the rhodamine-labelled carriers distributed

preferentially into deep structures of the inflamed colonic tissue compared to normal mice,

and the tissue concentrations of CsA-loaded counterparts were almost 5-fold higher than free

CsA. Moreover, there was no evidence of systemic delivery of CsA by either the entrapped or

even the free CsA delivered rectally over 7 days, a desirable outcome. No doubt, many other

nanoparticle constructs designed for colonic targeting will turn out to be suitable for

delivering CsA, but so far, they are limited to small molecules. One example of a receptor-

targeting approach was the demonstration that mannose-grafted PLGA nanoparticles could

deliver ovalbumin into the inflamed colonic mucosae from DSS-exposed mice in vitro [97].

Nanoparticle concepts for locally-delivering CsA to the colon of UC patients are still at a

relatively early stage and, so far, are largely predicated on excitement from data achieved in

mouse UC models. To develop the CsA particle concept further ultimately depends on

nanoparticles being protected in a suitably-coated capsule that can transit its way to the colon

intact before releasing the particulate epithelial targeting technology. Colonic targeting of

CsA in a solid-dose format containing self-emulsifying beads has been achieved in pigs using

Sublimity Therapeutics’ (Dublin, Ireland) SmPill™ technology [112]. It is based on an oil-in-

water emulsion that forms 1.5mm dried minisphere beads upon cooling in gelatin; the beads

are then coated with pectin over Opadry® white to release in the colon. In the pig study of

oral formulations, there was evidence of CsA concentrating in colonic tissue from the pectin-

coated system, again with reduced levels of plasma CsA compared to Neoral® (Fig. 4).

Using the same technology but with barium as payload, pectin-coated minispheres could be

seen on X-rays reaching the colon of rats intact following oral delivery, whereas ones coated

with ethyl cellulose dissolved in the small intestine [113]. While the rat model confirmed

colonic targeting with coated miniaturized minispheres, its use in developing a solid dosage

form dependent on coatings for regional targeting of the intestine is somewhat limited. Such

studies are technically very difficult to carry out, and, due to species differences in GI

physiology, the larger diameter minispheres require an alteration in coating composition to

achieve accurate release in the colons of higher animals and humans. A SmPill™ minisphere-

in-capsule formulation of CsA (STI-0529) with colon-targeted minispheres similar in design

to those used in the pig study completed a Phase II trial (NCT01033305) in 118 UC patients,

however results have not yet been published. A summary of studies to achieve colonic

targeting of CsA from oral- and (lower hurdle) rectal approaches is presented (Table 3).

Other peptides are being assessed as potential therapeutics for UC. Vasoactive-intestinal

peptide (VIP) is being examined as a potential therapeutic with the challenge to protect it

from intestinal metabolism and formulate it for local colonic delivery. In the mouse and

human colon, expression of the apical membrane-expressed VIP receptor, VIP receptor type

1 (VPAC1), seems to be higher than in other regions of the GI tract [114]. In a study

examining colonic epithelia from UC patients, levels of mRNA for VIP and

immunohistochemistry of the VPAC1 receptor were reduced compared to control epithelia

mucosae [115], but there is no consensus on VIP expression and its regulation as part of the

enteric nervous system in the colon ([116]). Nonetheless, recombinant VIP was injected by

the i.p. route on a daily basis and reduced inflammation in the TNBS rat model of colitis

[117]. VIP was also entrapped in sterically-stabilized micelles and was tested following a

single dose by the i.p. route in the DSS mouse model, where efficacy was again demonstrated

[118]. Data from i.p. administrations needs to be treated with caution as it may reflect an

effect on the colon from the peritoneal side of the colon. Nonetheless, rectal administration

of micelle-entrapped- but not free VIP reduced inflammation and diarrhoea in the DSS mouse

model of UC [119], thereby demonstrating an action from the mucosal side of the epithelium.

It is possible therefore that an oral controlled release of stabilized versions of VIP or VIP

analogues designed to release in the colon of UC patients might be efficacious.

The bioactive natural tripeptide, Lys-Pro-Val (KPV) is isolated from the C-terminus of α-

melanocyte stimulating hormone and has anti-inflammatory actions [120]. In an early attempt

to deliver it to the colon to treat colitis, it was formulated in PLA-based NPs with poly-vinyl

alcohol as a surfactant [121]. Subsequently, it was encapsulated in a hydrogel made of

alginate and chitosan, which was hypothesized to collapse in the colon upon degradation of

the polysaccharides. Daily gavage of the hydrogel was more efficacious than KPV in solution

in the DSS mouse model of UC and the conclusion was that far lower concentrations of the

peptide might be delivered to the colon in the hydrogel format. A further iteration was to

entrap KPV in a hyaluronic acid-based polymeric nanoparticle in a hydrogel, where it also

produced similar positive data following gavage [122]. KPV is transported into colonic

epithelia by human intestinal peptide transporter 1 (hPEPT1), which is upregulated in UC and

is associated with colorectal cancer. Studies in PepT1 knock-out mice or in transgenic mice

over-expressing hPEPT1 revealed a role for KPV in preventing inflammation-linked

carcinogenesis and that the beneficial effect was associated with hPepT1 down-regulation

[123]. New molecules to treat the different scales of UC must differentiate from the

established therapeutics in terms of efficacy or side-effects. Colonic delivery can assist in

this, but the concept of a peptide with additional potential to treat UC-associated colorectal

cancer via a molecular target is of special interest.

Another interesting example is the neuroprotective octapeptide, NAP (NAPVSIPQ,

davunetide). This peptide was administered by the i.p. route daily from day 3-6 after

induction by DSS and, while it did not alleviate macroscopic colonic inflammation, there was

a reduction in pro-inflammatory signaling and in local T cell numbers in the inflamed

intestine [124]. Though the data thus far does not suggest a major beneficial effect for UC

from this initial study nor does it confirm a direct action on colonic mucosae, part of the

interest is because NAP may have potential to be re-purposed from its main research area as a

neuroprotective agent in CNS. If it was to progress as an injectable for UC, options for local

colonic delivery could be examined. Another synthetic 37-mer peptide, gp96-II, was also

administered by repeat i.p. injections to two murine models of UC, the TNBS and the

IL-12/IL-18 models, representing acute and chronic inflammation scenarios respectively

[125]. Gp96-II is an antagonist of the heat-shock protein 90, GP96, which is associated with

inflammation via a selection of Toll-like receptors (TLR), including TLR-4. Disease activity

scores, weight loss, and reduced colon damage morphology was detected in both models

when the peptide antagonist was administered by daily i.p. injection to both murine models

and the data was on a par with that seen with prednisone. With the caveat that a direct

mucosal action of Gp-96-II is not yet confirmed, , if so, it is likely to be too large to cross the

intestinal wall, so perhaps local delivery to the colonic epithelium might be an interesting

option if it can be protected during GI transit.

L cells in the intestinal epithelium produce endogenous pleiotropic peptides including GLP-1,

GLP-2 and PPY. Apart from known benefits in managing Type II diabetes and obesity, there

could be additional potential for GLP-1 and GLP-2 analogues as anti-inflammatories in IBD.

Recently, the capacity of 200nm diameter lipid-based nanocarriers (in the absence of a

payload) to stimulate release of GLP-1 was demonstrated in mice [126]. Remarkably, the

same group has confirmed that increased plasma levels of GLP-2 can be induced at 60

minutes following oral administration of selected lipid-based nanocarriers (again without a

payload) to DSS-exposed mice [127]. This is of interest because GLP-2 can protect the

inflamed epithelium in the TNBS mouse model of UC it may do so by preventing a reduction

in VIP-expressing enteric neurons [128]. In addition, a peptidase-resistant injectable analogue

of GLP-2, teduglutide (Gattex®, Shire Pharma, Dublin), is approved to treat short-bowel

syndrome and intestinal failure in patients [129]. Perhaps combinations of stable analogues

of GLP-1 and GLP-2 can be delivered to the UC epithelium in a nanocarrier-protected format

encompassing a range of lipids that in turn have the capacity to stimulate release of

endogenous counterparts from L cells. A final point on the potential for nanoparticles to

deliver peptides to the colonic epithelium was suggested by a study in which the intestinal

fate of insulin-loaded nanocomplexes made from a range of excipients were studied using a

range of in vitro and in vivo imaging techniques [130]. The main conclusion offered was that,

although these particles did not deliver insulin to the circulation very well, one explanation

was that they may accumulate in intestinal tissue and could therefore have a niche for local

delivery to treat IBD with alternative payloads.

Efforts are also being made to improve the stability and efficacy of injectable small peptides

with potential to treat UC by prolonging their half-lives and reducing both the injection

burden and the concentration of peptide required. The anti-inflammatory tripeptide, MC-12

(Ac-Gln-Ala-Trp), is a motif derived from Annexin A1, and it down-regulates production of

TNF-α, IL-6, L-10, and NFkB, but it is sensitive to serine proteases. It was revealed as an

anti-inflammatory peptide following i.p., oral, and rectal administration to the DSS and

TNBS mice models with several doses of 5 and 25 mg/kg during the induction phases [131].

Cabos-Ceceres et al. [132] grafted it onto a cyclic 14-mer peptide scaffold made from

sunflower trypsin inhibitor. It was administered by a single i.p. injection of the stabilized

peptide at a single dose of 3mg/kg ahead of induction of murine intestinal inflammation by

TNBS. Due to the increased stability and potency, the cyclic version of MC-12 improved the

parameters of weight loss, colon length, and activity score in the model compared to free

MC-12. In this example, since it was resistant to trypsin, the modified peptide might be able

to resist small intestinal attack and could potentially be a candidate for oral colonic delivery.

4.2 Antibodies

As with peptides, therapeutic monoclonal IgG antibodies are unstable at acidic stomach pH

and are labile in the presence of intestinal peptidases. While systemic delivery from oral

formulations is not a realistic option for antibodies due the poor permeability of such

excessively-high molecular weight and hydrophilic moieties, there is still potential for local

delivery to the colon to treat inflammatory conditions and infections with the rationale of

inducing less side-effects than the injectable counterpart. For anti-TNF-α antibody injections,

potential side effects include an increase in the relative risk of common infections,

tuberculosis, and lymphomas [133]. To lay the groundwork for such formulation design, it

was recently shown that infliximab (Remicade®) and adalimumab (Humira®, Abbvie

Pharma, Chicago) could survive better in human colon fluid compared to the small intestine

and the stomach [134]. 50% or more of the two selected IgG1 antibodies remained intact in

the colonic buffer at 60 min, whereas metabolism occurred at 30 min and 1min in buffers

mimicking the small intestine and stomach respectively. The colonic stability advantage over

the stomach and small intestine for these antibodies was ascribed by Yadev et al [134] to low

levels of serine proteases (especially elastase) along with higher pH values of around 7.

Alternatively, IgG2 antibody appears to be more stable against pancreatic peptidases than

IgG1 [135], hence it may be a better starting point in terms of payload selection. Antibody

targets in the colon include TNF-α, clinically established as an inflammatory mediator in IBD

and CRC [136], Toxin A and B for C. diff., and Shigella for bacillary dysentery (reviewed in

[137]) For infection due to C. diff., a multivalent antibody (Ab1) was produced in CHO cells

and entrapped by spray-drying in mannitol beads coated with Eudragit® FS 30D [138].

Following a demonstration that the beads released 50% of the antibody at pH 7.4 and <20%

at pH 1.2, the multi-particulate formulation was tested by gavage in a hamster challenge

model and protected the animals against weight loss versus control.

With lack of propensity to induce tolerance and a capacity to be produced in a range of

species, non-humanized antibodies against GI targets can potentially be scaled up from milk

for oral delivery [137]. If the capacity for production of industrial quantities can be

addressed, outstanding issues are stability in the GI tract and release in the correct region,

along with enough specificity and potency against an accessible target. The development of

Avaxia Biologics’s (Lexington, Mass.) recombinant AVX-470 has been reviewed [3]. It is a

polyclonal antibody against TNF-α produced in cow milk and then formulated in an enteric

capsule, ultimately intended for treatment of pediatric UC [139]. It takes advantage of the

principle that antibodies produced in colostrum from mammary glands have increased

stability in the neonate GI tract, can concentrate in colonic tissue once it reaches there [140],

and has limited systemic exposure thereby reducing capacity to immunosuppress. A first-in-

man trial of AVX-470 (NCT01759056) comprised 37 patients with active moderate-to-severe

UC, who were administered ascending doses daily in capsules over a month period in a

randomized design [141]. The main outcome was a good safety profile, with some positive

trends for several biomarkers of efficacy seen at the top 3.5g/day dose. There was no

indication that the capsules used in this study were specifically optimized for colonic release

however, and it appears that AVX-470 is no longer in clinical development. On the other

hand, lower molecular weight Llama-derived domain antibodies against TNF-α are in Phase

II for CD using a colon-targeted oral formulation [142]. Using an alternative approach, this

time for systemic delivery, an attempt was made to design capsules from HPMC-coated

Intellicaps ® to release IgG2 conjugated to a ligand targeting the FcRn receptor in the small

intestine of cynomolgus monkey following weekly oral dosing [135]. While the study failed

to show systemic delivery from a receptor-targeting approach, it is the first indication that

more advanced formulations are being researched for regional release of antibodies in the GI

tract.

4.3 RNA therapeutics

There are two main strategies for RNA and related nucleotide therapeutics: inducing and

inhibiting protein production in cells. If warranted, mRNA can be delivered to further the

production of a desirable protein via translation by the ribosomes. However, in other cases

the DNA message and the resulting protein production give rise to disease and inhibiting the

translation would give rise to therapeutic benefit. The current main therapeutic approaches

used to degrade mRNA and to down-regulate protein production are: 1) small interfering

RNA (siRNA) and 2) antisense oligonucleotides (ASO). Relevant details on ASOs and

siRNA for their therapeutic action and their cellular mechanism are shown in Fig 5. and in

[143-144]. While ASOs may not need delivery enablement to get into the cell per se, delivery

systems may result in more productive uptake. Additionallym if the ASO chemistry is such

that it triggers immune responses, this may potentially be mitigated by formulation. larger

RNAs including siRNA and mRNA need a delivery system that would typically contain a

cationic polymer, lipid, or peptide to promote endosomal escape and cytosolic bioavailability.

In terms of local colonic delivery, the literature is focused on using siRNA and ASO

modalities to treat IBD, IBS, and CRC. Table 4 is an overview of ASO and siRNA delivery

approaches for these conditions citing ex/in vivo proof-of-concept (POC) in colon-relevant

rodent models.

TNF- α is a pro-inflammatory cytokine secreted by macrophages and it has a central role in

the pathogenesis of inflammatory disorders including IBD [145], and is a prime target to

inhibit via local colonic administration of siRNA/ASOs. Most of the studies cited in Table 4

have targeted TNF-α itself or in combination with inflammatory cytokines via siRNA. Study

1 (Table 4) reports that siRNA nanoparticles made from a mannosylated bioreducible cationic

polymer were complexed using sodium triphosphate as a cross-linker [146]. These particles

had reduced cytotoxicity in RAW264.7 macrophages and Caco2-BBE cells compared to

those made from branched polyethyleneimine (PEI) or oligofectamine™. The complexed

nanoparticles were also effective in reducing TNF-α secretion by macrophages and reduced

inflammation in the DSS-induced mouse UC model [146]. In Study 2 [147] the authors used

actively-targeted nanoparticles and reported that F4/80 Fab-grafted PLA-PEG particles

displayed improved targeting to RAW264.7 cells compared to non-grafted ones. Results

consistent with this were obtained when the nanoparticles containing the siRNA were

administered to the DSS-induced mouse model, where the targeted particles reduced

inflammation more efficiently than untargeted. Study 3 in Table 4 [148] investigated several

different anti-inflammatory cytokine siRNAs (in addition to TNF-α) in PEI-coated calcium

phosphate-PLGA nanoparticles and reported that the particles showed good epithelial uptake,

minimal cytotoxicity, and evidence of gene silencing in primary cultures of mouse colonic

crypt cells. Rectal administration of the particles to the DSS- mouse model led to a decrease

of the target gene expression in colonic biopsies, accompanied by a decrease in inflammation.

In Study 4 [149] a nanoparticles-in-microsphere oral system (NIMOS) system was

investigated using TNF-α siRNA , and the constructs resulted in decreased production of

TNF-α, interleukin (IL)-1β, interferon (IFN)-γ, and monocyte chemoattractant protein-1

(MCP-1 in the DSS mouse model. Study 5 [150] investigated particle-size effects and

binding affinities of siRNA-loaded galactosylated trimethyl chitosan cysteine nanoparticles.

They found that optimizing the particle diameter could provide low permeability across

Caco-2 monolayers along with high siRNA binding affinity, with a conclusion being that

large 450nm particles may have better anti-inflammatory potential in UC compared to 200nm

ones.

Study 6 in Table 4 [151] investigated TNF-α siRNA-loaded thioketal nanoparticles, which

were assessed following oral administration in the DSS mouse model. This polymer is

resistant to acidic and basic environments but degrades when exposed to reactive oxygen

species in inflamed tissues. The particles delivered their siRNA cargo selectively to the

inflamed colonic tissues and down-regulated TNF-α. TNF-α production can also be inhibited

via ASOs (Table 4). In Study 7 [152], a cationic konjac glucomannan (cKGM)/ ASO

nanocomplex was administered orally in the DSS mouse model, decreased local levels of

TNF- α, and alleviated inflammation. A physical transfection method was investigated by

[153] (Table 4, Study 8). Naked TNFα siRNA was administered as a simple solution locally

in the colon of mice with DSS-induced colitis followed by ultrasound transfection leading to

a 7-fold reduction in TNFα protein levels in colonic tissue compared to siRNA without

ultrasound, resulting in better control of colitis. The study also showed successful delivery of

firefly luciferase mRNA to colonic tissues using this approach. This group has also

successfully delivered microparticles to 100μm depth ex vivo in colonic tissues. Anionic and

cationic particles were delivered equally well using ultrasound as an enabler. The ultrasound

treatment appeared to strip away the mucus and this may overcome any negative impact of

particle surface charge [154].

There are other inflammatory mechanisms that can be targeted with siRNA in the colon.

CD98 is expressed on the surface of intestinal epithelial cells and plays a critical role in

controlling the innate immune system in the GI and is overexpressed in various colonic

inflammatory conditions. Table 4, Study 9 [155], investigated CD98-siRNA loaded PEI-

nanoparticles in the DSS mouse model. The PEI complexation aimed to protect the siRNA

against nucleases, and the particles were found to be biocompatible and non-toxic. CD98 was

effectively downregulated in colonic cells leading to decreased inflammation. In Table 4,

Study 10, CD98 siRNA PEI nanoparticles functionalized with a single-chain CD98 antibody

had affinity for CD98-overexpressing intestinal cells and reduced CD98 and cytokine

expression. In the DSS mouse model, these particles reduced CD98 expression in parallel

with a reduction in colitis severity [156]. Table 4, Study 11 investigated an anti-CD98 siRNA

and curcumin combination in chitosan nanoparticles with and without hyaluronic acid (HA)

functionalization. The HA functionalized particles were incorporated in a hydrogel and

administered to the DSS mouse model and they inhibited over-expression of TNF-α and CD-

98 and reduced inflammation more effectively than single agents [157]. Mitogen-activated

protein kinase kinase kinase kinase 4 (Map4k4) kinase is an upstream mediator of TNF-α

action. In Table 4, study 12, galactosylated trimethyl chitosan cysteine nanoparticles with the

cross linker, tri(poly)phosphate were compared to N-trimethyl chitosan-cysteine-conjugated

nanoparticles [158]. The in vitro (Raw 264.7 cells) and in vivo (DSS mouse model) oral data

demonstrated that the former more effectively mediated Map4k4 gene knockdown and

inhibited TNF-a production than the latter.

A decoy therapeutic system uses a double-stranded oligonucleotide (ODN) to competitively

inhibit binding of transcription factors to target genes. In Table 4, Study 13 [159], an ODN

targeted to NFkB was used to suppress cytokine gene expression. Studies in Caco-2 cells

showed that chitosan-PLGA nanoparticles were more effectively taken up than PLGA ones.

The chitosan-PLGA particles also efficiently reduced symptoms in the DSS rat model.

Intercellular Adhesion Molecule 1 (ICAM-1) (or CD54) is an inducible transmembrane

glycoprotein normally expressed on endothelial cells. Induction of ICAM-1 expression also

occurs in colonic epithelial cells in response to cytokines and other mediators of

inflammation. Table 4, Study 14 [160] described ICAM1/CD54 ASO (ISIS 9125)

administered rectally as a saline solution to the rat HLA-B27/2 microglobulin colitis model.

Decreased inflammation symptoms were evident along with reduced expression of TNF-α,

CD49d, and CD18 in colonic tissues. An ICAM-1 ASO for UC, formulated as a rectal enema,

has also advanced to the clinic (Table 4, Study 15 [161]). Alicaforsen/ISIS2303 was

administered in an open label study over six weeks to investigate colonic and systemic

pharmacokinetics in 15 subjects, who received nightly ASO enemas in solution. Systemic

bioavailability from the colon of ISIS2303 was < 0.6% and there was a decrease in disease

index and an increase in remissions.

Table 4 only contains a single siRNA study for CRC using an ex vivo model. Study 16 [162]

targeted the E2F1 transcription factor that plays a central role in cell cycle progression in

normal and CRC cells. Cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)

nano-liposomes downregulated the E2F1 target in HT29 colorectal cells and, in addition,

uptake and silencing were seen in cultured human colonic tissue mucosae. The cancer

xenograft model typically involves implanting the tumor by the sub-cutaneous (s.c.) route,

but it lacks physiological relevance for the colon. However, orthotopic models can be

established where the tumor is in a relevant organ, but these models are technically difficult

to establish, and this may explain why in vivo POC studies are not prevalent in the CRC

literature for local colonic administration. Several ASO/siRNA studies aiming at CRC are

instead using colonic cell lines and xenograft models, where the drug is administered by a

parenteral route. While those studies cannot guarantee that these delivery systems will

perform in the colon in vivo, a selected subset has been included in Table 5 for reference,

mostly in terms of probing the biology.

The Vascular Endothelial Growth Factor (VEGF) dsiRNA (i.e. a more potent and stable class

of siRNA) was entrapped in water soluble chitosan /pectin nanoparticles in Table 5, Study 1

[163]. It was demonstrated that physicochemical properties, particle size and zeta potential,

can be varied with dsiRNA/chitosan/pectin ratios. Release was low in simulated gastric fluid

(SGF, <15%) and intestinal fluid (SIF, <30%) but much higher in simulated colonic fluid

(SCF, >90%), due to presence of pectinases. Cytotoxicity studies in Caco-2 and CCD-18 CO

cells showed increasing cytotoxicity with the concentration of pectin-coated and uncoated

NPs, with the pectin-coated NP cytotoxic effect being slightly higher. In Table 5, Study 2

[164] pursued the β-catenin target, which is increased in 70–80% of CRCs and associated

with tumor progression. PEGylated chitosan NPs loaded with β-catenin siRNA were as

effective as Lipofectamine® 2000 in reducing protein levels in colon cancer cells. Two

studies in Table 5 (Study 3 and 4), are investigating siRNA-doxorubicin combinations in

carboxymethyl dextran /chitosan nanoparticles. Study 3 [165] used siRNA targeted against

SNAIL family transcriptional repressor 1 (SNAIL) in combination with doxorubicin. The

results demonstrated that treatment with the dual agent nanoparticles led to down regulation

of relevant genes, decreased cell migration, and cell death in HCT-116 cells. Study 4 (Table

5) [166] used High Mobility Group AT-Hook 2 (HMGA2) siRNA in combination with

doxorubicin and showed that the nanoparticles induced HT-29 cell death and reduced

expression of HMGA2. In Study 5 [167], four and a half LIM domains 2 (FHL2) siRNA

chitosan nanoparticles knocked down gene and protein expression at a similar level to siRNA

transfected by Lipofectamine® in Lovo cells. In study 6 (Table 5) [168], LoVo cells

transfected with VEGF-C siRNA CaCO3 nanoparticles conferred a reduction in VEGF-C

expression. These particles were administered by the s.c. route in a xenograft model, and

exhibited decreased tumor lymphangiogenesis, tumor growth, and lymph-node metastases. In

study 7 (Table 5) [169], Bcl-2 associated athanogene-1 (BAG-1) downregulation was

investigated in Lovo cells via a plasmid DNA (pDNA) loaded onto gold NPs.

Downregulation of BAG-1 gave rise to cell apoptosis in vitro and decreased tumor growth

when the cells were injected into nude mice.

Finally, a series of mechanistic studies elucidating the fate of nanomedicines entrapping

various types of RNA/DNA in the GI tract were conducted. Study 17 (Table 4, [170])

encapsulated enhanced green fluorescent protein pDNA in the NIMOS system. Both the

NIMOS and the NPs not incorporated in NIMOS were associated with 111In-labeled gelatin,

and were administered by gavage to mice where the NPs transited along the GI tract faster

than the NIMOS constructs. The NIMOS particles only transfected regions beyond the

stomach, thereby showing promise for local colonic delivery. Table 4, Study 18 [171]

studied the fate of GAPDH lipid nanoparticles (LNPs) containing siRNA in Caco-2 cells and

in mice. The LNP transfection efficacy decreased following exposure to fed-GI conditions in

the presence of pepsin, bile salts, and by mucin on Caco-2 epithelia. The latter effect could be

circumvented by increasing the amount of PEG in the LNPs. In the mouse study, the LNPs

were found to be retained in the GI tract for more than 8 hours, but gene silencing did not

occur despite confocal microscopy evidence of uptake by epithelia cells. The data confirm

that protection is needed in the upper GI tract for orally-delivered LNPs to deliver their gene

material to the colon. In summary, there is real potential for siRNA and ASOs to inhibit

production of the disease-causing proteins in colonic disease states if they can be delivered

appropriately. All siRNA studies to date used a nanoparticle formulation approach as

enablement to enable cell uptake, endosomal escape and protein knockdown, whereas many

ASOs were administered as simple solutions.

4.4 Cell-based systems

Probiotics are defined by the United Nations Food and Agriculture Association (FAO) and

the World Health Organization (WHO) as live bacteria or microorganisms, which give rise to

health benefits when dosed adequately [172]. There are currently no prescription probiotics

available, but several are in clinical development for local colonic disease states (Table 6).

The majority of probiotics are classified as nutritional supplements with or without health

claims, and colonic delivery of probiotics spans pharmaceutical dosage form design and food

science [173]. Naturally-occurring bacteria occur throughout the intestine, but they increase

in density as content is moved down the gastrointestinal (GI) tract. Species composition

patterns also modify from aerobic- in the upper GI tract to predominantly anaerobic in the

colon [174]. Most probiotic compositions are intended to concentrate in the colon, but also

for GI locations even outside of it. The main species are Lactobacillus and Bifidobacterium,

both naturally located in colon [175]. Changes in the colonic microbiome have been

implicated in IBS and IBD [176], and this adds to the rationale of rebalancing altered

bacterial ratios. Probiotics are also potential treatment options for dysbiosis associated with

inflammatory conditions, as well as infection-related childhood diarrhoea, antibiotic-

associated diarrhea [177], and C. diff. infection [178]. In addition, probiotics seem to

strengthen the host immune response, especially in neonates [179], and recent data

demonstrated that oral administration of Lactococcus lactis could reduce symptoms of acute

cholera infection in mice through production of lactic acid [180]. It is important to recognize

that there is no consensus on the merit of probiotic treatments, and a number of clinical trials

to test efficacy did not reach their primary end-point, for example, [181]. Regardless of their

as yet unproven therapeutic benefits, our focus here is on potential colonic formulation

strategies since lack of efficacy could be related to sub-optimal delivery.

There are several formulation challenges for a potential probiotic product. These include 1)

sufficient shelf-life; 2) protection against moisture, oxygen, and heat [182]; and 3) equipping

probiotics to survive the pH swings as well as digestion in the upper GI tract so that adequate

concentrations of live bacteria reach the colon [183]. The first step in the formulation process

is to reduce the bacteria to the solid-state using drying techniques such spray-, freeze- or

fluid-bed drying. However, these technologies induce bacterial stress, and protective

strategies are therefore needed to ensure survival. There are also economical aspects to be

considered. Freeze drying is the best process in terms of bacteria survival but also the most

expensive; it invariably requires addition of the cryoprotectants, trehalose or sucrose [184].

There are cheaper thermal drying alternatives (e.g., fluid bed drying, spray-drying), but they

result in lower viability. Options include controlling the drying process and including

protective excipients to reduce moisture in the final product and to preserve the viability.

Temperature is usually the main culprit, and therefore selecting equipment that allows for

temperature control (e.g., fluid bed drying) should be considered [182, 185]. Even when the

bacteria survive the drying process, the functionality of the produced dehydrated probiotics

can still be impacted. In a study measuring the impact of three drying processes (air-drying,

freeze-drying and spray-drying) on three types of probiotics (Bifidobacterium Bifidum,

Lactobacillus Plantarum, and Lactobacillus Zeae), the various drying processes gave rise to

varying viability and intestinal epithelial adherence. Adherence was promoted by air-drying,

but inhibited by spray-drying [186].

The next step after bringing the bacteria to the solid state is formulation development. In one

study Lactobacillus Gasseri and Bifidobacterium Bifidum, along with a prebiotic, quercetin,

were co-encapsulated in chitosan-coated alginate microspheres, but the yield of viable cells

was low. When the two probiotics were encapsulated separately in microspheres however,

they were stable in gastric and small intestinal conditions [187]. A dual coating approach has

been developed for lactobacilli that gives a 100-fold better protection over the uncoated

equivalent. The two layers include a pH-dependent protein layer (allowing the bacteria to

remain coated at stomach pH), but then to release in the intestine at neutral pH, and a second

polysaccharide matrix coating that protects the bacteria against environmental stressors

including pressure, temperature and humidity [183]. Microencapsulation (i.e., in

polysaccharides or protein matrices) and coating techniques can be used to keep bacteria

protected from the GI environment [172, 188]. Encapsulation of probiotics in size 0 capsules

coated with the Phloral® system comprising bacterial-activated starch and Eudragit S

components enabled 90% survival in simulated GIF, along with rapid release in a buffer at

pH 7.4, a pH likely to be present in the lower small intestine and colon [189]. The Phloral®

colonic targeting system is also being investigated for its capacity to deliver faecal material to

treat C. diff. infection as an alternative approach to achieving faecal transplantation [190].

Finally, other types of formulations are being researched for encapsulating probiotics so that

they can reach the colon intact. One example is a nanocellulose gel comprising gelatin and

lactobacillus rhamnosus GG [191].

Since bacteria are sensitive to physiological and environmental conditions, and there is a need

for quality control, typically by plate counting. Three probiotic dairy products (Imunele®,

Dannon®, Pomogayka®) contained Lactobacillus Bifidobacterium in a concentration of 107

CFU/ml, consistent with the label claim [192]. On the other hand, in the Dodoo et al study

[189], all of the commercial products tested were highly labile in GIF and the concentration

of viable bacteria reaching the colon and adhering to the epithelium bore no relation to the

starting concentrations in the product. Caution should also be taken when selecting quality

control methods, as the standard plate counting methods can underestimate cell numbers.

Drying and encapsulation may cause probiotics to enter a non-culturable, but still

metabolically-active state. Molecular biology assays to quantify viable bacteria include

polymerase chain reaction (PCR) and cell sorting techniques such as flow cytometry and

fluorescent activated cell sorting (FACS). These approaches to quantification have been

adopted in official guidelines and recently a new ISO guideline was launched to quantify

lactic acid bacteria via flow cytometry [193].

Recently, an additional application of probiotics has emerged: genetically engineering

bacteria to produce macromolecules in the colon. This presents additional delivery

challenges arising from the insertion of genetic material into bacteria and generation of stable

expression in the colon region. Macromolecules are too large to be internalize into cells

passively and therefore cannot normally locate to intracellular targets. To achieve uptake by

bacteria, electroporation and transfection agents can be used, and, to mediate delivery at the

desired location, a guide is needed (e.g., a plasmid or a transposases/recombinases) [194].

Recently, Lactococcus lactis was engineered to produce IL-10, which controls intestinal

inflammation by inhibiting activation of dendritic cells [195]. With respect to colon delivery

of anti-IgG antibodies against TNF-α, Lactococcus lactis was engineered to secrete murine

TNF-α heavy-chain camelid stable “Nanobodies” [196]. Daily oral administration of the

engineered probiotic achieved delivery of nanobodies against TNF-α in the colon of DSS-

induced mice and reduced inflammation both in that model, as well as in interleukin 10 (IL-

10) (-/-) mice. Similar data was also achieved in the DSS model using modified L. lactis to

release anti-inflammatory trefoil factor peptides in the colon following intra-gastric

administration [197].

The CRISPR cas-9 system that is currently being advanced for therapeutic gene editing (i.e. a

guide RNA plus a cas 9 protein, the latter functioning as the scissor editing the genes and the

guide RNA required to find the right location for CAS9 to cut) can also be used to

genetically-engineer probiotics to deliver macromolecules and enhance host immune

responses. The CRISPR Cas-9 system can also be used to modify probiotic strains to be more

amenable to formulation processing and GI survival such as acid and bile resistance [198].

Genetically -engineered probiotics for pharmaceutical purposes is an emerging field and

guidelines to fully ensure their safety will be required. The food industry has already built

significant knowledge in this area [199], which would serve as a good starting point for a

pharmaceutical guidance.

Finally, colonic delivery of vaccines represents another class of molecule that, like probiotics,

falls outside the traditional pharmaceutical macromolecule definition. One aim is to generate

oral colonic vaccination approaches specifically against viruses that invade via the rectal

route, e.g. HIV. Zhu et al.[200] entrapped a range of vaccinia-related antigens, as well as

luciferase-expressing DNA plasmids into PLGA microparticles coated with Eudragit®

FS30D and demonstrated antigen-specific T cells activation in the colon following particle

uptake by colonic epithelia following oral administration. Subsequent efforts at colonic

immunization using particle approaches for bacterial antigens include entrapment of hepatitis

B surface antigen in nanoparticles made of ratios of Eudragit® S-100 and L-100, along with

an adjuvant [201] The argument for colonic vaccination is that local mucosal protection can

be generated in respect of potent humoral and cellular immunity in the colon, rectum and

vagina through the common mucosal system.

5. Development challenges for colonic macromolecule delivery

Considering the therapeutic potential for local macromolecular colonic delivery, it is

surprising that there are not many candidates in clinical development. Relatively little has

been published on the selection, optimization and development of colonic delivery systems

designed for macromolecules pertaining to a realistic pathway for clinical development. To

achieve a successful colonic macromolecule product, it is necessary that the delivery system

is an integral part of the target product profile. Successfully translating colonic targeted

delivery systems, particularly those designed for local delivery from pre-clinical proof-of-

concept to demonstration in clinical trials is challenging. Therefore, it is important to learn

from other areas of drug development where target-driven design and patient stratification

have improved patient outcomes. Advanced drug delivery R & D strategies might therefore

build on the recent success of a drug discovery and development approaches, which have

been improved by applying a focused decision-making framework. For example, application

of AstraZeneca's (AZ) “5R” Framework uses five determinants: right target, right exposure,

right safety, right patient, and right commercial potential. It has had a significant impact

improving the success rate from AZ candidate nomination to Phase III from 4% in 2005-2010

to 19% in 2010-2015 [202]. A similar framework could be established for enhancing

advanced drug delivery systems translation and performance and it would be particularly

important for functional and targeted systems. Building informative data packages to answer

these questions for local colonic delivery systems require: (i) an improved understanding of

the heterogeneity of clinical disease and of the biological factors influencing the behaviour of

the delivery system and macromolecule in the patient disease setting, leading to “disease led

design,” (ii) the implementation of relevant pre-clinical models and testing protocols, (iii)

better techniques to measure exposure at site of action/in colonic tissue since plasma PK will

not be a surrogate for efficacy, and (iv) careful choice of patient population.

“Developability Assessment” work-flows are used in the pharmaceutical industry to guide

selection of a drug candidate and/or the delivery system in a serial or parallel fashion, an

exercise frequently taking place early in preclinical research at the interface between

Discovery and Development. Such work-flows have been designed for immediate release oral

formulations of small molecules and peptides intended for systemic delivery [203-207].

These assessments can be adapted to encompass local colonic delivery, an example here

being for nucleotide-based therapeutics where an early development delivery system

“developability” assessment has not yet been published to our knowledge. Due to the limited

progression of nucleotide-based therapeutics into clinical development, all publications

concentrated on the research and preclinical space. A summary of these studies based on

siRNA and DNA is provided (Table 7). Not all elements needed for a developability

assessment can be found in the nucleotide literature, and hence elements from the small-

molecule literature were also considered. As illustrated, a nanoparticle system is commonly

selected to enable colonic intracellular delivery for the major colonic-associated diseases. On

the product quality side, assays to confirm delivery system formation and entrapment

efficiency, along with chemical, physical and enzymatic characterisation, as well as stability

assessments are included in many publications.

The product performance assays in Table 7 were divided into in vitro assessment including

release assays, disease-relevant cell and tissue uptake studies, and in vivo studies. In addition

to information in Table 7 [208] is a recent review of colonic cell culture models. It is

noteworthy that data generated in differentiated cell lines may not always translate, and hence

primary cells, organoids or ex vivo tissues may be more appropriate. In vitro release assays

have been developed for assessing local colonic delivery of small molecules. In a study of a

dendrimer entrapping 5-ASA in a chitosan hydrogel designed for colon delivery, a staged

dissolution approach was used to simulate transit through the GI tract, with the first hour in

SGF (pH 1.2), the next two hours in mixed SGF and SIF to mimic the duodenum, then two

hours in SIF (pH 7.4), and finally three hours in simulated colonic fluid (SCF, pH 7) [209].

The study screen therefore selected the formulation that had the least intestinal 5-ASA release

in advance of reaching the colon. A related in vitro release assessment was used in a study of

HPMC-coated colonic release tablets of ketorolac tromethamine using buffers of various pH

values [210]. This study also illustrated the need to conduct appropriate stability studies on

solid dosage forms, whereby HPMC-coated colonic release tablets of ketorolac

tromethamine, were assessed conducted according to International Conference on

Harmonisation (ICH) guidelines at 40°C and 75% relative humidity for 6 months. In another

study, a SMEDDS (self-microemulsifying drug delivery system) containing prednisolone was

used for colon-specific delivery. USP II dissolution in low pH (pH 1) and in phosphate buffer

(pH 6.8) was used to measure release [211]. A standardised dissolution system for assessment

of colonic drug delivery systems was developed by Goyanes et al. [212] by using a

physiological bicarbonate buffer under dynamic pH conditions. It was established with an

Auto pH™ System, which modulates and maintains the buffer at preselected pH values

mimicking conditions along the GI tract. The performance of the model was validated with

an enteric dual layer prednisolone formulation (internal standard) where in vitro release

patterns correlated with the in vivo targeting performance in humans.

Given the diversity of such release assays, the composition of SCF was determined based on

analysis healthy human volunteer-aspirated colonic fluids [213]. The solubility of

ketoconazole, danazol, and felodipine was compared in SCF (fasted and fed) and standard

buffers: the solubility in SCF more closely predicted solubility in human colonic fluids than

in buffers. Hence, SCF constitutes a suitable vehicle for developability studies. TNO

(Leiden, NL) (gastro-) Intestinal Models, (“TIM”), also provide insight into the release and

availability of formulations along the GI tract. TNO has recently developed “TIM-2” as an

in vitro model of the colon, which can be used in conjunction with the TIM-1 model of upper

GI tract [214]. TIM-2 has been used to explore the site specificity and bioaccessibility to the

colon and to assess delivery system performance. The optimized system is inoculated with

microbes and metabolites from healthy- and diseased individuals and the resulting data has

correlated well with clinical trials. As an example of assessment of compatibility with local

drug delivery to the colon, Tannergren et al. [215] evaluated an in vitro method to investigate

bacterial- mediated luminal degradation of drugs in human colon as a tool for early

development assessment of drugs; this approach can also be applied to macromolecules. The

preclinical studies cited in Table 7 used rodent models, but for practical reasons the

nanoparticle formulations are not usually formulated into solid-dosage forms for these

species due to the constraints of rodent model physiology. In the small molecule literature,

additional stability studies are conducted on solid dosage forms designed for humans. Thus,

in the example of HPMC-coated colonic release tablets of ketorolac tromethamine, stability

was conducted according to ICH guidelines at 40°C and 75% relative humidity for 6 months

[210].

Regarding in vivo performance assessments for colonic delivery, aside from rare examples

using live whole animal imaging of electron-dense or fluorescent materials [114, 148], the

rodent studies cited in Table 7 were terminal and involved removing mucosae for analysis.

Conducting colonic biopsies from large animals and humans, while feasible, is slow and

expensive. Therefore, other in vivo testing options are needed to optimize local colonic

delivery systems for clinical development. Plasma PK feedback used to optimize oral

delivery systems intended for systemic exposure is not very informative for colonically-

acting macromolecules, except to confirm indirectly that they do not reach the blood. Several

approaches to monitoring intra-luminal behaviour of oral dosage forms can be leveraged for

studying the fate of colonic dosage forms (Table 8). Intra-luminal sampling is useful to

determine drug concentrations along the GI tract, as was exemplified in a recent study where

a catheter with ports was used to withdraw samples for quantification of mesalamine in the

various GI regions [216]. Telemetry is a related technology that can also be used for local

colonic developability studies, an example being a formulation of ziprasidone, an

antipsychotic small molecule [217]. In this study, the Enterion™ capsule with 111-indium was

used with scintigraphy to follow capsule progress throughout the GI tract. Telemetry capsules

have also been used for assessing macromolecule release in GI regions. The Heidelberg

capsule was tethered to an enteric-coated capsule formulation of salmon calcitonin. The pH

values measured by the capsule were correlated to systemic PK and used to determine the site

of release [218]. The Intellicap® system was also used to deliver an IgG2 monoclonal

antibody intended to bind to the FcRn receptor in the GI to mediate systemic uptake [135].

The study set out to use cynomolgus monkeys, however, in an initial screening study to

measure gastric retention, it was judged too variable and hence beagle dogs were used

instead. The capsule was used to dose the antibody and to measure intestinal PK profiles after

administration in an enteric-coated capsule.

The advantages of the intraluminal sampling technologies include the capacity to obtain real

time concentration measurements and to analyze the physicochemical status of the molecule

(e.g. precipitate, complexation). The disadvantages include the invasive nature of the

procedure: catheter approaches are perceived as unpleasant without sedation. Moreover,

many of the technologies cannot be used across species since many catheters and telemetry

devices are too large for rodent species and, in addition, many of the telemetry capsules have

limited volume. Lastly, the approaches do not allow study of kinetic aspects of dissolution

and they usually require the use of another technology for precise location in the GI tract.

With respect to technologies using radioactivity, examples where gamma scintigraphy was

used to monitor release of dosage forms in the colon includes several small molecules

intended for colonic were described earlier, while others are referenced in Table 8. Since

gamma scintigraphy can have issues with image resolution, other advanced imaging methods

have been developed, albeit assessed with small molecules to date. Lai et al. studied

prednisolone in colonic controlled release tablets for human volunteers, containing 99mTc, an

inner alginate layer, and an outer pH sensitive coat, in human volunteers using a hybrid

scanner combining SPECT (single-photon emission computed tomography) and CT

(computed tomography). The study recorded images of the disintegration process, and the

newer imaging technologies were judged superior over gamma scintigraphy as regards

locating the exact position of the dosage forms in the GI tract [219]. In summary, these

technologies are useful in recording kinetic dosage form behavior in the colonic lumen, but

on the other hand, they have disadvantages in that they require the use of radioactive labels

and advanced equipment.

While gamma scintigraphy and related technologies are by far the most common to visualize

the release of dosage forms intended for local or systemic exposure via the colon, other

imaging technologies focus on X-ray detection. Flurbiprofen tablets containing barium

sulphate as a contrast agent were prepared by direct compression and then coated with

Eudragit® 100 and hydroxypropyl methylcellulose. In vivo release in healthy volunteers

showed that the tablets reached the colon and did not disintegrate in the upper GI tract [220].

X-ray imaging using barium sulphate was also used to monitor release of flurbiprofen from

pH-sensitive colonic-targeted tablets [221] and ketorolac tromethamine from colonic release

hydroxypropyl methylcellulose tablets [210] . A useful technology to track regional delivery

in the GI tract is breath/urine analysis (Table 8). The concept measures radiolabeled additives

(e.g. urea or glucose) or their metabolites in the breath or urine. For example, a controlled

release ColoPulse™ tablet containing 13C-urea was ingested by each subject along with an

immediate release tablet containing 15N-urea. The ColoPulse™ is a pH-responsive tablet

technology that delivers a drug to the ileo-colonic region, a region rich in ureases that

metabolize [13C] -urea to 13CO2. The liberation of 15N -urea from uncoated tablets in the small

intestine led to excretion in urine, and hence the location of the two tablet forms can be

distinguished [222].

Finally, magnetic resonance imaging (MRI) monitors protons and is therefore best suited to

monitor organ and fluid volumes, but there are only a few examples describing the use of

contrast MRI to follow the progress of colonic dosage forms [223]. In one study the GI

distribution of a phosphatidylcholine liposome formulation containing the contrast agent

gadolinium tetraazacyclododecane tetraacetic acid (Gd-DOTA) was followed by MRI.

Liposomes were identified by the increased signal intensity provided by the contrast agent.

Stability studies were performed to ensure the intactness of the liposomes under GI relevant

conditions [224]. A similar strategy was used in a study evaluating three immediate release

tablets with different densities. In this case superparamagnetic Fe3O4 particles were added to

the tablets, and MRI used to analyze stomach tablet position and residence time in human

volunteers. In the same study a slow release tablet incorporated Gd-DOTA to allow the GI

locations to be followed by MRI [225]. Based on the review of the RNA research literature

(Table 7), the analysis of quality and product performance of selected colonic delivery

technologies for small molecules, and our own development experiences, we propose a

developability workflow to select colonic delivery systems for nucleotide- based therapeutics

(Fig. 6). The workflow can be used as a decision tree, where the researcher can revert to

earlier stages for additional evaluation. It is theoretical at this stage and should therefore be

verified with data from nucleotide-based formulations ear-marked for clinical development.

6. Conclusions and future perspectives

Developing dosage forms for local delivery of macromolecules to the colon to treat IBD, IBS,

colorectal cancer, and infections is not simply a leveraging of the strategies already used for

the limited range of small molecules that have been marketed as colonic release systems.

Neither is it a fallback position in response to any failure to achieve systemic delivery for

macromolecules, nor is it merely a life-cycle management approach for injectable

macromolecules. The advantages of the approach are many, including the opportunity to treat

early disease, avoid systemic toxicity of the candidate, the relatively accommodating

environment for macromolecules in the colon compared to the upper GI tract, as well as the

convenience for patients over injectable delivery systems.

Nonetheless, the technical hurdles to achieve accurate and reproducible colonic delivery of

macromolecules are high. Intra-subject and inter-disease variability in GI physiology,

overly-complex multi-functional component dosage form design, inadequate loading,

incompatibility between the macromolecule and the dosage form, and variability in the

triggering of release in the colon all combine, and the result is that there are relatively few

macromolecule examples in oral clinical development or on the market. Consequentlythere

is a lack of regulatory precedence and limited operational, scientific and clinical development

experience. There is clear potential in exploiting particulate technologies for many colonic

disease states discussed here. Much focus is on UC where there is evidence of selective

passive targeting and localization of nanoparticles containing macromolecules to the inflamed

epithelium, influenced by particle composition, diameter, and surface coating. The next phase

of this research requires delivery of the nanoparticles containing amino acid- based and

nucleotide-based medicines to be delivered to the colon in solid dosage forms because they

can only interact with the epithelium if they reach the region intact in the first place. These

dosage forms in turn will benefit from combination approaches, for example, the continued

use of Eudragit® coatings allied to exploitation of bacterial enzymes to ensure release of the

nanoparticles. As a companion to some of the research discussed in this article aimed at

macromolecules, we note that ADDR has just published a detailed analysis of how

biodegradable polysaccharide-coated systems can be used to target the inflamed colon [226].

For antibodies and probiotics, the challenge is not to achieve high colonic tissue levels, but to

act in the colonic lumen. This can also be achieved using solid dosage forms with

combination approaches to ensure accurate and reproducible release, however these payloads

have their own special needs related to ensuring entrapment of very high molecular weight

proteins (antibodies) and high cell viability (probiotics), and both at the required dose levels.

Finally, the developability aspects require earlier use of large animal distribution and imaging

studies to support the rodent models, accompanied by addressing the reproducibility, scale

up, and manufacturing questions around the selected colonic delivery technology. Colonic

delivery system assessments will also have to discover alternative read-outs to evaluate

preclinical and clinical performance, as the goal will not be addressed by measuring plasma

concentrations. We are confident that the significant advantages will outweigh the hurdles

and that a selection of macromolecules intended for local delivery to the colon will reach

clinical pipelines in the near term.

Acknowledgments

DB acknowledges grant support from the Science Foundation Ireland Centre for Medical

Devices (CURAM) and the European Regional Development Fund (Grant Number

13/RC/2073). We thank George Retsek for illustrating Fig. 2.

(http://technicalillustrators.org/2018/02/george-retseck/), Dr. Brendan Griffin of University

College Cork for use of the image in Fig. 4, and to Dr. Anders Holmen of AstraZeneca for

critical review of the article

Figure Legends

Fig. 1. Molecules developed for treatment of different clinical phenotypes of IBD since the

1950s. Macromolecules are in italics. Most are used for both UC and CD at different stages

of severity. Note that injectable biologics predominate as disease progresses. Fig. 2.

Physiological parameters in the colon that vary in the normal and diseased state. Many

parameters can impact dosage form release and interaction with the epithelium and some can

be exploited to trigger release. 1. Dosage form arrives intact and is affected by peristalsis (P).

2. Epithelial water flux impacts dosage form dissolution. 3. The potential difference (PD)

may differ along the colon and in diseased states. 4. Basal epithelial permeability to some

ions and drug molecules may be higher in IBD; it is ion- and molecule-dependent. 5. Mucus

composition and thickness may vary along the colon and in disease states thereby impacting

nanoparticle capacity to reach the epithelium. 6. pH values vary in colonic regions, with a

propensity for low values in the lumen of some UC patients. 7. The colon has low amounts

of fluid and this can mean variable dissolution. 8. Reactive oxygen species (ROS) can be

produced in colonic regions in IBD; this can be exploited to release drug molecules from

dosage forms. 9. The microbiome varies in disease and can be exploited to release drugs

from metabolism-sensitive polymers. 10. Colonic transit is highly variable, and this means

that release rates from dosage forms can be unpredictable if they rely on time. Note that these

factors occur throughout the colon and that the numbers do not denote precise locations.

Fig. 3. Cellular mechanisms accounting for colonic ion and water absorption and secretion.

Some overlap with those of the small intestine (SI). Factors that reduce the amount or

function of a specific transporter are shown in red boxes; those that increase levels of activity

are shown in green boxes. Long vertical arrows indicate paracellular absorption or secretion

of water. Glossary: DRA (PAT1): putative anion transporter; NHE3 (NHE2): sodium-

hydrogen exchanger; ENaC: epithelial sodium channel; CFTR: Cystic Fibrosis

Transmembrane Regulator; CaCC: calcium-activated chloride channel. LPA:

lysophosphatidic acid; TK, tyrosine kinase; MAPK: Mitogen-activated protein kinase; KCC:

potassium/chloride co-transporter; NKCC1: sodium/potassium/2-chloride co-transporter.

Reproduced with permission from [227].

Fig. 4. Investigation of novel SmPill® minispheres to enhance colonic delivery of

cyclosporine (CsA) to pigs after oral administration. Upper: CsA tissue concentration

profile. Middle: CsA luminal concentration profile after oral and i.v. administration of CsA.

Lower: CsA concentration in three subsections of porcine tissue taken from a biopsy of the

transverse colon (* p < 0.05). Data from [228] redrawn with permission of the authors

Fig. 5. Key features of ASOs and siRNA and their cellular uptake mechanism (adapted from

[142-144]).

Fig. 6. Proposed early development path for a local colonic delivery system developability

assessment for nucleotide-based therapeutics.

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Table 1. Clinical development status of selected orally-delivered macromolecules for pathologies expressed (at least in part) in the colon.

Name (MW, Da) Company Formulation Indication (Stage completed)Linaclotide, Linzess® (1525)

Allergan/Ironwood

Cyclic guanylate cyclase peptide agonist tablet

IBS-C, (market)

Plecanatide, Trulance® (1682)

Synergy Cyclic guanylate cyclase peptide agonist peptide tablet

IBS-C, CIC (market)

Doclanatide(1682) Synergy Cyclic amino acid substituted guanylate cyclase peptide agonist tablet

OIC (Phase II); UC (Phase I)

Vancomycin, Vancocin® (1449)

Generic (e.g. ViroPharma)

Cyclic modified macrolide peptide; capsule or tablet

CDAD; Staphylococcal enterocolitis including MRSA (market)

Fidaxomycin, Dificid® (1058)

Merck Macrocycle macrolide, tablet

CDAD (market)

Surtomycin (1681 Da)

Merck Cyclic lipopeptide, over-encapsulated tablet

CDAD (Phase III, discontinued in 2017)

LFF-571 (1367 Da) Novartis

Semi-synthetic thiopeptide, capsule

CDAD (Phase II)

AVX-470, Avaximab™-TNF, (160-900 kDa)

Circle33 LLC (Avaxia Biologics)

Polyclonal bovine antibody, enteric-coated capsule

UC (Phase 1B, terminated 2017)

V565 (12-15 kDa) VHsquared Protease-resistant anti-TNF-α domain antibody capsule

CD (Phase II Harbour Study, initiated in 2017)

Mongersen, GED-0301 (6952 Da)

Celgene 21-mer antisense oligonucleotide capsule targeting Smad7, mRNA

CD (Phase II, terminated 2017)

Alicaforsen (6368 Da)

Atlantic Healthcare

20-mer antisense oligonucleotide capsule targeting ICAM-1 and TLR-9 mRNA

Pouchitis (Phase II (enema), entering Phase 1 (tablet)

Sources (as of May 2016): PharmaCircle™ business reports, ClinicalTrials.gov; Press Releases. Abbreviations: MW = molecular weight; IBS-C: irritable bowel syndrome-with constipation; CIC: chronic idiopathic constipation; OIC: opioid-induced constipation; UC: ulcerative colitis; CD: Crohn’s disease; CDAD: Clostridium difficile-associated diarrhoea; TLR-9: toll-like receptor-9.

Table 2. Summary of colonic drug delivery systems as products or in clinical development for small molecules

Trigger for colonic site specificity

Polymer Repeat unit/architecture

Source Dosage forms

Products

Time Ethyl cellulose anhydroglucose

Polysaccharide from cellulose

Film-coated tablets, pellets or microspheres

Pentasa® 250, 500mg, microgranules in capsules/Shire Inc.

Hydroxypropyl methyl cellulose (HPMC)

Delayed release capsule with 4 coated tablets

Delzicol®

/Allergan

Geoclock™ timed release delivery system: hydrophilic shell coat compression coated over core tablet

Geoclock™/Skyepharma

pH Eudragit® and derivatives

acrylic acid and methacrylic acid and their esters

Polymethacrylate-based polymers

Tablet coated with Eudragit S® dissolves pH > 7

Asacol HD® / Allergan

Asacol™/Tillots Pharma

Capsules coated with Eudragit S® dissolves pH > 7

Entocort™ /Tillots Pharma/AstraZeneca in US

Cores with EUDRATEC®

sustained-release Eudragit® followed by a pH-dependent coating with Eudragit FS30®

COL/Evonik

pH and Time Eudragit® outer coat in three component matrix structure where amphiphilic and lipophilic matrices are dispersed in a hydrophilic matrix to provide prolonged release

acrylic acid and methacrylic acid and their esters

Multi Matrix MMX® technology

(Cosmo Pharma)

Modified release coated tablet/capsule

Lialda™ (Shire US Inc.)

UCERIS® (Salix)

Valent in US /

Cortiment®/Ferring, Rest of World

Rifamycin SV MMX® tablets

Methylene blue

Eudragit L® coated with Eudragit® core

Intellicor® Technology

Capsule with core granules coated with Eudragit L®,

Apriso/Salix Pharmaceuticals

Enteric coat plus ethyl cellulose

ECX™ controlled release technology

Ethyl cellulose /drug-coated pellets with

Tillotts Pharma

enteric coat

Enteric coat with release at pH6.8 plus oleo-gel matrix

Arachis oil, beeswax and colloidal silica

Oleo-matrix gel in enteric coated capsule

Colpermin™ (Tillots Pharma)

Bacterial enzymes

Pectin D-galacturonic acid and its methyl ester linked via α(1-4) glycosidic bonds

Extracted from peel of citrus fruits and apple pomace

Polysaccharide hydrogel coating around core of enteric- coated tablet

Prednisolone CSDS/Samyang biopharma

Pectin/ethyl cellulose

Coated onto drug beads

Glassy amylose/ethyl cellulose

Derived from starch

pellets coated with ethyl cellulose and glassy amylose

COLAL PRED®/Alizyme

Chitosan/Poly vinyl acetate (Kollicoat SR30D®)

Deacetylation of chitin

Azo polymers N=N Synthetic polymers

Film coated pellets

Enzymes and pH

Eudragit S® and resistant starch plus HPMC

OPTICORE™- coating system developed by Tillotts Pharma, based on the Phloral™ technology

Two trigger systems and accelerator coating layer HPMC designed to accelerate dissolution of the outer coating and release of drug, dual coating pH sensitive Eudragit S® and second

Opticore (Tillotts)

mixture of a resistant starch and Eudragit S 100®.

Pectin & Eudragit®

Pectin MP coated with Eudragit®

Mucoadhesives Carbomer® Acrylic acid Acrylic acid- based homopolymer

Hyaluronate D-glucuronic acid and N-acetyl-

D-glucosamine linked by

b-(1/3) linkages

Animal/microbial

Pellet-coated with drug and HA in multiple layers

IBD98-M/ Holystone multi-layer coating pellet technology

Alginate Alginic acid Saccharide distributed in cell walls of brown algae

Various

Thiomers Chitosan, acrylic polymers; alginates designed with thiol-bearing side chains

Chitosan/alginate MP

Nanoparticles Lactic acid or PLGA or PLA-PEG block co-polymers

Synthetic Polyesters of lactide and glycolide monomers

MP; NP

PLGA coated with Eudragit S®

Synthetic

Amphiphilic block copolymer comprising nitric oxide radicals at

Synthetic Self-assembled redox

the side chains of the hydrophobic segment

polymers

Table 3. Formulations being researched to achieve colonic regional and tissue delivery of CsA to treat moderate-severe UC.

Formulation approach/ route In vivo bioassay Reference

PLG MP-and NP /oral DSS mice [108]Eudragit FS30D®-coated PLG NP/oral DSS mice [109] mPEG hexyl-PLA micelles/intra- rectal TNBS mice [111]Solid lipid NP/oral DSS mice [229]SmPill™ minispheres-in-coated capsules (CyCol™) ™/oral Pigs and Phase II trial [112]Notes: Some formulations may also be leveraged for Graft-Versus-Host disease with intestinal complications. Approaches to increase systemic bioavailability of CsA in order to compete with Neoral ® were excluded.

Table 4. Nucleotide-based formulation survey for potential treatment of IBS, IBD, CD, UC, CRC, and general research studies investigating local colonic administration. Studies with colonic in vivo and ex vivo bioassays are included.

Study

Cargo Target Approach

Bioassay Route Composition Formulation description

1 TNFα-siRNA

IBD NP Mouse colonic tissues

NA TPP-PPM siRNA NPs

PPM was synthesized and spontaneously assembled into NPs assisted by TPP

2 TNFα-siRNA

IBD Active targeted NP

Mouse (DSS-induced colitis)

Oral F4/80 Fab grafted PLA-PEG NP

Enzymatic digestion of IgG antibody to get Fab fraction. Attached to PLA-PEG via disulphide bonds; NP formed by double emulsion/solvent evaporation [121]

3 TNF-α, KC, or IP-10 siRNA

IBD,

CD

NP Mouse (DSS-induced colitis)

Rectal CaP core coated with siRNA encapsulated into PLGA, and coated with PEI

A dispersion of CaP was mixed with siRNA; The CaP core was encapsulated in PLGA by a double emulsion technology and the PEI coat was applied

4 TNF-α siRNA

IBS

CD

UC

NP/MP Mouse (DSS-induced colitis)

Oral Lyophilized gelatin NPs encapsulated in PCL MS (NiMOS)

siRNAs were mixed withgelatin solutions followed by precipitation of gelatin solutions with ethanol; PCL encapsulation by the double emulsion technique

5 TNFα-siRNA

UC NP Mouse (DSS-induced colitis)

Intra colonic

GTC NP GTC synthesized as in minor modifications, and siRNA loaded NP prepared via ionic gelation

6 TNFα-siRNA

IBD NP Mouse (DSS-induced colitis)

Oral TKN NP formulated from the polymer PRADT. PLGA NP and DOTAP complexes as control

siRNA-DOTAP complexes were generated first and then a single-emulsion protocol was used to encapsulate these complexes in PRADT nanoparticles

7 TNFα ASO IBD NP Mouse (DSS-induced colitis)

Oral cKGM and ASO nano-complex

cKGM was synthesized was prepared mixing an aqueous solution of cKGM with the ASO

8 TNF siRNA Cholitis Ultrasound

Mouse (DSS-induced colitis)

Rectal siRNA solutions followed by ultrasound

No details

9 CD 98 siRNA

IBD NP Mouse (DSS-induced colitis)

Oral CD98 siRNA encapsulated in PEI and PLA

Double emulsion/solvent evaporation [121]

grafted by PVA

10 CD 98 siRNA

IBS

CD

UC

Active targeted NP

Mouse (DSS-induced colitis)

Oral Hydrogel that releases NP with CD98 antibodies on their surface, and loaded with CD98 siRNA.

A new polymer scCD98-PEG-UAD was synthesized with a single chain antibody grafted. The polymers and siRNA spontaneously assembled into NPs

11 CD98 siRNA, curcumin

combination

UC NP Mouse (DSS-induced colitis)

Oral Depolymerizedchitosan-coated NPs and the same NP functionalized with HA, imbedded in a hydrogel comprised of chitosan and alginate

NPs were prepared by a double emulsion solvent evaporation technique

12 Map4k4 siRNA

UC NP Mouse (DSS-induced colitis)

Oral Galactosylated trimethyl chitosan cysteine (GTC) NP

GTC was synthetized in 3 step process; siRNA loaded GTC NPs were prepared via ionic gelation of GTC with anionic crosslinkers TPP or HA

13 NF-kB ODN IBD

UC

NS Rat (DSS-induced colitis)

Oral Chitosan coated PLGA NS

Emulsion solvent diffusion method [231]

14 ICAM1/CD54 ASO (ISIS 9125)

IBD

CD

UC

Solution Rat (transgenic HLA-B27 / 2 microglobulin)

Rectal Saline solution of ASO

No details

15 ICAM-1 ASO (ISIS 2303)

UC Solution Human Rectal Rectal retention enema - HPMCaqueous formulation

No details

16 E2F1 siRNA CRC Nano-liposome

Human colonic biopsy tissues

NA PC, DOTAP and CHOL at 3:0.3:1; 1:260 (w/w) siRNA/total lipids ratio.

Thin film hydration method and ultrasound size reduction

17 EGFP-N1 pDNA

General research

NP/MP Mouse Oral Lyophilized gelatin NPs further encapsulated in PCL MS (NiMOS)

pDNA were mixed withgelatin solutions followed by precipitation of gelatin solutions with ethanol; PCL encapsulation by the double emulsion technique[234, 235]

18 GAPDH siRNA

General research

LNP Mouse Oral 10 siRNA; 50 lipidoid; 38.5 cholesterol; 10 DSPC; 1.5 PEG2000-DMG

Vortexing excipients dissolved in EtOH with siRNA dissolved in buffer

Glossary: Aq: Aqueous; ASO: Antisense oligonucleotide; CaP: Calcium phosphate; CD: Crohn's disease; CD98-PEG-UAC: (scCD98)-PEG-urocanic acid-modified chitosan; CHOl: Cholesterol; cKGM: cationic konjac glucomannan phytagel; CRC: Colorectal cancer; DDS colitis model: Dextran sodium sulphate induced colitis model; DsiRNA: Dicer small interfering ribonucleic acid; DNA: Deoxyribonucleic acid; DOTAP: Dioleoyloxypropyl-N,N,N-trimethylammoniumpropane; DSPC: distearoyl-sn-glycerol-3-phosphocholine; Fab: antigen binding region of an antibody; GAPDH siRNA: Positive Control human siRNA often used for developing and optimizing siRNA experiments; GTC: Galactosylated trimethyl chitosan cysteine; HA: hyaluronic acid; HPMC: hydroxypropyl-methylcellulose; IBD: Inflammatory bowel disease; IBS: Irritable bowel syndrome; ICAM1: intercellular adhesion molecule 1; IgG: Immunoglobulin G; Il-10: interleukin-10; IP-10: Interferon (IFN)-gamma inducible protein; KC: Keratinocyte chemoattractant; LNP: lipid NP; MP: Microparticle; MS: Microspheres; NA: Not applicable; NF-kB: nuclear factor kappa B; NIMOS: nanoparticles-in-microsphere oral system; NP: nanoparticle; NS: Nanosphere; ODN: decoy double-stranded oligonucleotide that competitively inhibit the binding of transcription factors; PLA: Poly lactic acid; PLGA: poly(lactic-co-glycolic acid; PPM: polymer p (cystamine bisacrylamide - branched PEI)-PEG- Mannose; pDNA: Plasmid DNA; PEI: Polyethyleneimine; PC: L-a-phosphatidylcholine; PCL: biodegradable poly(epsilon-caprolactone); PEG2000-DMG: 1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene;PLGA: poly(D,L-lactide-co-glycolide acid); PPADT: poly-(1,4-phenyleneacetone dimethylene thioketal) (PPADT); PVA: Polyvinyl alcohol; siRNA: Small interfering ribonucleic acid; TKN: Thioketal; TNF: Tumor necrosis factor; TNF-α: Tumor necrosis factor alpha; TPP: Sodium triphosphate; UC: Ulcerative Colitis; VEGF: Vascular endothelial growth factor; WSC: water soluble chitosan.

Table 5. Nucleotide-based formulation survey within CRC investigating local colonic NP administration. Selected studies with in vitro confirmation in CRC relevant cell-lines and intra-tumoral administration to Xenograft models are included.Study

Active cargo

In vitro assay

In vivo assay

Composition Formulation Ref.

1 VEGF DsiRNA

CCD-18 CO and Caco-2

- WSC-DsiRNA complexed NP coated with pectin

Low Mw WSC made by enzymatic reaction, complexed with DsiRNA, and coated with pectin in Aq

[163]

solution

2 β-catenin siRNA

HCT-116 - PEGylated chitosan/siRNA nanoparticles, chitosan/siRNA NPs

PEG was grafted onto chitosan using a carbodiimide-mediated reaction; NPs were prepared by ionotropic gelation technique [236, 237]

[164]

3 snail siRNA, Dox combination

HCT-116 - CMD/Chitosan NP

The chitosan NPs were prepared by a precipitation method (Eivazy_2017). CMD, DOX and/or snail siRNA was added by mixing and incubating

[165]

4 HMGA2 siRNA, Dox combination

HT-29 - CMD/Chitosan NP

Lower Mw chitosan was made by a pH swing/Na3NO3 reaction. The chitosan /CMD NPs leaded with DOX and/or HMGA2 siRNA were prepared by precipitation [238]

[166]

5 FHL2 siRNA LoVo - Chitosan NP Chitosan/siRNA formed by complexation [239]

[167]

6 VEGF-C siRNA/pDNA

LoVo LoVo cell tumor xenograft model/Itu

CaCO3 NP CaCO3 NP synthesized from CaCl2 and Na2CO3 in micro-emulsions; siRNA via a pDNA was complexed with the nanoparticles

[168]

7 Bag-1 siRNA (via pDNA)

LoVo LoVo cell tumour xenograft model/Itu

Gold NP Gold NP was combined with the pDNA in medium

[169]

Glossary: Aq: Aqueous; Bag-1: Bcl-2-associated athanogene; Caco-2 cells: colorectal adenocarcinoma cells; CCD-18 CO cells: normal colon cells, CMD: Carboxymethyl dextran; CRC: Colorectal cancer; Dox: Doxorubicin; DsiRNA: Dicer small interfering ribonucleic acid; HCT116 cells: Human colon carcinoma cell line; HT-29 cells: Human Caucasian colon

adenocarcinoma cell line; ITu: Intertumoral administration; LoVo cells: human colon(supraclavicular lymph node metastasis cells; NP: nanoparticle; pDNA: Plasmid DNA; PEG: Polyethylene glycol;siRNA: Small interfering ribonucleic acid; Snail: Transcriptional repressor 1; VEGF: Vascular endothelial growth factor; VEGF-C: Vascular endothelial growth factor C; WSC: water soluble chitosan.

Table 6. Clinical studies using probiotics relevant for colonic disease states. NCT# Disease Agent Sponsor Status Phase Last

update Study country

03482765 Acute GET 2 PB strains Biocodex R 4 2018 Argentina

02989350 Acute GET L. reuteri DSM 17938

Szpital im. Św. Jadwigi Śląskiej

R 2/3 2018 Poland

02765217 Antibiotic- Associated Diarrhoea

L. Reuteri DSM 17938

Eskisehir Osmangazi University

R 4 2017 Turkey

02993419 Antibiotic-associated diarrhoea

B. Licheniformis

Jiangsu Famous Medical Technology Co., Ltd.

NR NA 2016  

03271138 Coeliac Disease

START®2 Global Institute of Probiotics

R 2 2017 Argentina

03477669 Colic in infants

PB undefined University of Nebraska

NR NA 2018  

03415711 Colic in infants

PB, 1 or 2 strains

Biosearch S.A. R 2 2018 Spain

03106285 Colic in infants

L. reuteri DSM17938

BioGaia AB NR NA 2017  

02865564 Colic in infants

L. reuteri DSM 17938

Uni Medical Centre Ljubljana

R NA 2017 Slovenia

00893711 Colic in infants

L reuteri DSM 17938

Azienda Ospedaliera Ospedale Infantile Regina Margherita Sant'Anna

R NA 2018 Italy

03030664 Constipation L.reuteri Centre Hospitalier Intercommunal Creteil

R NA 2018 France

03333070 Constipation L.reuteri HaEmek Medical Center

NR NA 2018  

01992497 Diarrhoea PB undefined Nestlé R NA 2018 Germany

03334604 Diarrhoea, Antibiotic associated

8 PB strains Medical University of Warsaw

R NA 2018 Poland

02819960 Diarrhoea, PROBIO-FIX S&D Pharma R 3 2016 Slovakia

Chemo associated

INUM SK s.r.o.

02949882 Diarrhoea, Constipation

Yakult Athena Institute, Netherlands

NR 4 2016  

02803827 Diarrhoea, GET

L.reuteri Hamilton Health Sciences Corporation

R 3 2018 Botswana

03266315 GI disorder FloraBaby University of Calgary

NR NA 2017  

03403387 GI Symptoms GlutenShield pre/probiotic

East Tennessee State University

R NA 2018 USA

02351089 Gynecological cancer, GI tox

Probiotic undefined

Probi AB R NA 2018 Sweden

03150394 Helicobacter Pylori Infection

Gastrus DS Hospital Clínico Universitario de Valladolid

R 4 2017 Spain

03297242 Helicobacter Pylori Infection

L Acidophilus Changhai Hospital

NR NA 2017  

03220542 Helicobacter Pylori infection

Bioflor 250 Kyunghee Uni Medical Center

R 4 2017 Thailand

02953171 IBS Mutaflor University of Copenhagen

R NA 2016 Denmark

03482765 IBS 2 PB strains Vedic Lifesciences Pvt. Ltd.

R NA 2018 India

03150212 IBS S cerevisiae CNCM I-3856

Lesaffre International

R NA 2017 France

03266484 IBS, UC, CD 8 strains Mass General Hospital

R NA 2017 USA

02431533 IBS/Diarrhoea PX0612, 2 PB strains

Pharmabiotix Inc

R 2 2016 Canada

02796703 Infant Necrotizing Enterocolitis

Heat inactivated probiotics

Shaare Zedek Medical Center

NR 2 2016  

03531606 Sigmoid Colon Cancer, surgical

Mechnicov probiotics

Kye Bong-Hyeon, The Catholic

R NA 2018 Korea

outcome University of Korea

03415711 UC VSL#3 VSL Pharmaceuticals

R NA 2018 Italy

03136419 UC L casei University of Padova

R NA 2017 Italy

03524352 UC Fecal microbiota

Nantes University Hospital

NR 3 2018  

03444311 UC 3 PB strains AB Botics R NA 2018 Spain

01765439 UC, CD VSL#3 Charles University

R NA 2017 Czech Republic

Glossary: PB: Probiotics; DS: Dietary supplement; GET: Gastroenteritis; GI: Gastrointestinal; NA: Not applicable; NR: Not recruiting yet; R: Recruiting. 1132 studies were found under the key word Probiotics, but only studies that are recruiting or not yet recruiting were investigated in more details [224]. The table includes studies investigating probiotics as a dietary supplement or a drug, as defined in the record or as judged by the examination, but not studies investigating diets or intestinal microbiome without intervention [69] (accessed June 6, 2018).Table 7. Local colonic delivery system developability assessment, derived preclinical nucleotide-therapeutic literature.

Category Cargo(s) Indication(s) Delivery System(s)

Assays Consideration

Delivery System Formation

siRNA Colorectal cancer, IBD/IBS

Nano-liposomes, Polymeric NP

Gel electrophoresis Product quality, to qualitatively assess the cargo association with the delivery system

Assay; entrapment efficiency

siRNA, dsRNA, DNA

Colorectal cancer, IBD/IBS,

Nano-liposomes, Polymeric NP, Polymeric MP, lipid NP

UV-Vis spectrophotometry, Pico and Ribo-green dye assays

Product quality, to quantitatively assess the cargo association with the delivery system

Morphology siRNA, dsRNA, DNA

Colorectal cancer, IBD/IBS,

Polymeric NP, Polymeric MP

Scanning electron microscopy; Atomic force microscopy

Product quality, to assess morphology of NP and MP

Physical characterization/ stability

siRNA, dsRNA, DNA

Colorectal cancer, IBD/IBS,

Nano-liposomes, Polymeric NP, Polymeric MP

Particle size, zeta potential

Product quality, to confirm size and charge

Stability - chemical, physical,

siRNA IBD/IBS, Polymeric NP, Polymeric MP, lipid NP

pH stability ± enzymes and bile salts, RNAse

Product quality, to ensure stability against chemical and biological agents

enzymatic treatment, incubation in aspired fluids

important for performance

In vitro assessment, release

DsiRNA; DNA

CRC Polymeric NP Release in buffers w/wo changing pH, enzymes

Product performance, to ensure appropriate release for desired effect

In vitro assessment, cells/tissues

siRNA CRC Nano-liposomes; Polymeric NP

HT29 cells, HCT116 cells; Patient donor tissues

Product performance, to assess uptake and efficacy in disease relevant cells and donor tissues

In vitro assessment, cells/tissues

siRNA Lipid NP Caco-2 cells Product performance, to assess uptake in cells

In vitro assessment, cells/tissues

siRNA IBD/IBS Polymeric NP Mode-K cells, Colon-26 cells, RAW 264.7 macrophages, colon explants, organoids

Product performance, to assess uptake and efficacy in disease relevant cells and tissues

In vivo assessment

siRNA; DNA

Lipid NP; Polymeric NP

Mice/rats studies, intestines/colons removed and analyzed

Product performance, to assess efficacy in vivo

In vivo assessment

siRNA IBD/IBS Polymeric NP, Polymeric MP

Mouse w/wo colitis, colon removed an analyzed or in life imaging

Product performance, to assess efficacy in vivo

Selected definitions: DsiRNA: Dicer small interfering ribonucleic acid; MP: Microparticle; NP: Nanoparticle; siRNA: Small interfering ribonucleic acid.

Table 8. Overview of technologies that can be used to track intra-luminal dosage forms in large animals and humans (adapted from [223, 240-242])

Technology Description Examples of studies using technology

Intraluminal sampling

Intubation with catheters (humans, non-rodent experimental animals)

Permanent ports (experimental animals)

[216]

Telemetry Sampling devices: Heidelberg capsule, Smart Pill®, Intellicap®:

Delivery devices: Enterion®,

[135, 218, 243]

IntelliSite®, Intellicap®:

Visualise in vivo behaviour: PillCam®;

Radioactive Gamma scintigraphy

Tomography

X-ray imaging with contrast agents

[210, 219, 220, 244-247]

Non-radioactive

Breath/urine tests after ingestion of stable isotopically labelled compounds (13C and 15N)

Magnetic resonance imaging (MRI)

[222, 224, 225, 248]

94

95