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Peptide drug delivery: Colonic and rectal absorption
advanced
drug delivery reviews
ELSEVIER
Advanced
Drug Delivery
Reviews
28 (1997) 253-273
Peptide drug delivery: Colonic and rectal absorption Martin Mackay”‘“, Judy Phillipsb, John Hastewell” “CNS Research, “Drug Discovry, ‘Accel
Prwtnrrs.
Cihn
Pharmacruticuls.
Ciho
Bask
Phanwcruticals,
Embnrcndero
Cmtre.
CH-4002,
Horrharn
Switzerland
RH 12 4BT.
Son Frmcisco.
UK
CA 9411 I, USA
Abstract To date, a limited number of peptides and proteins have been used therapeutically. However, this number is growing rapidly. Some have been synthesised chemically or extracted from tissue and have been used in the clinic for decades. Recently, advances in cell and molecular biology have led to a much greater perception of the therapeutic value of many other peptides and proteins. Recombinant DNA technology has enabled high level expression and the biotechnology industry has made available large scale production for clinical use. In order to ensure that maximal use is made of this class of drug. scientists will need to devise patient compliant formulations. Clearly, these formulations will have to be safe and pharmacologically relevant. In addition, the route of administration will have to bc acceptable to the patient given any particular indication. Almost all therapeutic peptidex and proteins are administered by injection. It has been recognised that specific regions of the gastrointestinal tract may offer potential in terms of macromolecular absorption. This paper will address the attractiveness of the colon and rectum as routes 0 1997 Elsevier Science B.V. of administration for peptide and protein drugs. Keywords:
Drug absorption;
Drug delivery;
Therapeutic
peptidcs
and proteins
Contents I. Introduction _............................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... .__..._..., ,.,... 254 2. The colon . .._......................................................................................................................................................... .__..._........,. 254 ..................... ................. 2.54 2.1. Colonic environment.. ........................................................................................................... 2.2. Colonic epithelium.. ......................................................................................................................................................... 256 2.3. Delivery to the colon.. ...................................................................................................................................................... 257 .......................................................................... 2.4. &Ionic absorption.. ............................................................................ 258 2.4.1. Absorption studies.. ................................................................................................................................................ 25Y 2.4.2. Colonic delivery of insulin.. .................................................................................................................................... 261 ... .......................................................................... ‘2.4.3. Colonic delivery of calcitonin ................................................ 262 2.4.4. Advantages of colonic peptide and protein drug delivery.. ......................................................................................... 263 3. The rectum............................................................................................................................................................................. 263 263 3.1. Rectal absorption ............................................................................................................................................................ 3.2. Advantages of rectal peptide and protein drug delivery ....................................................................................................... 264 4. Human studies ........................................................................................................................................................................ 264 265 4.1. Calcitonin: A case study ............................................................................................................................................... 4. I. I. Colonic absorption ................................................................................................................................................. 2h5 1. I .2. Rectal absorption ................................................................................................................................................... 266 .................................................. 267 5. Impact of GI tract delivery of therapeutic peptides and protein\ .............................................. “:Corresponding
author. Pfizer Central Research,
Ol69-409X/97/$32.00 0 PI/ SOlh9.409X(97)00076-8
Sandwich.
Kent CTI 3 YNJ, England.
1997 Elsevier Science B.V. All rights reserved
Tel.:
+ 44 1304 8333: fax.
+ 44 I304 X2 18.
S. 1. Pharmacokinetic 5.2. Pharmacoeconomic 6. Challenges
of colonic
impact of GI tract deliver)
267
.._....
impact of GI tract delivery and rectal delivery
of peptides and protein drug>...
..,,.,.,..._.... .._................_...................,..........
26X 26X
Acknowledgements
26X
References
26X
1. Introduction The delivery of therapeutic peptides and proteins remains a priority for many pharmaceutical companies. In a recent review [I] of delivery strategies for peptides and proteins it was found that over 40% of all pharmaceutical companies were active in this area. Significantly, 27. representing 37%’ of the companies surveyed. are pursuing research programmes in the oral delivery of peptides and proteins. This has resulted from the advances in molecular biology and biotechnology, which between them have identified and made commercially available many peptides and proteins with valuable therapeutic properties [2,3]. It is recognised that biotechnology makes the discovery process for therapeutic peptides and proteins more efficient than traditional drug discovery. The success of therapeutic peptides and proteins is shown by the fact that three recombinant products are in the top IO best selling drugs list for 1993. Erythropoietin (for example, Am&en, Johnson and Johnson), human and animal insulin (for example, Lilly, Novo Nordisk) and o-interferon (for example. Schering-Plough. Sumitomo, Roche) achieved sales of $1806 million, $1610 million and $466 million. respectively. Other products such as growth hormone. B-interferon and granulocyte colony stimulating factor are expected to achieve increased sales over the next few years. In addition, many peptides and proteins are still in clinical development and the forecast is promising for agents such as bovine myehn protein. hirudin and transforming growth factor-B. Table I lists peptides and proteins. in clinical development or already marketed. However. the biopharmaceutical challenges that these agents present are huge. At present. the vast majority of therapeutic peptides and proteins are formulated for injection independent of the indication, the chronicity of dosing or the need for selfadministration. This severely limits the attractiveness of peptide and proteins as therapeutic agents in all
but the most serious indications or where there is no opportunity for alternative therapies. The need to find non-injectable routes of administration for peptides has focused attention on the oral route; the traditional method for patient compliant drug administration. However, the desire to achieve oral availability and the reality are somewhat apart. In considering the oral route of administration there are several factors that have to be addressed. These are illustrated in Table 2. This review will highlight peptide and protein absorption studies with two specitic areas of the gastrointestinal (GI) tract, namely the colon and rectum.
2. The colon 2. I. Colonic rnvironment The structure and function of the colon have been extensively reviewed 14-71. However, to date, their are few data on correlations between structure/function and drug transport across the colonic epithelium. Conventional wisdom suggests that many drugs are not absorbed from the colon or show little absorption. Nevertheless. studies have been reported where the absorption of some drugs across different regions of the GI tract is similar. Brockmeier et al. [Xl reported that the absorption of glibenclamide across the stomach, duodenum or colon was similar. Given that the environment of the colon is different to the stomach and small intestine in terms of proteolytic degradation the potential of the colon as a site for peptide and protein absorption has been studied. Cummings and co-workers [9] have shown that the colon in man contains few contents under normal conditions. They reported that the total amount of content in 46 adults was 222?21 g (wet) with only 93 g in the caecum and ascending regions. Moreover, other studies using cell culture models have assessed the barrier function of the colon. The development of
255
Table
I
Examples and application of peptides and proteins in clinical use or undergoing clinical trial Therapeutic peptide or protein
Application
Tissue necrosis factor
Carcinoma
Proleukin
Carcinoma
y-Interferon
Carcinoma
Epidermal growth factor
Wound healing
Transforming
Wound healing
growth factors
Fibroblast growth factor
Wound healing
Insulin-like
Wound healing
growth factors
Hirudin
Fibrinolytic
Tissue plasminogen activator
Fibrinolytic
Streptokinase
Fibrinolytic
Erythropoietin
Erythropoieais stimulation
Factor VIII
Haemophilia
Factor IX
Christmas disease
Triproamylin
Glucose regulation
Insulin
Glucose regulation
Somatostatm
Glucose regulation
Proinsulin
Glucose regulation
a-Interferon
Viral diseases/hairy cell leukemia
P-Interferon
Multlple sclerosis
Glucocerebrosidase
Gaucher’\
CereLyme
Type I Gaucher‘s direase
Pulmo7yme
Cystic fibrosis
disease
Calcitoninh
Bone disen\e
Oxytocin
Labour induction
Growth hormone
Short stature
cr.1 Antitrypsin (aat)
aat deticiency
Superoxide dismutase
Respiratory disorders
Table 2 Physical and physiological factors affecting the extent of peptide and protein absorption from the GI tract Physical factors
Physiological factors
Solubility
pH, regional and micro-climate
Dissolution rate
Binding and complexation
Stability
Proteases
Sire and conformation
Mucosal barrier
Partition coefticient
Intestinal permeability
Charge
Intestinal metabolism
Delivery
system
Hepatic metabolism
primary colonic epithelial cultures [ 10,l I], cell lines derived from colonic adenocarcinomas [ 12- 141, and cell lines derived from rectal adenocarcinomas [ 1S181 have given greater insight into the barrier function of the colonic epithelium. The microbiology of the colon is complex [1922]. Over 400 different species of aerobic and anaerobic bacteria reside in the colon. However, the anaerobic flora predominate [23]. The number of
anaerobic bacteria is approximately 10” per ml resulting in almost one-third of the dry weight of faeces in man [24]. Colonic bacteria secrete many enzymes usually involved in the fermentation of substrates for energy production. Some of these enzymes have been used as ‘triggers’ for colonspecific drug delivery (see later). In addition, it is believed that some residual pancreatic proteases are also present. To date, there are few data on the relative amounts of pancreatic enzymes in the small intestine and colon. The evidence that is available suggests that the amount is reduced in the colon. For example, Gibson et al. [25] highlighted the significance of microflora on proteolysis in the human colon. They showed that proteolytic activity was an order of magnitude greater in small intestinal fluid than in faeces. Interestingly, faecal proteolysis was qualitatively different from ileal proteolysis, as shown by the range of proteins hydrolysed and the susceptibility of the enzymes to some protease inhibitors, for example chymostatin.
Therefore, drug absorption in the colon is a consequence of the epithelial structure. Nevertheless, receptors for peptides and pl-oteins such as insulinlike growth factor I (IGF-I) 1261 and insulin 1271 exist on the apical membrane of proximal colon epithelial cells of the rabbit. It remains to be seen whether these receptors can be exploited IO transport peptide and protein drugs specifically. The general properties of the colon which may be of signiticance terms of peptide and protein absorption are: less luminal proteolytic activity compared with the small intestine: the epithelial cells of the mature organ do not express membrane associated peptidases: The luminal milieu has been shown to be suitable for prodrug metabolism as with salicylazosulphapyridine or offer an environment suitable fat drug delivery or site-specific drug release.
In the mammalian colon the epithelium contains absorptive. goblet. endocrine and a small number of Paneth cells. The absorptive cells possess microvilli. However, the colonic microvilli are much shorter than the microvilli of small intestinal absorptive cells. It is believed that the absorptive cells are the most important in terms of nutrient and drug transport. The monolayer of columnar epithelial cells is similar throughout the colon. However, towards the rectum there is a transition between this columnar epithelium to the keratinised stratified squamous epithelium of the anal skin. The anatomy of this area of transition. sometimes referred to as the anal transitional zone. is complex 1281. Goblet cells are present in the colonic epithelium in keeping with many other types of epithelia 1291. They represent the most abundant cell type in the colon and display marked differences in their degree of differentiation. Goblet cell secretions consist of water, mucin and various electrolytes [30]. The structure of colonic mucus and the physiology of colonic mucus secretion have been described by Allen (311 and Filipe 1321. Endocrine cells are less abundant in the colon than in the small intestine. They can occur singly or in clusters and are often found in the lamina propria.
Colonic endocrine cells are small, clear cells with a basal cytoplasm that contains electron-dense granules. They are most prominent deep in the crypts and methods have been developed to study endocrine cells in this location (33.341. Regulatory peptides $uch as enteroglucagon, pancreatic polypeptide, substance P. peptide YY and somatostatin are present in colonic endocrine cells 1351. These peptides have been shown to display a variety of functions which influence colonic secretion, motility and blood flow. Very few Paneth cells are found in the colon. They are found in the caecum and ascending colon. A wide variety of functions have been attributed to Pnneth ceils including secretion of digestive enr.ymes. production of trophic factors and elimination of heavy metals [Ml. They have abundant zinc-rich secretory granules which lysozyme, contain crlycoproteins and other proteins. They display irEgular microvilli and do not possess a terminal web. Due to their scarcity in the colon it is doubtful whether they play a major physiological function in this part of the Gl tract. The gut associated lytnphoid tissue (GALT) is unevenly distributed throughout the GI tract. The lymphoepithelial regions of the colon are known as lymphoglandular complexes (LGC). Little is known about the LGC. although their structure and composition bear a strong resemblance to sites in the small intestine associated with antigen sampling. In addition. it has been noted that LGC epithelium displays diminished mucin secretion 137). Colonic LGC demonstrates differences in size, cell numbers 1181 and regional variation in frequency 1.391. although the significance of these differences is not known. The lamina propria which delineates the basement membrane of the epithelial crypts and the muscularis mucosae contains many cell types including lymphocytes. plasma cells. macrophages, eosinophils, neutrophils and peripheral nerves. Lymphocytes and eosinophils are often observed between epithelial cells. The structure of the proximal colonic epithelium is well suited to one of its primary functions, namely the resorption of water. although it does not possess tht: curface area of the small intestine. In addition, the mucus which acts as a lubricant produced by the colonic goblet cells protects against abrasion from solid matter particularly in the distal colon and
M. Mackq
enables the excretion the tissue. 2.3. Delivery
et al. I Adanced
of faeces without
damage
Drug
to
to the colon
An excellent review of the specific delivery of drugs to the colon has been published recently [40] and Table 3 shows examples of the approaches. A novel timed-release dosage form has been developed by Scherer DDS. The ‘Pulsincap’ device can be modified such that its contents are released in the GI tract after a definite time period. Notwithstanding the vagaries of GI transit times 1511, it has been shown that the device can reproducibly release it’s payload in vitro and in the colon of man. The device consists of hydrogel components which when exposed to an aqueous environment causes swelling and release of a plug and subsequent release of drug. Application of an enteric coating prevents hydration in the gastric fluid. After stomach emptying the coat dissolves and hydrogel hydration occurs. The timing of the release is determined by the configuration of the hydrogel components [41]. This system is simple in construction and is produced from only pharmaceutically acceptable polymers. Devices have been produced which rely on bacterial enzyme breakdown to specifically release drug in the colon. Saffran developed a gelatin capsule coated with an impermeable polymer containing insulin or vasopressin [44]. The coating is resistant to degradation in the stomach and small intestine. The preparation is based on azo functional crosslinking agents. In a similar reaction to sulphasalazine the azoaromatic bonds (R-C,H,-N=N-C,H,-R) are reduced to form a pair of aromatic amines (R-
Delicry
Reviews 28 (1997)
257
2_%-27-J
C,H,-NH, + H,N-C,H,-R) by bacterial azoreductases. The capsule is broken down in the colon and released. With insulin, drug is specifically azopolymer coated capsules were given per OS to diabetic dogs and a hypoglycaemic response resulted. Blood glucose was reduced from 4 to In the 2.9 mg ml- ‘, 3 h after oral administration. case of vasopressin, azopolymer coated capsules were delivered to the stomach of an anaesthetised rat and a pharmacodynamic response in terms of antidiuresis determined. Maximum antidiuresis resulted 3 h following administration of the capsule. Both the insulin and vasopressin data were subject to a high degree of variability, probably due to the vagaries of coating the capsules. Other workers have attempted to devise more reproducible systems. Kopecek et al. [45] developed elegant hydrogel capsules, based on acrylic acid, N,N-dimethylacrylamide and N-tert-butylacrylamide crosslinked with 4,4’-di(methacryloylamino)azobenzene. These hydrogels do not swell significantly in the pH conditions of the stomach. In transit through the small intestine, swelling increases due to an increase in pH. In the colon. the degree of swelling exposes the cross-links to the bacterial azoreductases and this results in breakdown of the hydrogel and release of the drug. The capsules have been tested both in vitro and in vivo where they display a high degree of reproducibility. This group are also developing protease triggered systems. To date, few data have been published demonstrating the effectiveness of this type of system for colonic release of peptide or protein drugs and no studies exist in man. Davies and co-workers have patented a capsule for targeted delivery of peptides to the colon 1521. The
Table 3 Colon specific drug d&very
approaches
concept Timed-release Delayed-release
Examples delivery system delivery systems
Pulsincap [4 I ] Eudragit coated capsule\ [42] Cellulose acetate phthalate coating 1431 Azopolymer
coated capsules 1441
Hydrogels with azopolymer links [4S] Polysaccharide cross-linked capsules 1231 Sustained-release delivery systems
Ethyl cellulose membranes (461
Particulate-based delivery systems
Silica-drug preparations 1471
Prodrugs
Low MWt aro bond prodrug
(481
Polymeric aro bond prodrug
[49]
Glycoaidic bond prodrugs [XI]
capsule is coated with a 60- 150 pm thick layer of a commercially available anionic polymerpolyacrylic polymer. Eudragit S. The coating is insoluble in the stomach and small intestine below pH 7.0 but soluble in the colonic environment. Insulin, porcine calcitonin and human growth hormone have been encapsulated as formulations which also contain absorption enhancers, wetting agents and surfactants. The reproducibility of such a system for colonic delivery in man has not been demonstrated and the effects of the specific formulations used not determined. Touitou and Rubinstein [ 53 1 used a similar type of capsule to deliver porcine insulin. The formulation also contained an absorption enhancer. sodium laurate/cetyl alcohol, 2:s. A hypoglycaemic response was demonstrated in rats. although relative bioavailability was still low. pH-sensitive polymers have also been reported that rely on the belief that the proximal colon has a pH of 8.0-8.4. The polymer only dissolves at high pH. It has been shown, however, that high pH can be found in other regions of the GI tract rendering these polymers unreliable to specific colonic drug release 1541. The concept of specific drug release in the colon has been tested with low molecular weight drugs by making prodrugs. The prodrugs survive transit through the small intestine where they are not absorbed and whose active moiety is released by bacterial enzymes produced in the colon. The most notable example is sulphasalazine (salicylazosulphapyridine) which is not absorbed in the small intestine and is reduced to S-amino salicylic acid and sulphapyridine by azoreductdses produced by anaerobic bacteria in the colon 1481. In support of the mechanism of action. the cleavage of sulphasalazine is signiticantly reduced in patients receiving antibiotic treatment or that have had colonostomy. A colon-specitic drug-delivery system has been described based on the use of steroid glycosides. This system relies on glycosidasea produced by colonic bacteria [SO]. Steroid glycosides are poorly absorbed from the small intestine. However. colonic bacteria produce glycosidases which releases free steroids such as dexamethasone. The free steroids are then absorbed from the colon. To date. little evidence exists which suggests that a prodrug approach can be used for the delivery of therapeutic peptides and proteins. However, the exploitation of bacterial
enzymes to degrade delivery devices which potentially can contain peptides and proteins has been shown. It is apparent that specific release of peptide and protein drugs in the colon is possible. The possibility of polymeric delivery systems that degrade when exposed to enzymes produced by the colonic flora has been shown and awaits appropriate peptide formulation for clinical trials. Moreover, the development of timed release devices augurs well for achieving reproducible release of drugs such as therapeutic peptides and proteins. The challenge remains, however, to achieve adequate absorption across the epithelium following specific release.
The transport pathways of the colon provide for rapid and specific active bi-directional transport of ions across the epithelial layer. Unlike the small intestine, there are no documented active transporters for organic nutrients in the mature organ and, therefore, no chances for drug molecules to be absorbed in a piggy-back fashion. In the small intestine examples of this kind of active absorption of drugs are seen for 5-fluorouracil [.55.56] on the pyrimidine transporter and antibiotics on the peptide transporters [S7-591. The lack of such transporters limits the scope for drug design with respect to mediated transport across the epithelial barrier and. therefore, drug absorption at this site is a consequence of the general properties and features of the colon. The active transport pathways of the colon have been reviewed 171. The apparent lack of organic nutrient transporters may limit the potential for drug design with respect to carrier mediated transport across the colon. The following of which may be of significance: l
l
l
The transmucosal and membrane potential differences may be of signiticance in the absorption of ionised or ionisable drugs [60,61]. The colon is considered to be less of a barrier to macromolecules than the small intestine and thus may offer opportunities for both peptide and protein absorption. The bulk water absorption in this region of the intestine will provide scope for solvent drag and possibly improved drug absorption [62].
M. Muck+
et 01. I Advunced
Drug Delivery
The luminal milieu may be suitable for prodrug metabolism as with salicylazosulphapyridine [63,64] or offer an environment suitable for drug delivery [23] or site-specific drug release. Experiments using absorption promoters such as formulations Ca’+ chelators or lipid/detergent have proved that the colon is susceptible to enhancement strategies [65].
These factors may influence the choice of the colon as the region for drug absorption after oral administration. However, in the main they are not as a consequence of the physiological or biochemical properties of the colonic epithelia. Finally, any drug which is able to initiate or inhibit any of the intracellular cascades that regulate the absorptive versus the secretory response of the colon may not be good candidates for this site of absorption. The oral absorption of peptides and proteins has been reviewed [66] and the mechanisms of peptide and protein absorption have been documented in terms of paracellular intestinal transport [67], transcellular transport 1681 and endocytic pathways [69]. There are reports of peptides crossing the colonic mucosa in both animals 1701 and man 17I]. Moreover, this absorption can be enhanced by a series of agents such as salicylates 1721, water-oil-water emulsions 1731, surfactants [74], bile salts 17.51, combinative promotion effects of azone and fusogenic fatty acids 1761 and lipids [77,78] (for more details see Yamamoto et al. 1791). However, as shown earlier, to take advantage of these observations it is necessary for the drug to be delivered to the colon. The mode of delivery must either ensure that the peptide is protected from the digestive functions of the stomach and small intestine or circumvent them entirely by accessing the large intestine via the rectum (see below). Research into the large intestinal absorption of peptides has, therefore, been concerned with three main areas:
methodologies to deliver/release peptides in the large intestine after oral administration; rectal formulations to provide release in the distal colon; formulation and mechanistic studies to enable absorption across the colonic mucosa.
Reviews 28 (1997)
253-273
259
The aim of such research is to achieve therapeutically relevant peptide delivery. To illustrate these issues the genera1 absorption across the colon and the peptide hormones insulin and calcitonin will be considered with respect to the value of colonic delivery for therapeutic purposes. These latter peptides are particularly relevant as they have been in the vanguard of many peptide and protein drug delivery approaches and as such represent the challenge that peptides present to the pharmaceutical industry. 2.4.1. Absorption studies The GI absorption of drugs has been reviewed [80-841. It has been known for decades that peptides and proteins cross the GI epithelium, albeit in low amounts 185-891. These peptides and proteins include chymotrypsin, elastase, antigens [90,9 I ] horseradish peroxidase (HRP) [92], insulin [93] and Clostridium bofulinum type A toxin [94]. It is also apparent that more than one mechanism exists by which peptides enter the vascalature via the lumen of the GI tract and these include both passive and specific pathways. The question as to whether these physiological processes can be used for the colonic absorption of therapeutic peptides and proteins remains a challenge. It is accepted that these processes only enable the transport of low amounts of macromolecules and therefore would be insufficient to achieve therapeutic effects of most peptides or proteins. In addition, pharmacoeconomic issues would arise from delivering peptides and proteins with known poor bioavailability. Recently, human cell lines have been used as intestinal models to investigate the transport of a range of drugs. These cells have been grown in culture as monolayers on semi-permeable filters and shown to display many characteristics associated with differentiated intestinal epithelial cells. For example, an adenocarcinoma cell line. Caco-2, derived from human colon was used to study the transport of HRP [ 131. Only very low concentrations of apically applied HRP were seen in the basolateral chamber (0.0001%) demonstrating the tightness of the cell model barrier. Using the same cell line. the absorption profiles of the vasopressin analogues, arginine-vasopressin (AVP) and 1-deamino-8-D-arginine-vasopressin dDAVP 19.5], were determined. Both these hydrophilic peptides displayed linear
transport kinetics and were absorbed at very slow rates by a non-saturable, non-polar process across the Caco-2 monolayers. Neither peptide showed degradation after a 4 h incubation in the apical medium ot the cells. The use of cell models such as the Caco-2 line to study peptide and protein transport is at an early stage but results to date indicate their potential as a tool to discover new pathways and novel formulations. Other in vitro methods have been used to assess the importance of the epithelial layer as a physical barrier to peptide absorption. Isolated intestinal sheets have been used. In two recently reported studies. Quadros et al. [96,97] demonstrated the permeability of colonic sheets from different species to CGS 16617, a novel ACE inhibitor. and insulinlike Growth Factor 1 (IGF-I ). CGS 16617, a trihas a permeability coefticient of peptide, I. 12-CO.09 cm s ’ X IO (’ across rat colon sheets. compared to that of 8.6252.1 1 cm s ’ X IO ’ foi IGF- 1, 70 amino acid residues. As expected. molecular volume has a signiticant effect on transepithelial permeability. To date, only a few studies have been reported where a peptide or protein has been administered intracolonically in the absence of protease inhibitors or absorption enhancers. Lundin and Vilhardt 1981 determined the absorption of dDAVP from different regions of the GI tract of rats. The regions were stomach, duodenum. mid-part of the ileum. the ileocaecal junction and the mid-part of the colon. The absorption of intact dDAVP occurred from each site and was rapid with peak plasma levels after I o20 min. Less absorption was observed across the stomach and colon. The authors suggested that the
low surface area associated with these regions compared to the small intestine was the reason for less absorption. Langhuth et al. 1991 compared metabolism of metkephamide. a synthetic pentapeptide resistant to peptidase activity, by the brush border of the enterocytes in different regions of the rat GI tract using a single pass perfusion of isolated sections, in situ. They reported maximum degradation in the jejunum. decreasing down the intestine to no detectable metabolism in the colon. Values reported for metabolism per cm intestine are: duodenum 14.9%3.050/o, proximal 8.14-t2.32%, jejunum middle jejunum 3.08-+0.51%, ileum 1.72?0.31% and colon - not detectable. Metabolic barriers to peptide absorption and methods of overcoming them have been reviewed in detail by Zhou [ IOO]. A number of groups have compared absorption from different regions of the GI tract of rats in vivo using the in situ perfused loop method. The results are summarised in Table 4. Each group assessed absorption by a different method. Table 4 shows that the authors have looked at the absorption of peptides ranging in molecular weight from 661 to 6000. The ileal epithelium is shown to be slightly more permeable to the peptides in this model, confirming evidence from in vitro models that the permeability of the intestinal epithelium increases down the GI tract. In these studies, the differences between regions disappear in the presence of intestinal contents, suggesting that in the colon the lower luminal levels of metabolism compensate for its decreased permeability compared to the ileum. It is clear that the few experimental data that exist
Table 4 Comparative absorption of peptides from different regions of the GI tract 111rat5
[ 1011
[ 102 I
Author:
Geary and Schlameus
Langhuth et al. [Y91
Morishita et al.
Peptide:
Vancomycin
Metkephamide
Insulin (porcine)
MWr:
I so0
661
6000
Wall permeability
Efficacy (J glucose)
Bioabnilability
Duodenum
(‘k)
Not detectable
Jejunum
+ wash
~ wash
0. I
0
I .641-0.31
0
0
Ileum
3.5-tl.l
(xi)
I .c)Ito.21
0.3
0.I
Colon
3.1+0.9
(xl)
0.67ZO.38
0. I
0. I
M. Mucka! et cd. I Advanced Drug Drli~vt:v Rersiews 2X (1997) 2.5%27.7
show that therapeutic peptides and proteins are poorly absorbed from the GI tract. Although the colon may be a preferable site for peptide delivery because of the relative lack of proteases, this fact alone does not lead to effective bioavailability. As a result, various strategies have been applied to elevate absorption levels and include: protection against presystemic degradation; chemical derivatisation of the drug (prodrugs); and co-administration of socalled ‘absorption enhancers’. In a few instances these have been successfully used to promote the absorption of peptides and proteins. 2.4.2. Colorzic delivery of insulin The discovery of insulin rapidly led to its clinical application for treating insulin dependent diabetes melitus (IDDM). The therapeutic impact of insulin on the treatment of IDDM is dramatic. Patients suffering from this disease are able to control their blood glucose to a point where they can lead normal lives. The need to deliver the drug by injection is acceptable when compared with the consequences of not taking insulin. Current therapy consists of once or twice daily injections of insulin, including mixed intermediate or rapid-acting insulins [103]. Even so it is recognised that this therapy is not a complete solution as, for example, with aging, a variety of conditions become prevalent in diabetic patients. The most common of these are retinopathy, nephropathy, neuropathy and cardiovascular disease [ 1041. These conditions are a consequence of incomplete control of blood glucose levels leading to long-term adverse side effects I10.5,106]. Recent clinical studies have shown that careful monitoring of blood glucose coupled with intensive insulin administration can lead to a significant reduction in these side effects [ 1031. To be successful oral insulin at the very least must provide therapeutic equivalency to the current therapies but ideally would provide the ability to tightly control glucose levels. To achieve this, control of insulin administration is key to both achieving the correct acute hypoglycaemic response and reducing the chronic morbidity associated with the disease. The large intestine can be reached either by oral or rectal administration. The former requires that the formulation or device protects the peptide from the proteolytic conditions found in the small intestine,
261
specifically the stomach, duodenum. jejunum and ileum. There are many approaches to this, however the element constant to all these is the time taken to achieve passage to the large intestine and the factors influencing this passage. Whilst passage along the small intestine is considered to be relatively constant at 3-5 h under most conditions 11071. Retention in the stomach can delay the passage by several hours dependent upon stomach contents. Therefore, it is extremely difficult to predict when an orally administered dose of insulin would be delivered to the colon for absorption. The consequence of this is that it would be virtually impossible for the oral dosing of insulin to be co-ordinated with ingestion of meals such that the systemic insulin levels corresponded to the post-prandial glucose peak. This lack of control in the timing of the insulin after oral administration will rarely achieve the fine control for optimal therapy. The best that could be hopecl for is a sustained delivery that could achieve similar levels of control to current once a day parenteral formulations. However, timing the ingestion of the dose to provide day to day control of glucose levels will be a challenge. In this case rectal delivery offers the greatest opportunity for delivery via the large intestine. However, this is still non-trivial. In the case of insulin the dose delivered must be reproducible and quantifiable. Atchison et al. [ 1081 studied the colonic absorption of radiolabelled insulin using non-everted sacs of rat colon. The percentage of intraluminal insulin degradation and the transport of insulin into the surrounding media was determined. They showed that transepithelial flux of insulin was consistently less than 0.3% of the dose. In addition, significant degradation of insulin (64%) was found within 15 min of exposure. The effects of intracolonically administered human insulin, and human insulin-DEAE-dextran complex entrapped in liposomes, on blood glucose in rats has been reported [ 1091. In both cases, a hypoglycaemic response was noted which lasted for several hours. No indication of relative bioavailability was given and it can only be presumed that insulin was transported across the colon. Given the difficulty in predicting when an insulin dose would reach the colon and be transported across the epithelium it is difficult to imagine an oral dosage form reaching the market soon. Nevertheless,
activity in this tield is enormous. More practical approaches to oral delivery may result in a diabetic patient taking fewer injections and thus lead to an improved quality of life rather than complete respite from the needle.
The rationale for -considering oral delivery of peptides for therapeutic purposes is based on patient compliance. In the case of diabetes. the disease characteristics and the consequences of not taking insulin force the patient into accepting the injectable formulations. In contrast to this, peptides indicated for prophylactic treatment of a non-terminal disease are poorly tolerated by the patients because of the need for injections to dose the drug. This is well illustrated by calcitonin. Historically, calcitonin has had a relatively narrow usage in Paget’s disease and control of hypercalceamia associated with cancers. both treated with injectable formulations. Although it has been recognized for many years that it i4 effective in retarding the progress of osteoporosis. current formulations limit the use of calcitonin in this indication. In responding to market needs many delivery routes have been considered. Nasal delivery has had some success showing therapeutically signiticant endpoints whilst avoiding injectable administration. However. it is considered that ultimately an orally administered formulation will be the optimal solution. The therapeutic regimen for calcitonin in treating osteoporosis differs markedly from the treatment of IDDM with insulin. Calcitonin has a large therapeutic window. In the treatment of osteoporosis. calcitonin is influencing the outcome of a long onset disease by down-regulating osteoclast activity. There is no requirement for precise dosing either regarding the amount absorbed or the timing of the administration. The requirement is that a therapeutic dose is delivered in the order of once a day. This therapeutic profile greatly simplifies the dosing requirements and makes calcitonin a particularly attractive peptide fog GI tract delivery. Hastewell et al. 1701 used direct administration of human calcitonin into a colonic loop in anaesthetised rats to examine the bioavailability of human calcitonin. This was compared to the pharmacodynamic effect. detectable in normal juvenile animals, of a reduction in plasma calcium levels in response to
human calcitonin. They demonstrated the bioavailability achieved after intracolonic dosing of three different doses compared to an i.v. dose. These were: 0.5% at 5.0 mg kg ’ , 0.9% at I .O mg kg- ’ and 0.2% at 0. I mg kg ‘. In addition, intracolonically administered human calcitonin at doses of 0. I-5.0 mg kg -’ resulted in a dose-dependent reduction in plasma calcium levels. These doses achieved reductions in plasma calcium levels of 12?6 to 382.5%. The reference i.v. dose of 1.25 kg kg ’ achieved a calcium reduction of 2924%. Moreover. immunohistochemistry showed that human calcitonin transport across the rat colon was rapid and a significant amount was via a transcellular pathway. In a subsequent report, Hastewell et al. 1781 studied the influence of eyuimolar monoolein/sodium taurocholate enhancer formulations on the absorption of human calcitonin and two markers of intestinal permeability, HRP and polyethylene glyco], MWt 4000 (PEG 4000). Human calcitonin, HRP and PEG 4000 were all absorbed across the colonic mucosa to a limited extent. The use of a 40 mM monoolein/40 mM sodium taurocholate mixed micellar formulation significantly ( p < 0.001) enhanced (9.0t I.O-fold) the absorption of all three molecules with no acute damage to the mucosal tissue as judged after light microscopy. At concentrations of 20 mM and below, the monooleini sodium taurocholate formulation did not enhance the absorption of human calcitonin. HRP or PEG 4000. HRP immunohistochemistry showed an intracellular localisation suggesting that a transcellular pathway was involved in absorption across the epithelium. The increased absorption of human calcitonin in the presence of the 40 mM enhancer formulation was able to elicit a maximal hypocalcaemic response. whereas no signiticant effect was observed in the absence of the enhancer. The authors concluded that the absorption enhancer used in this study can increase intestinal absorption of a range of molecules without causing major tissue damage, albeit after acute treatment. These formulations may offer advantages as they enable pharmacodynamic responses to be elicited from reduced doses of therapeutic peptides and proteins. It is important to understand the relevance of these results to the potential pharmacokinetic and pharmacodynamic effects after dosing to the unligated colon of conscious man (see below).
Fukunaga et al. [l lo] showed that liposomally entrapped salmon calcitonin produced a hypocalcaemic effect in rats when dosed orally, but did not determine the mechanism or intestinal location of the absorption. 2.4.4. Advantages of colonic peptide and protein drug deliver? The advantages of colonic peptide delivery can be summarised as follows: l l l l
l
l
low metabolic activity; longer residence time; responsive to absorption enhancers; colonic bacterial enzymes present may offer targeting opportunities; the transmucosal and membrane potential differences may be of significance in the absorption of ionised or ionisable drugs; the bulk water absorption in this region of the intestine may provide scope for solvent drag.
Nevertheless, despite the colon’s apparent attractiveness as a route for the oral delivery of peptides, only very limited bioavailability can be demonstrated for this route in vivo. In addition, delivery systems to achieve oral delivery to the colon remain to be resolved.
3. The rectum 3.1. Rectul chorption The rectal route has been used to deliver many drugs and offers similar advantages over oral delivery to those enjoyed by the nasal route [ 11 l-l 131. It can be an extremely useful route for delivery of drugs to babies and young children where difficulties can arise using per OS administration. Similar disadvantages are also evident, for example limited surface area and dissolution problems. Interruption of drug absorption during defecation is another concern and patient acceptability is variable. However, the rectal route does offer a more convenient way to control drug release using osmotic pumps and hydrogel cylinders, although to date they have only been tested with low molecular weight drugs such as propranolol [ 1141 and nifedipine [ 1 I5,l 161.
Historically, the rectum has been an accepted site of drug delivery. Its principal applications have been for local therapy, e.g. haemorrhoids, and for systemic delivery of drugs to groups presenting practical problems for parenteral or oral dosing, for example infants, epileptics. Some populations are less willing to accept this route as a standard method for drug delivery, but it is the most easily accessible area of the lower GI tract. As such it offers the potential for delivery of peptides without the need for targeted devices, and it’s efficiency is simpler to test in vivo in both animals and humans. The advantages offered by the rectum for peptide and protein drug delivery have been discussed [ 1171. However, it is generally agreed that absorption enhancers are required to achieve therapeutic plasma levels of rectally dosed peptides. Many groups have looked at the bioavailability of a variety of peptides in the rat after rectal administration. Mikaye et al. [l 181 and Morimoto et al. [ 1191 detected low levels of absorption of eel calcitonin (MWt 3415) after rectal administration to rats. Only in the presence of an enhancer was a hypoglycaemic response detectable. Rectal absorption of desglycinamide arginine vasopressin (dGAVP, MWt 1100) has been investigated by van Hoogdalem et al. [ 1171. They achieved only negligible bioavailability with dGAVP alone, but with sodium tauro-24,25dihydrofusidate (STDHF), an absorption enhancer, they reported a bioavailability of 27-C6%. After rectal administration of insulin (MWt 5800), the same group demonstrate a bioavailability of 0.2?0.2%, increasing to 4.2?3.2% and 6.7-C3.2% following co-administration of different doses of STDHF [ 1201. The importance of absorption from the rectum into the lymphatic system has been investigated by Yoshikawa et al. [ 1211. They demonstrate negligible levels of human p-interferon (MWt 17 000) in serum and plasma after rectal administration. Co-administered mixed micelles achieved selective lymphatic uptake. These data indicate that rectal absorption of even large peptides is feasible. The generally low bioavailability by simple rectal absorption has resulted in more recent work concentrating on the optimisation of enhancement strategies. An extensive array of enhancers have been used to overcome the difficulties associated with poor and erratic availability after rectal administration with
some limited success [ 1221. Non-ionic surfactants [ 1231 and glycerol esters [ 1241 have been used to deliver therapeutic amounts of insulin and non-ionic surfactants in a polyacrylic acid gel [125,126]. Bile salts have been used to deliver interferon-a 11271 and insulin [ 128). However, given the damaging effects of bile salts on the mucosa [ 1291 and the possible carcinogenic effects of rectally administered have been debile salts [ 1301, safer enhancers veloped. Yoshikawa and co-workers [ 120.13 11 have demonstrated the successful enhancement of recombinant p-interferon using suppositories containing lipid-surfactant mixed micelles comprised of linoleic acid (0.5%) and HCO-60 (0.4%. a polyoxyethylene castor oil derivative). In addition to a signilicant increase in absorption. P-interferon entered the lymph circulation in greater proportion to the blood compartment. The same group have investigated the enhancing effects of another mixed micelle formulation, monoolein/sodium taurocholate, with dextran sulphate on bleomycin absorption [ 1321. Once again they demonstrated the preferential uptake into lymph and a significant degree of absorption enhancement. In anaesthetised rats, Miyake et al. [ 1 1X] showed that eel calcitonin (5 U kg ’ ) dosed rectally achieved a bioavailability of 0.6% relative to intramuscular (i.m.) administration. However, co-administration of an enhancer (PheEtAA) increased the area under the curve (AUC) by IXO-fold. The same group looked at the pharmacodynamic response to an equivalent dose. 5 U kg ‘, and could detect no decrease in plasma calcium. However. the presence of an enhancer, polyacrylic acid gel. increased absorption from the rectum, producing a significant hypocalcaemic response [ 119 1. For some forms of treatment, the rectal route could provide a more acceptable alternative to multiple injections. It is evident from the studies cited that peptide and protein delivery is strongly influenced by numerous formulation factors and does require the judicious use of enhancing agents. However. given that this mode of delivery shares many problems with the oral and nasal route. it is difficult to envisage widespread use of peptide and protein formulations suitable for rectal delivery. This is especially so within the US and UK markets where patient acceptability is considerably lower than in some other countries such as France and Italy.
.J.Z Advantuges deliver\!
of’ rectal peptide and protein drug
The advantages for rectal delivery summarised below:
of drugs are
transition from columnar to stratified epithelium allows rapid absorption of many low molecular weight drugs: middle and inferior rectal veins drain into the inferior vena cava, thus avoiding first pass metabolism; potential for absorption into the lymphatic system. due to large pore radii: retention of large volumes ( IO-25 ml); potential for time controlled release; constant environment aids reproducible absorption. Specitically, the advantages peptides are also summarised: l
l
l
for rectal delivery
of
low levels of protease activity, particularly of pancreatic origin; large surface area, potentiated by using spreading/ foaming agents: avoidance of first pass metabolism.
However. similar to the situation with the colon. the bioavailability of therapeutic peptides and proteins following rectal administration in the absence of absorption enhancers is low.
4. Human studies Only recently have there been attempts to examine the absorption of therapeutic peptides and proteins from the human colon in vivo. Much can be learned by considering the comparative results of different human colonic delivery studies with both the relevant animal data and the results of human rectal studies. Studies with calcitonin, a polypeptide hormone with measurable pharmacokinetics and pharof macodynamic effects, offer the opportunity rationalising the choice of the optimal region for absorption.
4.1. Culcitonin:
A cuse study
Human calcitonin is a 32 amino acid hormone produced by C-cells of the thyroid gland. It lowers blood calcium levels by inhibition of bone resorption and increasing urinary calcium excretion in animal models and in human diseases where the rate of bone turnover is high [ 1331. Calcitonin from several sources, for example human, salmon, porcine and an analogue from eel, are marketed in several countries. They have proved effective therapeutic agents used in the management of several disorders identified with accelerated bone resorption. These disorders include Paget’s disease [ 1341, hereditary bone dysplasia [ 1351 and post menopausal osteoporosis [ 136J. The major challenge associated with current therapy is that subcutaneous (s.c.) and i.m. injections were the only routes of administration until recently [ 1371. In addition, side effects (nausea and facial flushing) are common following injection of salmon and human calcitonins [138]. The efficacy of intranasal administration of human calcitonin and salmon calcitonin to healthy volunteers [ 139,140], patients with Paget’s disease [141-1451 and post menopausal osteoporosis [ 146- 1481 has been demonstrated. Moreover, intranasal human calcitonin was shown to be better tolerated than parenteral administrations [ 1451. Nasal salmon calcitonin is now marketed is several countries successfully. Nevertheless, oral dosage forms would still be preferred by patient and physician. 4. I. I. Colonic obsorptim Antonin et al. [7 I ] used the technique of colonoscopy to dose human calcitonin to the distal colon of volunteers in a cross-over bioavailability study. Human calcitonin plasma profiles obtained, after a 90 min iv. infusion, showed little variability between subjects with a rapid decay at the end of the infusion period. After colonic dosing, in which the dosed was flushed into the colon with 20 ml saline, the results divided into two distinct groups. Group A (5/8 subjects), in which plasma human calcitonin was detected, and Group B (3/8), in which no plasma human calcitonin was measurable. Group B reflected the subjects in whom the pre-dosing micro-enema failed to clear the distal colon of faecal material. In Group A the C,,,;,, of 8 10% 165 pg ml-’ induced by
colonically administered human calcitonin, at a dose of 10 mg, represented a bioavailability of 0.118-+0.028% compared to an i.v. infusion of 0.5 mg. Over all eight subjects the bioavailability was 0.076-+0.026%. Clearly, there is limited absorption of human calcitonin from the distal colon of conscious humans. However, the presence of faecal material, as to be expected at the point of release of a targeted oral colonic dosing formulation, appears to have a deleterious effect on absorption. The previous study used the relatively easily accessible descending colon for administration of a colonic dose. However, the colonic mucosa is known to decrease in absorptive capacity from ileal-caecal junction to anus. In addition, delivery of an oral dose is most likely to result in release in the proximal colon. Therefore, Antonin et al. [ 1491 used the opportunity offered by loop stoma patients of dosing human calcitonin directly to the transverse colon. The protocol was similar to the initial study, in which 10 mg human calcitonin was dosed directly to the target area and washed through with 20 ml saline. In a group of eight patients, a C,,,,, of 1242?346 pg ml-’ was achieved, giving a bioavailability of 0.22+-0.06%, compared to 0.12t0.03% (C,,,,, of 810t 165 pg ml-‘) shown by the responders dosed to the descending colon. This indicates that absorption may be favoured in the more proximal colon in vivo. More recently, Mackay et al. [150] have conducted further studies in humans to test hypotheses concerning the optimisation of colonic delivery of peptides. As already discussed, metabolism, both luminal and epithelial, is a major barrier to efficient absorption of orally administered peptides. Although the colon has been shown to be lower in peptidase activity than the proximal gastrointestinal tract, it retains traces of pancreatic enzymes. Mackay and co-workers [ 1501 have demonstrated in vitro that low concentrations of human calcitonin are rapidly degraded by human faecal material. At 37°C there was a dose dependent effect on the time taken for 50% degradation. This varied from 2.0-+0.1 min at 0.1 mg ml-’ to 25.9-tl.8 at 2.0 mg ml ‘. These values were extended by approximately 50-75% in the presence of 16 000 units of aprotinin, a protease inhibitor. The previously reported colonoscopy study was repeated in nine healthy volunteers. again after
an enema. The applied dose of 10 mg was flushed into the colon with either 5 ml of saline or 5 ml of aprotinin solution (20 000 units ml- ‘). In the absence of aprotinin a bioavailability of 0.22+-0.07% was achieved. Surprisingly, the presence of the aprotinin caused a decrease in bioavailability to 0.11?0.03%. Aprotinin delayed the T ,,,,,, from 5 min to IO min after dosing, but the C,,,,, was reduced three-fold, from 12082357 pg ml ’ to 4402 1 16 pg ml ‘. The difference in experimental design between this and the first study, i.e. a fourfold reduction in the volume used to flush the dose. resulted in a four-fold increase in the intracolonic concentration of human calcitonin achieved in the absence of aprotinin. This apparently resulted in an increase of 2.5 in bioavailability from 0.08+0.03% to 0.22t0.07%. It can be deduced that a high concentration of dose increases the level of absorption. The apparent inhibitory effect of aprotinin on the colonic absorption of human calcitonin is more difticult to explain. One suggestion is that the aprotinin may have caused precipitation of the dose from solution within the colonic lumen. It appears that although metabolism within the colonic lumen may restrict absorption of intact peptide, care must be taken in co-administration of compounds which may affect the intraluminal physicochemical environment of the dose. Interestingly, Geary and Schlameus [ IO1 1 looked at the effect of aprotinin on the absorption of porcine insulin in the presence of sodium glycocholate. an absorption enhancer from the in situ perfused colonic loop. They found that although the aprotinin produced an increase in the AUC for plasma insulin. from 28.6-+3.1 to 92.6-t 17.8, the reduction in glucose observed was identical in each group, 33%. In a separate study into the bioavailability of colonically administered human calcitonin [ 15 I] the dose was administered through a gastrointestinal tube into the unflushed lumen of the descending colon. In
addition to serum human calcitonin levels, serum calcium levels were analysed and urinary excretion of salts was monitored. In this study 0.5 mg human calcitonin was infused i.v. over 30 min and 10 mg was administered to the colon. In the group of six subjects, a bioavailability of 2.7% was reached in one volunteer, with much lower values from the other five, 0.034~0.015%. After i.v. dosing, a reduction in serum calcium levels and a stimulation in fractional urinary excretion of calcium, phosphorus, sodium and chloride was measured. Although after intracolonic administration no change in serum calcium was observed, a marginal increase in fractional urinary salt excretion was detected. Again, limited absorption of human calcitonin from the human colon in vivo has been demonstrated, which was not totally impaired by the presence of faecal material. More importantly there is an indication from the pharmacodynamic data that the peptide is ‘absorbed in a bioactive form. Table 5 shows comparative data from four human studies. A number of conditions have been defined for achieving the optimal absorption of human calcitonin from the human colon in vivo: l l l
high concentration; absence of faecal material; proximal regions of the colon.
Still the challenge remains to translate these interesting data into a dosage form that would benefit patients and allow easier administration of this therapeutically valuable peptide.
Due to its greater accessibility, more studies have been reported on the potential of the rectal route for the administration of systemically active peptides in vivo in humans. Most of the clinical studies on the rectal absorption of calcitonin have concentrated on
Table 5 Comparative data from sItdie\ of the in viva abaorptwn of human calcltonin tram human colon Author Antonin et al. [7
I]
Region of colon
Concentration (mg ml
’)
Bioavailability
Descending
0.4
0.08 +0.03
Antonin et al. [I491
Transverse
0.4
0.22rfrO.06
Mackay et al. [ I.501
Descending
1.7
0.2250.07
1.7
0.
0.4
(0.02~0.01
Beglinger
[ IS II
Dewding
I I -1-0.03 )
(o/r)
salmon calcitonin and eel calcitonin. Both salmon calcitonin and eel calcitonin have very slow clearance rates compared to human calcitonin because of their comparative resistance to metabolism. Buclin et al. [140] compared the efficiency of the nasal and rectal routes for the dosing of salmon calcitonin in human volunteers. Nine subjects received 200 IU salmon calcitonin as a suppository. The bioavailability achieved was 75% of the nasal value with similar biological responses recorded in both groups. These were transient hypocalcaemia, increased urinary salt excretion and decreased urinary hydroxyproline. Pagani et al. [ 1.521 looked at the hypocalcaemic effect of rectal salmon calcitonin compared to an i.m. dose. In a double crossover study in 10 healthy volunteers they measured a decrease in plasma calcium after 100 IU dosed rectally equivalent to that induced by SO IU i.m. In a more recent study, Fiore et al. [ 1531 compared both the pharmacokinetic profile and the biological response to eel calcitonin administered i.m. and rectally. They looked at the effects of a single dose and also of repeated administration. A single dose of 100 U administered rectally to 12 volunteers produced a C,,,;,, of 153.9&10.4 pg ml-’ compared to 177.956.5 pg ml ’ after an i.m. dose of 100 U. This represents a comparative bioavailability of just under SO%. Both doses induced a significant increase in plasma CAMP levels 30 and 60 min after dosing. Two groups of six subjects were dosed rectally daily with either placebo or 100 U eel calcitonin for 21 days. Significant increases in plasma CAMP levels were observed in the treated subjects over the 21 days. No changes were detectable in the placebo controls. Nuti et al. [ 1.541 also examined the biological responses to rectally administered eel calcitonin. Nine normal subjects and 11 with Paget’s disease received eel calcitonin rectally (100 or 200 IU). Both groups registered a greater response to the dose in terms of hypocalcaemia, which was higher in the group with Paget’s disease. Normal subjects showed a slight increase in plasma CAMP levels. The most significant study of the clinical relevance of the rectal route of administration of calcitonin has been carried out by Overgaard and co-workers [ 1551. They report a 2 year study involving 36 elderly women with moderate osteoporosis. The study in-
volved 1 year of observation, with no treatment, followed by 1 year of treatment comprising 100 IU salmon calcitonin dosed rectally daily and 500 mg calcium supplement. During the year’s observation a significant loss of bone (1.5%) was recorded in the forearm whilst spinal bone mass remained constant. Over the subsequent year of treatment, bone loss from the forearm was arrested and a significant increase (2%) of mass was observed in the spine. Parameters of bone turnover measured concomitantly showed no change during the first year, but declined significantly by lo-30% during the year of treatment. Tolerance was good. Eleven of the 36 subjects did not complete the study. Six dropped out because of symptoms associated with systemic calcitonin, four dropped out for reasons unrelated to the treatment and only two because of local reactions (changes in bowel pattern). In interpreting the relative results of systemic delivery of calcitonin by colonic and rectal routes the relative stability to metabolism of the rectal doses must be remembered. The last study demonstrates unequivocally the feasibility of the rectal route as a non-parenteral route for the long-term systemic dosing of a peptide or protein drug. It confirms the conditions for which this route is likely to offer the most realistic alternative to parenteral dosing: l l l l l
chronic treatment; moderate disease states; maximum dosing regimen of once daily; potential for reducing uncomfortable side effects; large population.
5. Impact of GI tract delivery peptides and proteins 5. I. Pharmacokinetic
of therapeutic
impuct of GI truct delivery
The administration of drugs as S.C. or i.m. formulations has a profound effect of the pharmacokinetics. Often enabling the active to be released for many hours and reducing the impact of first pass clearance. Standard formulations of drugs administered by the GI tract do not show these characteristics. Therefore, it is likely that it will not be a simple matter substituting one delivery route for the other when peptides are considered. It will be
necessary to consider the release characteristics ot the formulation in the GI tract, the impact this will have on the local metabolism in the lumen and the influence on the absorption. In the case of insulin the pharmacological profile could also be altered as a result of enhanced action on the liver as a consequence of initial drainage and distribution via the hepatic portal vein after delivery via the GI tract.
The impact of oral or rectal formulations of peptides is not simply and issue of patient compliance. Data on intestinal absorption of peptidea suggests that the absolute bioavailability is in the order of 1%. Even if therapeutically relevant formulations can be prepared the implication is that approximately two orders of magnitude more drug will have to be used in GI tract formulations compared with an in.jection. This could have serious repercussions in two areas. Firstly. it is likely that GI tract formulations will be considerably more expensive than current therapies because of the need for large quantities of active ingredient. Secondly, if availability of the active ingredient is limited then companies providing formulations for delivery by the GI tract could be reducing the pool of patients that could be treated if in.jectable formulations were used.
ib not in question and would be significant in terms of patient compliance. One can envisage that multidisciplinary approaches by scientists in academia and within the pharmaceutical industry will lead to targeting of peptide and protein drugs to specific regions of the GI tract, such as the colon, and lead to both better compliance and therefore improved therapies. Moreover, the selection of the peptide and the disease indication is critical. The major opportunity rests with peptides that will need to be self-administered by the patient for disease indications where there is little competition from traditional pharmaceutical agents. The success of nasal salmon calcitonin showed the value of non-injectable formulations opening up markets. Market forces will continue to drive the need for GI tract formulations. It should be remembered that dDAVP. a small peptide, is already available as an oral formulation in some countries. This may represent the bridgehead that will expand as more therapeutic peptides and protein are identified and GI tract research makes breakthrough discoveries enabling safe and efficient delivery after oral or rectal administration.
Acknowledgements Many thanks to Natalie Schneiders ration of the references.
6. Challenges of colonic and rectal delivery peptides and protein drugs
for the prepa-
of
Achieving therapeutically relevant Gl tract deli\)ery of peptides and proteins is still a major challenge. The current state of the art is limited such that it is important to optimise the opportunity by careful selection of peptide, mode of delivery. and the site of absorption. Conventional wisdom suggests that the colon offers a window of opportunity. Attempts to achieve therapeutic levels of peptide and protein drugs in the vascular compartment following intracolonic administration have mostly relied on absorption enhancers. The use of such enhancers for chronic administration of drug is doubtful given the epithelial damage that many enhancers have been shown to cause. However, the need for more acceptable methods to achieve effective peptide absorption
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tal administration
[ 12x1
Nakamoto,
synthetic
and treatment
aclmini\tration
Pharmuceuttcal
pel hasz
J. Phnrmacohiol.
[ 177)
01. msulin and msulin in rahhit\. Y.
Fisher,
IS.
Clink Pharmacol.
T.
esters of acetoacetrc
280--2X6.
Hama.
K. MoriTaka.
acid aqucoua
( 1980)
I.
M.T.
pp. 207-2
[13YJ A.E. Pontiroli,
\up
A. Knmada. N. Y&I.
of glvccrol ,
effects
physiology
and Paget’s Disease. Hans Huher,
rprav
T. Ni\hihata.
( 19x1 )
( I981 ) 269-278.
I. Maclntyre,
tn the treatment
McDermott,
B~func-
of bleomycln
( IYX.3) SY4&59S.
issue).
S. Kawamura.
absorption
Doyle,
867-X7
S.M.
J. Phar-
H. SeLaki.
(‘alcitonin
rffccts
31X.
[ 1241
M. Horuith,
of
transfer
H. Heath, Calcitomn.
( 1973)
N.
route, Int. J. Pharm. X9
N. Engl. J. Med. 304
u\c ot calcitonin.
in rat\. Pharm.
1I.181 .Ah\orptlon
C. Kato,
via enteral
F.H.
Y. Satoh, absorption
SY%62.
system for selective
Greenberg,
19
into lymphatics.
tional
delivery
ih
Kinet.
( 19x7 1377~3YO. [ I371
interferon
enteral
S. Muranishl.
development
I
suppoGtorlc5.
tihrohlast
delivery
delivery
C19X4)
Tissue
S. Muranishi,
H. Yoshikawa.
[13hJM.T. N.
interferon
M.
Dyn. 7
rat colorectum
Cell
potentiate
I t~ypel-pho~phatasarmia),
unfused
120.
1. Ohata.
macohiol.
,Med. Sh
J.C. Verhoet.
of rectallq
Satoh.
to
and selective
calcitonin
of human
Adc. Drug Deliv.
Ichrkawa.
Maeno,
of
H. Ryttlng.
administration.
I 17.31 K.
1’.
lipid via Iqmphotropic
Res. 3 (1986)
[ 1271
in rat\.
I X0-- I X3.
Takadn.
Takada.
disease of bone with
?cI babe on
&lJllgX\-f'eiJcll.
K.
method
interferon
I1311F?B.
Karnad~~.
on
metronidarole.
G.F. Joplin. J. Pennock, A.
hy sodium tauro-2-t.25.tiihydrofusidate
Yoshikaw:!.
A
physiology.
( I98517SY-760. C.D.
rats. Pharm. Kc\. 7
[ I21 ]
in
deoxycholate
by oral
‘9 I ~708. ( I.l.31h.. Austin,
A.
calcitonln
Moriaaha.
A.G. de Boer. D.D. Breimel-. Absorption insulin
intrnrectal
into lymphatics
rat\. J
1’. Kyohahl.
111;I polyacrylic
of [AU 37
ban Hoogdalrm.
\aop~-r\\~n
730&71S.
K.
of no1i-1omc surfactants
the rectal
[ I1101E.J.
of
33
I I.121
731-7-14.
Nagantr.
Naruse.
enhance-
in consciou
A.
absorption
rat\. Chem. Pharm
1I IY]
of
( 1986) 4x.5-4YO. 1I31 1 H. Yoshikawa,
K.A.A.
arginine
( 1989)
Nishlhata.
Rectal
effect
35
van Brec, J.C Verhorl.
and de&cinamide
T.
Ther.
Hei,jilieerh-FriJell,
J.B.M.M.
Exp. Ther. 2.5 I
Miyake,
and dy-
Pharmacol.
by sodium taoro-2-1.25.dihydrofusidate
( I IX]
36
J.A. van de Linde.
Nifedlpme:
sub.jectlr, Clin.
( 19X4) 742~739. 1I 171E.J. van Hoogdalem. Math8t.
Ther.
unaffected
D.D.
in
Pharmacol.
( 1092)
35-39.
M. Wouters.
N.
The effect of nasal hC1‘
disease of hone - implication\ bone diseases. Br. J.
[ 1461 R.D. Dal Negro, P. Turco, C. Pomari, F. Trevisan, Calcitonin nasal spray in patients with chronic asthma: A double-blind crossover study vs placebo, Int. J. Clin. Pharmacol. Ther. Toxicol. 29 (1991) 1444146. [ 1471 K. Overgaard, B.J. Riis, C. Christiansen. M.A. Hansen, Effect of salcatonin given intranasally on early postmenopausal bone loss, Br. Med. J. 299 ( 1989) 477-479. ] 1481 E. Vega, D. Gonzalez, G. Ghiringheli, C. Mautalen, Acute effect of the intranasal administration of salmon calcitonin in osteoporotic women, Bone Miner. 7 ( 1989)267-273. [I491 K.H. Antonm, R. Rak, P.R. Bieck, R. Preiss, U. Schenker. J. Hastwell, R. Fox. M. Mackay, The absorption of human calcitonin from the transverse colon of man, Int. J. Pharm. (submitted). [ISO] M. Mackay, K. Antonin, J. Hastewell. Colonic delivery of human calcitonin. Int. J. Pharm. (submitted). [ISI] C. Beglinger. W. Born, R. Muff, J. Drewe. J.L. Dreyfuss, A. Bock. M. Mackay, J.A. Fischer, Intracolonic bioavailability of human calcitonin in man, Eur. J. Pharmacol. 43 (1992) 527-53 I.
[IS21 G. Pagani, M.D. Pagani, D. Gianola. A. Pedroncelli, L. Cortesi, F. Gherardi, L. Gualteroni, F. Tentgattini, M. Montini. Hypocalcemic effects of rectal and intramuscular administration of synthetic salmon calcitonin, Int. J. Clin. Pharmacol. Ther. Toxicol. 29 (1991) 3299332. [ 1531 C.E. Fiore, S. Fiorito, R. Foti, M. Motta, C. Incognito, G. Grasso. Evaluation of the biological activity of unmodilied synthetic eel ct rectal capsules. Comparison with intramuscular administration and placebo, Int. J. Clin. Pharmacol. Res. X11 ( 1992) IV- 189. [IS41 R. Nuti. G. Martini, B. Frediani. S. Giovnnt, R. Valenti. A. Marcocci, A. Ciappi, Metabolic effects of rectal administration of unmodified eel calcitonin in humans, Drugs Exp. Clin. Res. XVIII ( 1992) 395-300. [ 1551 K. Overgaard, M.A. Hansen, K-L. Dirksen. C. Chriatian\en. Rectal salmon calcitonin for the treatment of postmenopausal osteoporosis, Calcif. Tissue Int. 51 (1992) 184-18X.
drug delivery reviews
ELSEVIER
Advanced
Drug Delivery
Reviews
28 (1997) 253-273
Peptide drug delivery: Colonic and rectal absorption Martin Mackay”‘“, Judy Phillipsb, John Hastewell” “CNS Research, “Drug Discovry, ‘Accel
Prwtnrrs.
Cihn
Pharmacruticuls.
Ciho
Bask
Phanwcruticals,
Embnrcndero
Cmtre.
CH-4002,
Horrharn
Switzerland
RH 12 4BT.
Son Frmcisco.
UK
CA 9411 I, USA
Abstract To date, a limited number of peptides and proteins have been used therapeutically. However, this number is growing rapidly. Some have been synthesised chemically or extracted from tissue and have been used in the clinic for decades. Recently, advances in cell and molecular biology have led to a much greater perception of the therapeutic value of many other peptides and proteins. Recombinant DNA technology has enabled high level expression and the biotechnology industry has made available large scale production for clinical use. In order to ensure that maximal use is made of this class of drug. scientists will need to devise patient compliant formulations. Clearly, these formulations will have to be safe and pharmacologically relevant. In addition, the route of administration will have to bc acceptable to the patient given any particular indication. Almost all therapeutic peptidex and proteins are administered by injection. It has been recognised that specific regions of the gastrointestinal tract may offer potential in terms of macromolecular absorption. This paper will address the attractiveness of the colon and rectum as routes 0 1997 Elsevier Science B.V. of administration for peptide and protein drugs. Keywords:
Drug absorption;
Drug delivery;
Therapeutic
peptidcs
and proteins
Contents I. Introduction _............................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... .__..._..., ,.,... 254 2. The colon . .._......................................................................................................................................................... .__..._........,. 254 ..................... ................. 2.54 2.1. Colonic environment.. ........................................................................................................... 2.2. Colonic epithelium.. ......................................................................................................................................................... 256 2.3. Delivery to the colon.. ...................................................................................................................................................... 257 .......................................................................... 2.4. &Ionic absorption.. ............................................................................ 258 2.4.1. Absorption studies.. ................................................................................................................................................ 25Y 2.4.2. Colonic delivery of insulin.. .................................................................................................................................... 261 ... .......................................................................... ‘2.4.3. Colonic delivery of calcitonin ................................................ 262 2.4.4. Advantages of colonic peptide and protein drug delivery.. ......................................................................................... 263 3. The rectum............................................................................................................................................................................. 263 263 3.1. Rectal absorption ............................................................................................................................................................ 3.2. Advantages of rectal peptide and protein drug delivery ....................................................................................................... 264 4. Human studies ........................................................................................................................................................................ 264 265 4.1. Calcitonin: A case study ............................................................................................................................................... 4. I. I. Colonic absorption ................................................................................................................................................. 2h5 1. I .2. Rectal absorption ................................................................................................................................................... 266 .................................................. 267 5. Impact of GI tract delivery of therapeutic peptides and protein\ .............................................. “:Corresponding
author. Pfizer Central Research,
Ol69-409X/97/$32.00 0 PI/ SOlh9.409X(97)00076-8
Sandwich.
Kent CTI 3 YNJ, England.
1997 Elsevier Science B.V. All rights reserved
Tel.:
+ 44 1304 8333: fax.
+ 44 I304 X2 18.
S. 1. Pharmacokinetic 5.2. Pharmacoeconomic 6. Challenges
of colonic
impact of GI tract deliver)
267
.._....
impact of GI tract delivery and rectal delivery
of peptides and protein drug>...
..,,.,.,..._.... .._................_...................,..........
26X 26X
Acknowledgements
26X
References
26X
1. Introduction The delivery of therapeutic peptides and proteins remains a priority for many pharmaceutical companies. In a recent review [I] of delivery strategies for peptides and proteins it was found that over 40% of all pharmaceutical companies were active in this area. Significantly, 27. representing 37%’ of the companies surveyed. are pursuing research programmes in the oral delivery of peptides and proteins. This has resulted from the advances in molecular biology and biotechnology, which between them have identified and made commercially available many peptides and proteins with valuable therapeutic properties [2,3]. It is recognised that biotechnology makes the discovery process for therapeutic peptides and proteins more efficient than traditional drug discovery. The success of therapeutic peptides and proteins is shown by the fact that three recombinant products are in the top IO best selling drugs list for 1993. Erythropoietin (for example, Am&en, Johnson and Johnson), human and animal insulin (for example, Lilly, Novo Nordisk) and o-interferon (for example. Schering-Plough. Sumitomo, Roche) achieved sales of $1806 million, $1610 million and $466 million. respectively. Other products such as growth hormone. B-interferon and granulocyte colony stimulating factor are expected to achieve increased sales over the next few years. In addition, many peptides and proteins are still in clinical development and the forecast is promising for agents such as bovine myehn protein. hirudin and transforming growth factor-B. Table I lists peptides and proteins. in clinical development or already marketed. However. the biopharmaceutical challenges that these agents present are huge. At present. the vast majority of therapeutic peptides and proteins are formulated for injection independent of the indication, the chronicity of dosing or the need for selfadministration. This severely limits the attractiveness of peptide and proteins as therapeutic agents in all
but the most serious indications or where there is no opportunity for alternative therapies. The need to find non-injectable routes of administration for peptides has focused attention on the oral route; the traditional method for patient compliant drug administration. However, the desire to achieve oral availability and the reality are somewhat apart. In considering the oral route of administration there are several factors that have to be addressed. These are illustrated in Table 2. This review will highlight peptide and protein absorption studies with two specitic areas of the gastrointestinal (GI) tract, namely the colon and rectum.
2. The colon 2. I. Colonic rnvironment The structure and function of the colon have been extensively reviewed 14-71. However, to date, their are few data on correlations between structure/function and drug transport across the colonic epithelium. Conventional wisdom suggests that many drugs are not absorbed from the colon or show little absorption. Nevertheless. studies have been reported where the absorption of some drugs across different regions of the GI tract is similar. Brockmeier et al. [Xl reported that the absorption of glibenclamide across the stomach, duodenum or colon was similar. Given that the environment of the colon is different to the stomach and small intestine in terms of proteolytic degradation the potential of the colon as a site for peptide and protein absorption has been studied. Cummings and co-workers [9] have shown that the colon in man contains few contents under normal conditions. They reported that the total amount of content in 46 adults was 222?21 g (wet) with only 93 g in the caecum and ascending regions. Moreover, other studies using cell culture models have assessed the barrier function of the colon. The development of
255
Table
I
Examples and application of peptides and proteins in clinical use or undergoing clinical trial Therapeutic peptide or protein
Application
Tissue necrosis factor
Carcinoma
Proleukin
Carcinoma
y-Interferon
Carcinoma
Epidermal growth factor
Wound healing
Transforming
Wound healing
growth factors
Fibroblast growth factor
Wound healing
Insulin-like
Wound healing
growth factors
Hirudin
Fibrinolytic
Tissue plasminogen activator
Fibrinolytic
Streptokinase
Fibrinolytic
Erythropoietin
Erythropoieais stimulation
Factor VIII
Haemophilia
Factor IX
Christmas disease
Triproamylin
Glucose regulation
Insulin
Glucose regulation
Somatostatm
Glucose regulation
Proinsulin
Glucose regulation
a-Interferon
Viral diseases/hairy cell leukemia
P-Interferon
Multlple sclerosis
Glucocerebrosidase
Gaucher’\
CereLyme
Type I Gaucher‘s direase
Pulmo7yme
Cystic fibrosis
disease
Calcitoninh
Bone disen\e
Oxytocin
Labour induction
Growth hormone
Short stature
cr.1 Antitrypsin (aat)
aat deticiency
Superoxide dismutase
Respiratory disorders
Table 2 Physical and physiological factors affecting the extent of peptide and protein absorption from the GI tract Physical factors
Physiological factors
Solubility
pH, regional and micro-climate
Dissolution rate
Binding and complexation
Stability
Proteases
Sire and conformation
Mucosal barrier
Partition coefticient
Intestinal permeability
Charge
Intestinal metabolism
Delivery
system
Hepatic metabolism
primary colonic epithelial cultures [ 10,l I], cell lines derived from colonic adenocarcinomas [ 12- 141, and cell lines derived from rectal adenocarcinomas [ 1S181 have given greater insight into the barrier function of the colonic epithelium. The microbiology of the colon is complex [1922]. Over 400 different species of aerobic and anaerobic bacteria reside in the colon. However, the anaerobic flora predominate [23]. The number of
anaerobic bacteria is approximately 10” per ml resulting in almost one-third of the dry weight of faeces in man [24]. Colonic bacteria secrete many enzymes usually involved in the fermentation of substrates for energy production. Some of these enzymes have been used as ‘triggers’ for colonspecific drug delivery (see later). In addition, it is believed that some residual pancreatic proteases are also present. To date, there are few data on the relative amounts of pancreatic enzymes in the small intestine and colon. The evidence that is available suggests that the amount is reduced in the colon. For example, Gibson et al. [25] highlighted the significance of microflora on proteolysis in the human colon. They showed that proteolytic activity was an order of magnitude greater in small intestinal fluid than in faeces. Interestingly, faecal proteolysis was qualitatively different from ileal proteolysis, as shown by the range of proteins hydrolysed and the susceptibility of the enzymes to some protease inhibitors, for example chymostatin.
Therefore, drug absorption in the colon is a consequence of the epithelial structure. Nevertheless, receptors for peptides and pl-oteins such as insulinlike growth factor I (IGF-I) 1261 and insulin 1271 exist on the apical membrane of proximal colon epithelial cells of the rabbit. It remains to be seen whether these receptors can be exploited IO transport peptide and protein drugs specifically. The general properties of the colon which may be of signiticance terms of peptide and protein absorption are: less luminal proteolytic activity compared with the small intestine: the epithelial cells of the mature organ do not express membrane associated peptidases: The luminal milieu has been shown to be suitable for prodrug metabolism as with salicylazosulphapyridine or offer an environment suitable fat drug delivery or site-specific drug release.
In the mammalian colon the epithelium contains absorptive. goblet. endocrine and a small number of Paneth cells. The absorptive cells possess microvilli. However, the colonic microvilli are much shorter than the microvilli of small intestinal absorptive cells. It is believed that the absorptive cells are the most important in terms of nutrient and drug transport. The monolayer of columnar epithelial cells is similar throughout the colon. However, towards the rectum there is a transition between this columnar epithelium to the keratinised stratified squamous epithelium of the anal skin. The anatomy of this area of transition. sometimes referred to as the anal transitional zone. is complex 1281. Goblet cells are present in the colonic epithelium in keeping with many other types of epithelia 1291. They represent the most abundant cell type in the colon and display marked differences in their degree of differentiation. Goblet cell secretions consist of water, mucin and various electrolytes [30]. The structure of colonic mucus and the physiology of colonic mucus secretion have been described by Allen (311 and Filipe 1321. Endocrine cells are less abundant in the colon than in the small intestine. They can occur singly or in clusters and are often found in the lamina propria.
Colonic endocrine cells are small, clear cells with a basal cytoplasm that contains electron-dense granules. They are most prominent deep in the crypts and methods have been developed to study endocrine cells in this location (33.341. Regulatory peptides $uch as enteroglucagon, pancreatic polypeptide, substance P. peptide YY and somatostatin are present in colonic endocrine cells 1351. These peptides have been shown to display a variety of functions which influence colonic secretion, motility and blood flow. Very few Paneth cells are found in the colon. They are found in the caecum and ascending colon. A wide variety of functions have been attributed to Pnneth ceils including secretion of digestive enr.ymes. production of trophic factors and elimination of heavy metals [Ml. They have abundant zinc-rich secretory granules which lysozyme, contain crlycoproteins and other proteins. They display irEgular microvilli and do not possess a terminal web. Due to their scarcity in the colon it is doubtful whether they play a major physiological function in this part of the Gl tract. The gut associated lytnphoid tissue (GALT) is unevenly distributed throughout the GI tract. The lymphoepithelial regions of the colon are known as lymphoglandular complexes (LGC). Little is known about the LGC. although their structure and composition bear a strong resemblance to sites in the small intestine associated with antigen sampling. In addition. it has been noted that LGC epithelium displays diminished mucin secretion 137). Colonic LGC demonstrates differences in size, cell numbers 1181 and regional variation in frequency 1.391. although the significance of these differences is not known. The lamina propria which delineates the basement membrane of the epithelial crypts and the muscularis mucosae contains many cell types including lymphocytes. plasma cells. macrophages, eosinophils, neutrophils and peripheral nerves. Lymphocytes and eosinophils are often observed between epithelial cells. The structure of the proximal colonic epithelium is well suited to one of its primary functions, namely the resorption of water. although it does not possess tht: curface area of the small intestine. In addition, the mucus which acts as a lubricant produced by the colonic goblet cells protects against abrasion from solid matter particularly in the distal colon and
M. Mackq
enables the excretion the tissue. 2.3. Delivery
et al. I Adanced
of faeces without
damage
Drug
to
to the colon
An excellent review of the specific delivery of drugs to the colon has been published recently [40] and Table 3 shows examples of the approaches. A novel timed-release dosage form has been developed by Scherer DDS. The ‘Pulsincap’ device can be modified such that its contents are released in the GI tract after a definite time period. Notwithstanding the vagaries of GI transit times 1511, it has been shown that the device can reproducibly release it’s payload in vitro and in the colon of man. The device consists of hydrogel components which when exposed to an aqueous environment causes swelling and release of a plug and subsequent release of drug. Application of an enteric coating prevents hydration in the gastric fluid. After stomach emptying the coat dissolves and hydrogel hydration occurs. The timing of the release is determined by the configuration of the hydrogel components [41]. This system is simple in construction and is produced from only pharmaceutically acceptable polymers. Devices have been produced which rely on bacterial enzyme breakdown to specifically release drug in the colon. Saffran developed a gelatin capsule coated with an impermeable polymer containing insulin or vasopressin [44]. The coating is resistant to degradation in the stomach and small intestine. The preparation is based on azo functional crosslinking agents. In a similar reaction to sulphasalazine the azoaromatic bonds (R-C,H,-N=N-C,H,-R) are reduced to form a pair of aromatic amines (R-
Delicry
Reviews 28 (1997)
257
2_%-27-J
C,H,-NH, + H,N-C,H,-R) by bacterial azoreductases. The capsule is broken down in the colon and released. With insulin, drug is specifically azopolymer coated capsules were given per OS to diabetic dogs and a hypoglycaemic response resulted. Blood glucose was reduced from 4 to In the 2.9 mg ml- ‘, 3 h after oral administration. case of vasopressin, azopolymer coated capsules were delivered to the stomach of an anaesthetised rat and a pharmacodynamic response in terms of antidiuresis determined. Maximum antidiuresis resulted 3 h following administration of the capsule. Both the insulin and vasopressin data were subject to a high degree of variability, probably due to the vagaries of coating the capsules. Other workers have attempted to devise more reproducible systems. Kopecek et al. [45] developed elegant hydrogel capsules, based on acrylic acid, N,N-dimethylacrylamide and N-tert-butylacrylamide crosslinked with 4,4’-di(methacryloylamino)azobenzene. These hydrogels do not swell significantly in the pH conditions of the stomach. In transit through the small intestine, swelling increases due to an increase in pH. In the colon. the degree of swelling exposes the cross-links to the bacterial azoreductases and this results in breakdown of the hydrogel and release of the drug. The capsules have been tested both in vitro and in vivo where they display a high degree of reproducibility. This group are also developing protease triggered systems. To date, few data have been published demonstrating the effectiveness of this type of system for colonic release of peptide or protein drugs and no studies exist in man. Davies and co-workers have patented a capsule for targeted delivery of peptides to the colon 1521. The
Table 3 Colon specific drug d&very
approaches
concept Timed-release Delayed-release
Examples delivery system delivery systems
Pulsincap [4 I ] Eudragit coated capsule\ [42] Cellulose acetate phthalate coating 1431 Azopolymer
coated capsules 1441
Hydrogels with azopolymer links [4S] Polysaccharide cross-linked capsules 1231 Sustained-release delivery systems
Ethyl cellulose membranes (461
Particulate-based delivery systems
Silica-drug preparations 1471
Prodrugs
Low MWt aro bond prodrug
(481
Polymeric aro bond prodrug
[49]
Glycoaidic bond prodrugs [XI]
capsule is coated with a 60- 150 pm thick layer of a commercially available anionic polymerpolyacrylic polymer. Eudragit S. The coating is insoluble in the stomach and small intestine below pH 7.0 but soluble in the colonic environment. Insulin, porcine calcitonin and human growth hormone have been encapsulated as formulations which also contain absorption enhancers, wetting agents and surfactants. The reproducibility of such a system for colonic delivery in man has not been demonstrated and the effects of the specific formulations used not determined. Touitou and Rubinstein [ 53 1 used a similar type of capsule to deliver porcine insulin. The formulation also contained an absorption enhancer. sodium laurate/cetyl alcohol, 2:s. A hypoglycaemic response was demonstrated in rats. although relative bioavailability was still low. pH-sensitive polymers have also been reported that rely on the belief that the proximal colon has a pH of 8.0-8.4. The polymer only dissolves at high pH. It has been shown, however, that high pH can be found in other regions of the GI tract rendering these polymers unreliable to specific colonic drug release 1541. The concept of specific drug release in the colon has been tested with low molecular weight drugs by making prodrugs. The prodrugs survive transit through the small intestine where they are not absorbed and whose active moiety is released by bacterial enzymes produced in the colon. The most notable example is sulphasalazine (salicylazosulphapyridine) which is not absorbed in the small intestine and is reduced to S-amino salicylic acid and sulphapyridine by azoreductdses produced by anaerobic bacteria in the colon 1481. In support of the mechanism of action. the cleavage of sulphasalazine is signiticantly reduced in patients receiving antibiotic treatment or that have had colonostomy. A colon-specitic drug-delivery system has been described based on the use of steroid glycosides. This system relies on glycosidasea produced by colonic bacteria [SO]. Steroid glycosides are poorly absorbed from the small intestine. However. colonic bacteria produce glycosidases which releases free steroids such as dexamethasone. The free steroids are then absorbed from the colon. To date. little evidence exists which suggests that a prodrug approach can be used for the delivery of therapeutic peptides and proteins. However, the exploitation of bacterial
enzymes to degrade delivery devices which potentially can contain peptides and proteins has been shown. It is apparent that specific release of peptide and protein drugs in the colon is possible. The possibility of polymeric delivery systems that degrade when exposed to enzymes produced by the colonic flora has been shown and awaits appropriate peptide formulation for clinical trials. Moreover, the development of timed release devices augurs well for achieving reproducible release of drugs such as therapeutic peptides and proteins. The challenge remains, however, to achieve adequate absorption across the epithelium following specific release.
The transport pathways of the colon provide for rapid and specific active bi-directional transport of ions across the epithelial layer. Unlike the small intestine, there are no documented active transporters for organic nutrients in the mature organ and, therefore, no chances for drug molecules to be absorbed in a piggy-back fashion. In the small intestine examples of this kind of active absorption of drugs are seen for 5-fluorouracil [.55.56] on the pyrimidine transporter and antibiotics on the peptide transporters [S7-591. The lack of such transporters limits the scope for drug design with respect to mediated transport across the epithelial barrier and. therefore, drug absorption at this site is a consequence of the general properties and features of the colon. The active transport pathways of the colon have been reviewed 171. The apparent lack of organic nutrient transporters may limit the potential for drug design with respect to carrier mediated transport across the colon. The following of which may be of significance: l
l
l
The transmucosal and membrane potential differences may be of signiticance in the absorption of ionised or ionisable drugs [60,61]. The colon is considered to be less of a barrier to macromolecules than the small intestine and thus may offer opportunities for both peptide and protein absorption. The bulk water absorption in this region of the intestine will provide scope for solvent drag and possibly improved drug absorption [62].
M. Muck+
et 01. I Advunced
Drug Delivery
The luminal milieu may be suitable for prodrug metabolism as with salicylazosulphapyridine [63,64] or offer an environment suitable for drug delivery [23] or site-specific drug release. Experiments using absorption promoters such as formulations Ca’+ chelators or lipid/detergent have proved that the colon is susceptible to enhancement strategies [65].
These factors may influence the choice of the colon as the region for drug absorption after oral administration. However, in the main they are not as a consequence of the physiological or biochemical properties of the colonic epithelia. Finally, any drug which is able to initiate or inhibit any of the intracellular cascades that regulate the absorptive versus the secretory response of the colon may not be good candidates for this site of absorption. The oral absorption of peptides and proteins has been reviewed [66] and the mechanisms of peptide and protein absorption have been documented in terms of paracellular intestinal transport [67], transcellular transport 1681 and endocytic pathways [69]. There are reports of peptides crossing the colonic mucosa in both animals 1701 and man 17I]. Moreover, this absorption can be enhanced by a series of agents such as salicylates 1721, water-oil-water emulsions 1731, surfactants [74], bile salts 17.51, combinative promotion effects of azone and fusogenic fatty acids 1761 and lipids [77,78] (for more details see Yamamoto et al. 1791). However, as shown earlier, to take advantage of these observations it is necessary for the drug to be delivered to the colon. The mode of delivery must either ensure that the peptide is protected from the digestive functions of the stomach and small intestine or circumvent them entirely by accessing the large intestine via the rectum (see below). Research into the large intestinal absorption of peptides has, therefore, been concerned with three main areas:
methodologies to deliver/release peptides in the large intestine after oral administration; rectal formulations to provide release in the distal colon; formulation and mechanistic studies to enable absorption across the colonic mucosa.
Reviews 28 (1997)
253-273
259
The aim of such research is to achieve therapeutically relevant peptide delivery. To illustrate these issues the genera1 absorption across the colon and the peptide hormones insulin and calcitonin will be considered with respect to the value of colonic delivery for therapeutic purposes. These latter peptides are particularly relevant as they have been in the vanguard of many peptide and protein drug delivery approaches and as such represent the challenge that peptides present to the pharmaceutical industry. 2.4.1. Absorption studies The GI absorption of drugs has been reviewed [80-841. It has been known for decades that peptides and proteins cross the GI epithelium, albeit in low amounts 185-891. These peptides and proteins include chymotrypsin, elastase, antigens [90,9 I ] horseradish peroxidase (HRP) [92], insulin [93] and Clostridium bofulinum type A toxin [94]. It is also apparent that more than one mechanism exists by which peptides enter the vascalature via the lumen of the GI tract and these include both passive and specific pathways. The question as to whether these physiological processes can be used for the colonic absorption of therapeutic peptides and proteins remains a challenge. It is accepted that these processes only enable the transport of low amounts of macromolecules and therefore would be insufficient to achieve therapeutic effects of most peptides or proteins. In addition, pharmacoeconomic issues would arise from delivering peptides and proteins with known poor bioavailability. Recently, human cell lines have been used as intestinal models to investigate the transport of a range of drugs. These cells have been grown in culture as monolayers on semi-permeable filters and shown to display many characteristics associated with differentiated intestinal epithelial cells. For example, an adenocarcinoma cell line. Caco-2, derived from human colon was used to study the transport of HRP [ 131. Only very low concentrations of apically applied HRP were seen in the basolateral chamber (0.0001%) demonstrating the tightness of the cell model barrier. Using the same cell line. the absorption profiles of the vasopressin analogues, arginine-vasopressin (AVP) and 1-deamino-8-D-arginine-vasopressin dDAVP 19.5], were determined. Both these hydrophilic peptides displayed linear
transport kinetics and were absorbed at very slow rates by a non-saturable, non-polar process across the Caco-2 monolayers. Neither peptide showed degradation after a 4 h incubation in the apical medium ot the cells. The use of cell models such as the Caco-2 line to study peptide and protein transport is at an early stage but results to date indicate their potential as a tool to discover new pathways and novel formulations. Other in vitro methods have been used to assess the importance of the epithelial layer as a physical barrier to peptide absorption. Isolated intestinal sheets have been used. In two recently reported studies. Quadros et al. [96,97] demonstrated the permeability of colonic sheets from different species to CGS 16617, a novel ACE inhibitor. and insulinlike Growth Factor 1 (IGF-I ). CGS 16617, a trihas a permeability coefticient of peptide, I. 12-CO.09 cm s ’ X IO (’ across rat colon sheets. compared to that of 8.6252.1 1 cm s ’ X IO ’ foi IGF- 1, 70 amino acid residues. As expected. molecular volume has a signiticant effect on transepithelial permeability. To date, only a few studies have been reported where a peptide or protein has been administered intracolonically in the absence of protease inhibitors or absorption enhancers. Lundin and Vilhardt 1981 determined the absorption of dDAVP from different regions of the GI tract of rats. The regions were stomach, duodenum. mid-part of the ileum. the ileocaecal junction and the mid-part of the colon. The absorption of intact dDAVP occurred from each site and was rapid with peak plasma levels after I o20 min. Less absorption was observed across the stomach and colon. The authors suggested that the
low surface area associated with these regions compared to the small intestine was the reason for less absorption. Langhuth et al. 1991 compared metabolism of metkephamide. a synthetic pentapeptide resistant to peptidase activity, by the brush border of the enterocytes in different regions of the rat GI tract using a single pass perfusion of isolated sections, in situ. They reported maximum degradation in the jejunum. decreasing down the intestine to no detectable metabolism in the colon. Values reported for metabolism per cm intestine are: duodenum 14.9%3.050/o, proximal 8.14-t2.32%, jejunum middle jejunum 3.08-+0.51%, ileum 1.72?0.31% and colon - not detectable. Metabolic barriers to peptide absorption and methods of overcoming them have been reviewed in detail by Zhou [ IOO]. A number of groups have compared absorption from different regions of the GI tract of rats in vivo using the in situ perfused loop method. The results are summarised in Table 4. Each group assessed absorption by a different method. Table 4 shows that the authors have looked at the absorption of peptides ranging in molecular weight from 661 to 6000. The ileal epithelium is shown to be slightly more permeable to the peptides in this model, confirming evidence from in vitro models that the permeability of the intestinal epithelium increases down the GI tract. In these studies, the differences between regions disappear in the presence of intestinal contents, suggesting that in the colon the lower luminal levels of metabolism compensate for its decreased permeability compared to the ileum. It is clear that the few experimental data that exist
Table 4 Comparative absorption of peptides from different regions of the GI tract 111rat5
[ 1011
[ 102 I
Author:
Geary and Schlameus
Langhuth et al. [Y91
Morishita et al.
Peptide:
Vancomycin
Metkephamide
Insulin (porcine)
MWr:
I so0
661
6000
Wall permeability
Efficacy (J glucose)
Bioabnilability
Duodenum
(‘k)
Not detectable
Jejunum
+ wash
~ wash
0. I
0
I .641-0.31
0
0
Ileum
3.5-tl.l
(xi)
I .c)Ito.21
0.3
0.I
Colon
3.1+0.9
(xl)
0.67ZO.38
0. I
0. I
M. Mucka! et cd. I Advanced Drug Drli~vt:v Rersiews 2X (1997) 2.5%27.7
show that therapeutic peptides and proteins are poorly absorbed from the GI tract. Although the colon may be a preferable site for peptide delivery because of the relative lack of proteases, this fact alone does not lead to effective bioavailability. As a result, various strategies have been applied to elevate absorption levels and include: protection against presystemic degradation; chemical derivatisation of the drug (prodrugs); and co-administration of socalled ‘absorption enhancers’. In a few instances these have been successfully used to promote the absorption of peptides and proteins. 2.4.2. Colorzic delivery of insulin The discovery of insulin rapidly led to its clinical application for treating insulin dependent diabetes melitus (IDDM). The therapeutic impact of insulin on the treatment of IDDM is dramatic. Patients suffering from this disease are able to control their blood glucose to a point where they can lead normal lives. The need to deliver the drug by injection is acceptable when compared with the consequences of not taking insulin. Current therapy consists of once or twice daily injections of insulin, including mixed intermediate or rapid-acting insulins [103]. Even so it is recognised that this therapy is not a complete solution as, for example, with aging, a variety of conditions become prevalent in diabetic patients. The most common of these are retinopathy, nephropathy, neuropathy and cardiovascular disease [ 1041. These conditions are a consequence of incomplete control of blood glucose levels leading to long-term adverse side effects I10.5,106]. Recent clinical studies have shown that careful monitoring of blood glucose coupled with intensive insulin administration can lead to a significant reduction in these side effects [ 1031. To be successful oral insulin at the very least must provide therapeutic equivalency to the current therapies but ideally would provide the ability to tightly control glucose levels. To achieve this, control of insulin administration is key to both achieving the correct acute hypoglycaemic response and reducing the chronic morbidity associated with the disease. The large intestine can be reached either by oral or rectal administration. The former requires that the formulation or device protects the peptide from the proteolytic conditions found in the small intestine,
261
specifically the stomach, duodenum. jejunum and ileum. There are many approaches to this, however the element constant to all these is the time taken to achieve passage to the large intestine and the factors influencing this passage. Whilst passage along the small intestine is considered to be relatively constant at 3-5 h under most conditions 11071. Retention in the stomach can delay the passage by several hours dependent upon stomach contents. Therefore, it is extremely difficult to predict when an orally administered dose of insulin would be delivered to the colon for absorption. The consequence of this is that it would be virtually impossible for the oral dosing of insulin to be co-ordinated with ingestion of meals such that the systemic insulin levels corresponded to the post-prandial glucose peak. This lack of control in the timing of the insulin after oral administration will rarely achieve the fine control for optimal therapy. The best that could be hopecl for is a sustained delivery that could achieve similar levels of control to current once a day parenteral formulations. However, timing the ingestion of the dose to provide day to day control of glucose levels will be a challenge. In this case rectal delivery offers the greatest opportunity for delivery via the large intestine. However, this is still non-trivial. In the case of insulin the dose delivered must be reproducible and quantifiable. Atchison et al. [ 1081 studied the colonic absorption of radiolabelled insulin using non-everted sacs of rat colon. The percentage of intraluminal insulin degradation and the transport of insulin into the surrounding media was determined. They showed that transepithelial flux of insulin was consistently less than 0.3% of the dose. In addition, significant degradation of insulin (64%) was found within 15 min of exposure. The effects of intracolonically administered human insulin, and human insulin-DEAE-dextran complex entrapped in liposomes, on blood glucose in rats has been reported [ 1091. In both cases, a hypoglycaemic response was noted which lasted for several hours. No indication of relative bioavailability was given and it can only be presumed that insulin was transported across the colon. Given the difficulty in predicting when an insulin dose would reach the colon and be transported across the epithelium it is difficult to imagine an oral dosage form reaching the market soon. Nevertheless,
activity in this tield is enormous. More practical approaches to oral delivery may result in a diabetic patient taking fewer injections and thus lead to an improved quality of life rather than complete respite from the needle.
The rationale for -considering oral delivery of peptides for therapeutic purposes is based on patient compliance. In the case of diabetes. the disease characteristics and the consequences of not taking insulin force the patient into accepting the injectable formulations. In contrast to this, peptides indicated for prophylactic treatment of a non-terminal disease are poorly tolerated by the patients because of the need for injections to dose the drug. This is well illustrated by calcitonin. Historically, calcitonin has had a relatively narrow usage in Paget’s disease and control of hypercalceamia associated with cancers. both treated with injectable formulations. Although it has been recognized for many years that it i4 effective in retarding the progress of osteoporosis. current formulations limit the use of calcitonin in this indication. In responding to market needs many delivery routes have been considered. Nasal delivery has had some success showing therapeutically signiticant endpoints whilst avoiding injectable administration. However. it is considered that ultimately an orally administered formulation will be the optimal solution. The therapeutic regimen for calcitonin in treating osteoporosis differs markedly from the treatment of IDDM with insulin. Calcitonin has a large therapeutic window. In the treatment of osteoporosis. calcitonin is influencing the outcome of a long onset disease by down-regulating osteoclast activity. There is no requirement for precise dosing either regarding the amount absorbed or the timing of the administration. The requirement is that a therapeutic dose is delivered in the order of once a day. This therapeutic profile greatly simplifies the dosing requirements and makes calcitonin a particularly attractive peptide fog GI tract delivery. Hastewell et al. 1701 used direct administration of human calcitonin into a colonic loop in anaesthetised rats to examine the bioavailability of human calcitonin. This was compared to the pharmacodynamic effect. detectable in normal juvenile animals, of a reduction in plasma calcium levels in response to
human calcitonin. They demonstrated the bioavailability achieved after intracolonic dosing of three different doses compared to an i.v. dose. These were: 0.5% at 5.0 mg kg ’ , 0.9% at I .O mg kg- ’ and 0.2% at 0. I mg kg ‘. In addition, intracolonically administered human calcitonin at doses of 0. I-5.0 mg kg -’ resulted in a dose-dependent reduction in plasma calcium levels. These doses achieved reductions in plasma calcium levels of 12?6 to 382.5%. The reference i.v. dose of 1.25 kg kg ’ achieved a calcium reduction of 2924%. Moreover. immunohistochemistry showed that human calcitonin transport across the rat colon was rapid and a significant amount was via a transcellular pathway. In a subsequent report, Hastewell et al. 1781 studied the influence of eyuimolar monoolein/sodium taurocholate enhancer formulations on the absorption of human calcitonin and two markers of intestinal permeability, HRP and polyethylene glyco], MWt 4000 (PEG 4000). Human calcitonin, HRP and PEG 4000 were all absorbed across the colonic mucosa to a limited extent. The use of a 40 mM monoolein/40 mM sodium taurocholate mixed micellar formulation significantly ( p < 0.001) enhanced (9.0t I.O-fold) the absorption of all three molecules with no acute damage to the mucosal tissue as judged after light microscopy. At concentrations of 20 mM and below, the monooleini sodium taurocholate formulation did not enhance the absorption of human calcitonin. HRP or PEG 4000. HRP immunohistochemistry showed an intracellular localisation suggesting that a transcellular pathway was involved in absorption across the epithelium. The increased absorption of human calcitonin in the presence of the 40 mM enhancer formulation was able to elicit a maximal hypocalcaemic response. whereas no signiticant effect was observed in the absence of the enhancer. The authors concluded that the absorption enhancer used in this study can increase intestinal absorption of a range of molecules without causing major tissue damage, albeit after acute treatment. These formulations may offer advantages as they enable pharmacodynamic responses to be elicited from reduced doses of therapeutic peptides and proteins. It is important to understand the relevance of these results to the potential pharmacokinetic and pharmacodynamic effects after dosing to the unligated colon of conscious man (see below).
Fukunaga et al. [l lo] showed that liposomally entrapped salmon calcitonin produced a hypocalcaemic effect in rats when dosed orally, but did not determine the mechanism or intestinal location of the absorption. 2.4.4. Advantages of colonic peptide and protein drug deliver? The advantages of colonic peptide delivery can be summarised as follows: l l l l
l
l
low metabolic activity; longer residence time; responsive to absorption enhancers; colonic bacterial enzymes present may offer targeting opportunities; the transmucosal and membrane potential differences may be of significance in the absorption of ionised or ionisable drugs; the bulk water absorption in this region of the intestine may provide scope for solvent drag.
Nevertheless, despite the colon’s apparent attractiveness as a route for the oral delivery of peptides, only very limited bioavailability can be demonstrated for this route in vivo. In addition, delivery systems to achieve oral delivery to the colon remain to be resolved.
3. The rectum 3.1. Rectul chorption The rectal route has been used to deliver many drugs and offers similar advantages over oral delivery to those enjoyed by the nasal route [ 11 l-l 131. It can be an extremely useful route for delivery of drugs to babies and young children where difficulties can arise using per OS administration. Similar disadvantages are also evident, for example limited surface area and dissolution problems. Interruption of drug absorption during defecation is another concern and patient acceptability is variable. However, the rectal route does offer a more convenient way to control drug release using osmotic pumps and hydrogel cylinders, although to date they have only been tested with low molecular weight drugs such as propranolol [ 1141 and nifedipine [ 1 I5,l 161.
Historically, the rectum has been an accepted site of drug delivery. Its principal applications have been for local therapy, e.g. haemorrhoids, and for systemic delivery of drugs to groups presenting practical problems for parenteral or oral dosing, for example infants, epileptics. Some populations are less willing to accept this route as a standard method for drug delivery, but it is the most easily accessible area of the lower GI tract. As such it offers the potential for delivery of peptides without the need for targeted devices, and it’s efficiency is simpler to test in vivo in both animals and humans. The advantages offered by the rectum for peptide and protein drug delivery have been discussed [ 1171. However, it is generally agreed that absorption enhancers are required to achieve therapeutic plasma levels of rectally dosed peptides. Many groups have looked at the bioavailability of a variety of peptides in the rat after rectal administration. Mikaye et al. [l 181 and Morimoto et al. [ 1191 detected low levels of absorption of eel calcitonin (MWt 3415) after rectal administration to rats. Only in the presence of an enhancer was a hypoglycaemic response detectable. Rectal absorption of desglycinamide arginine vasopressin (dGAVP, MWt 1100) has been investigated by van Hoogdalem et al. [ 1171. They achieved only negligible bioavailability with dGAVP alone, but with sodium tauro-24,25dihydrofusidate (STDHF), an absorption enhancer, they reported a bioavailability of 27-C6%. After rectal administration of insulin (MWt 5800), the same group demonstrate a bioavailability of 0.2?0.2%, increasing to 4.2?3.2% and 6.7-C3.2% following co-administration of different doses of STDHF [ 1201. The importance of absorption from the rectum into the lymphatic system has been investigated by Yoshikawa et al. [ 1211. They demonstrate negligible levels of human p-interferon (MWt 17 000) in serum and plasma after rectal administration. Co-administered mixed micelles achieved selective lymphatic uptake. These data indicate that rectal absorption of even large peptides is feasible. The generally low bioavailability by simple rectal absorption has resulted in more recent work concentrating on the optimisation of enhancement strategies. An extensive array of enhancers have been used to overcome the difficulties associated with poor and erratic availability after rectal administration with
some limited success [ 1221. Non-ionic surfactants [ 1231 and glycerol esters [ 1241 have been used to deliver therapeutic amounts of insulin and non-ionic surfactants in a polyacrylic acid gel [125,126]. Bile salts have been used to deliver interferon-a 11271 and insulin [ 128). However, given the damaging effects of bile salts on the mucosa [ 1291 and the possible carcinogenic effects of rectally administered have been debile salts [ 1301, safer enhancers veloped. Yoshikawa and co-workers [ 120.13 11 have demonstrated the successful enhancement of recombinant p-interferon using suppositories containing lipid-surfactant mixed micelles comprised of linoleic acid (0.5%) and HCO-60 (0.4%. a polyoxyethylene castor oil derivative). In addition to a signilicant increase in absorption. P-interferon entered the lymph circulation in greater proportion to the blood compartment. The same group have investigated the enhancing effects of another mixed micelle formulation, monoolein/sodium taurocholate, with dextran sulphate on bleomycin absorption [ 1321. Once again they demonstrated the preferential uptake into lymph and a significant degree of absorption enhancement. In anaesthetised rats, Miyake et al. [ 1 1X] showed that eel calcitonin (5 U kg ’ ) dosed rectally achieved a bioavailability of 0.6% relative to intramuscular (i.m.) administration. However, co-administration of an enhancer (PheEtAA) increased the area under the curve (AUC) by IXO-fold. The same group looked at the pharmacodynamic response to an equivalent dose. 5 U kg ‘, and could detect no decrease in plasma calcium. However. the presence of an enhancer, polyacrylic acid gel. increased absorption from the rectum, producing a significant hypocalcaemic response [ 119 1. For some forms of treatment, the rectal route could provide a more acceptable alternative to multiple injections. It is evident from the studies cited that peptide and protein delivery is strongly influenced by numerous formulation factors and does require the judicious use of enhancing agents. However. given that this mode of delivery shares many problems with the oral and nasal route. it is difficult to envisage widespread use of peptide and protein formulations suitable for rectal delivery. This is especially so within the US and UK markets where patient acceptability is considerably lower than in some other countries such as France and Italy.
.J.Z Advantuges deliver\!
of’ rectal peptide and protein drug
The advantages for rectal delivery summarised below:
of drugs are
transition from columnar to stratified epithelium allows rapid absorption of many low molecular weight drugs: middle and inferior rectal veins drain into the inferior vena cava, thus avoiding first pass metabolism; potential for absorption into the lymphatic system. due to large pore radii: retention of large volumes ( IO-25 ml); potential for time controlled release; constant environment aids reproducible absorption. Specitically, the advantages peptides are also summarised: l
l
l
for rectal delivery
of
low levels of protease activity, particularly of pancreatic origin; large surface area, potentiated by using spreading/ foaming agents: avoidance of first pass metabolism.
However. similar to the situation with the colon. the bioavailability of therapeutic peptides and proteins following rectal administration in the absence of absorption enhancers is low.
4. Human studies Only recently have there been attempts to examine the absorption of therapeutic peptides and proteins from the human colon in vivo. Much can be learned by considering the comparative results of different human colonic delivery studies with both the relevant animal data and the results of human rectal studies. Studies with calcitonin, a polypeptide hormone with measurable pharmacokinetics and pharof macodynamic effects, offer the opportunity rationalising the choice of the optimal region for absorption.
4.1. Culcitonin:
A cuse study
Human calcitonin is a 32 amino acid hormone produced by C-cells of the thyroid gland. It lowers blood calcium levels by inhibition of bone resorption and increasing urinary calcium excretion in animal models and in human diseases where the rate of bone turnover is high [ 1331. Calcitonin from several sources, for example human, salmon, porcine and an analogue from eel, are marketed in several countries. They have proved effective therapeutic agents used in the management of several disorders identified with accelerated bone resorption. These disorders include Paget’s disease [ 1341, hereditary bone dysplasia [ 1351 and post menopausal osteoporosis [ 136J. The major challenge associated with current therapy is that subcutaneous (s.c.) and i.m. injections were the only routes of administration until recently [ 1371. In addition, side effects (nausea and facial flushing) are common following injection of salmon and human calcitonins [138]. The efficacy of intranasal administration of human calcitonin and salmon calcitonin to healthy volunteers [ 139,140], patients with Paget’s disease [141-1451 and post menopausal osteoporosis [ 146- 1481 has been demonstrated. Moreover, intranasal human calcitonin was shown to be better tolerated than parenteral administrations [ 1451. Nasal salmon calcitonin is now marketed is several countries successfully. Nevertheless, oral dosage forms would still be preferred by patient and physician. 4. I. I. Colonic obsorptim Antonin et al. [7 I ] used the technique of colonoscopy to dose human calcitonin to the distal colon of volunteers in a cross-over bioavailability study. Human calcitonin plasma profiles obtained, after a 90 min iv. infusion, showed little variability between subjects with a rapid decay at the end of the infusion period. After colonic dosing, in which the dosed was flushed into the colon with 20 ml saline, the results divided into two distinct groups. Group A (5/8 subjects), in which plasma human calcitonin was detected, and Group B (3/8), in which no plasma human calcitonin was measurable. Group B reflected the subjects in whom the pre-dosing micro-enema failed to clear the distal colon of faecal material. In Group A the C,,,;,, of 8 10% 165 pg ml-’ induced by
colonically administered human calcitonin, at a dose of 10 mg, represented a bioavailability of 0.118-+0.028% compared to an i.v. infusion of 0.5 mg. Over all eight subjects the bioavailability was 0.076-+0.026%. Clearly, there is limited absorption of human calcitonin from the distal colon of conscious humans. However, the presence of faecal material, as to be expected at the point of release of a targeted oral colonic dosing formulation, appears to have a deleterious effect on absorption. The previous study used the relatively easily accessible descending colon for administration of a colonic dose. However, the colonic mucosa is known to decrease in absorptive capacity from ileal-caecal junction to anus. In addition, delivery of an oral dose is most likely to result in release in the proximal colon. Therefore, Antonin et al. [ 1491 used the opportunity offered by loop stoma patients of dosing human calcitonin directly to the transverse colon. The protocol was similar to the initial study, in which 10 mg human calcitonin was dosed directly to the target area and washed through with 20 ml saline. In a group of eight patients, a C,,,,, of 1242?346 pg ml-’ was achieved, giving a bioavailability of 0.22+-0.06%, compared to 0.12t0.03% (C,,,,, of 810t 165 pg ml-‘) shown by the responders dosed to the descending colon. This indicates that absorption may be favoured in the more proximal colon in vivo. More recently, Mackay et al. [150] have conducted further studies in humans to test hypotheses concerning the optimisation of colonic delivery of peptides. As already discussed, metabolism, both luminal and epithelial, is a major barrier to efficient absorption of orally administered peptides. Although the colon has been shown to be lower in peptidase activity than the proximal gastrointestinal tract, it retains traces of pancreatic enzymes. Mackay and co-workers [ 1501 have demonstrated in vitro that low concentrations of human calcitonin are rapidly degraded by human faecal material. At 37°C there was a dose dependent effect on the time taken for 50% degradation. This varied from 2.0-+0.1 min at 0.1 mg ml-’ to 25.9-tl.8 at 2.0 mg ml ‘. These values were extended by approximately 50-75% in the presence of 16 000 units of aprotinin, a protease inhibitor. The previously reported colonoscopy study was repeated in nine healthy volunteers. again after
an enema. The applied dose of 10 mg was flushed into the colon with either 5 ml of saline or 5 ml of aprotinin solution (20 000 units ml- ‘). In the absence of aprotinin a bioavailability of 0.22+-0.07% was achieved. Surprisingly, the presence of the aprotinin caused a decrease in bioavailability to 0.11?0.03%. Aprotinin delayed the T ,,,,,, from 5 min to IO min after dosing, but the C,,,,, was reduced three-fold, from 12082357 pg ml ’ to 4402 1 16 pg ml ‘. The difference in experimental design between this and the first study, i.e. a fourfold reduction in the volume used to flush the dose. resulted in a four-fold increase in the intracolonic concentration of human calcitonin achieved in the absence of aprotinin. This apparently resulted in an increase of 2.5 in bioavailability from 0.08+0.03% to 0.22t0.07%. It can be deduced that a high concentration of dose increases the level of absorption. The apparent inhibitory effect of aprotinin on the colonic absorption of human calcitonin is more difticult to explain. One suggestion is that the aprotinin may have caused precipitation of the dose from solution within the colonic lumen. It appears that although metabolism within the colonic lumen may restrict absorption of intact peptide, care must be taken in co-administration of compounds which may affect the intraluminal physicochemical environment of the dose. Interestingly, Geary and Schlameus [ IO1 1 looked at the effect of aprotinin on the absorption of porcine insulin in the presence of sodium glycocholate. an absorption enhancer from the in situ perfused colonic loop. They found that although the aprotinin produced an increase in the AUC for plasma insulin. from 28.6-+3.1 to 92.6-t 17.8, the reduction in glucose observed was identical in each group, 33%. In a separate study into the bioavailability of colonically administered human calcitonin [ 15 I] the dose was administered through a gastrointestinal tube into the unflushed lumen of the descending colon. In
addition to serum human calcitonin levels, serum calcium levels were analysed and urinary excretion of salts was monitored. In this study 0.5 mg human calcitonin was infused i.v. over 30 min and 10 mg was administered to the colon. In the group of six subjects, a bioavailability of 2.7% was reached in one volunteer, with much lower values from the other five, 0.034~0.015%. After i.v. dosing, a reduction in serum calcium levels and a stimulation in fractional urinary excretion of calcium, phosphorus, sodium and chloride was measured. Although after intracolonic administration no change in serum calcium was observed, a marginal increase in fractional urinary salt excretion was detected. Again, limited absorption of human calcitonin from the human colon in vivo has been demonstrated, which was not totally impaired by the presence of faecal material. More importantly there is an indication from the pharmacodynamic data that the peptide is ‘absorbed in a bioactive form. Table 5 shows comparative data from four human studies. A number of conditions have been defined for achieving the optimal absorption of human calcitonin from the human colon in vivo: l l l
high concentration; absence of faecal material; proximal regions of the colon.
Still the challenge remains to translate these interesting data into a dosage form that would benefit patients and allow easier administration of this therapeutically valuable peptide.
Due to its greater accessibility, more studies have been reported on the potential of the rectal route for the administration of systemically active peptides in vivo in humans. Most of the clinical studies on the rectal absorption of calcitonin have concentrated on
Table 5 Comparative data from sItdie\ of the in viva abaorptwn of human calcltonin tram human colon Author Antonin et al. [7
I]
Region of colon
Concentration (mg ml
’)
Bioavailability
Descending
0.4
0.08 +0.03
Antonin et al. [I491
Transverse
0.4
0.22rfrO.06
Mackay et al. [ I.501
Descending
1.7
0.2250.07
1.7
0.
0.4
(0.02~0.01
Beglinger
[ IS II
Dewding
I I -1-0.03 )
(o/r)
salmon calcitonin and eel calcitonin. Both salmon calcitonin and eel calcitonin have very slow clearance rates compared to human calcitonin because of their comparative resistance to metabolism. Buclin et al. [140] compared the efficiency of the nasal and rectal routes for the dosing of salmon calcitonin in human volunteers. Nine subjects received 200 IU salmon calcitonin as a suppository. The bioavailability achieved was 75% of the nasal value with similar biological responses recorded in both groups. These were transient hypocalcaemia, increased urinary salt excretion and decreased urinary hydroxyproline. Pagani et al. [ 1.521 looked at the hypocalcaemic effect of rectal salmon calcitonin compared to an i.m. dose. In a double crossover study in 10 healthy volunteers they measured a decrease in plasma calcium after 100 IU dosed rectally equivalent to that induced by SO IU i.m. In a more recent study, Fiore et al. [ 1531 compared both the pharmacokinetic profile and the biological response to eel calcitonin administered i.m. and rectally. They looked at the effects of a single dose and also of repeated administration. A single dose of 100 U administered rectally to 12 volunteers produced a C,,,;,, of 153.9&10.4 pg ml-’ compared to 177.956.5 pg ml ’ after an i.m. dose of 100 U. This represents a comparative bioavailability of just under SO%. Both doses induced a significant increase in plasma CAMP levels 30 and 60 min after dosing. Two groups of six subjects were dosed rectally daily with either placebo or 100 U eel calcitonin for 21 days. Significant increases in plasma CAMP levels were observed in the treated subjects over the 21 days. No changes were detectable in the placebo controls. Nuti et al. [ 1.541 also examined the biological responses to rectally administered eel calcitonin. Nine normal subjects and 11 with Paget’s disease received eel calcitonin rectally (100 or 200 IU). Both groups registered a greater response to the dose in terms of hypocalcaemia, which was higher in the group with Paget’s disease. Normal subjects showed a slight increase in plasma CAMP levels. The most significant study of the clinical relevance of the rectal route of administration of calcitonin has been carried out by Overgaard and co-workers [ 1551. They report a 2 year study involving 36 elderly women with moderate osteoporosis. The study in-
volved 1 year of observation, with no treatment, followed by 1 year of treatment comprising 100 IU salmon calcitonin dosed rectally daily and 500 mg calcium supplement. During the year’s observation a significant loss of bone (1.5%) was recorded in the forearm whilst spinal bone mass remained constant. Over the subsequent year of treatment, bone loss from the forearm was arrested and a significant increase (2%) of mass was observed in the spine. Parameters of bone turnover measured concomitantly showed no change during the first year, but declined significantly by lo-30% during the year of treatment. Tolerance was good. Eleven of the 36 subjects did not complete the study. Six dropped out because of symptoms associated with systemic calcitonin, four dropped out for reasons unrelated to the treatment and only two because of local reactions (changes in bowel pattern). In interpreting the relative results of systemic delivery of calcitonin by colonic and rectal routes the relative stability to metabolism of the rectal doses must be remembered. The last study demonstrates unequivocally the feasibility of the rectal route as a non-parenteral route for the long-term systemic dosing of a peptide or protein drug. It confirms the conditions for which this route is likely to offer the most realistic alternative to parenteral dosing: l l l l l
chronic treatment; moderate disease states; maximum dosing regimen of once daily; potential for reducing uncomfortable side effects; large population.
5. Impact of GI tract delivery peptides and proteins 5. I. Pharmacokinetic
of therapeutic
impuct of GI truct delivery
The administration of drugs as S.C. or i.m. formulations has a profound effect of the pharmacokinetics. Often enabling the active to be released for many hours and reducing the impact of first pass clearance. Standard formulations of drugs administered by the GI tract do not show these characteristics. Therefore, it is likely that it will not be a simple matter substituting one delivery route for the other when peptides are considered. It will be
necessary to consider the release characteristics ot the formulation in the GI tract, the impact this will have on the local metabolism in the lumen and the influence on the absorption. In the case of insulin the pharmacological profile could also be altered as a result of enhanced action on the liver as a consequence of initial drainage and distribution via the hepatic portal vein after delivery via the GI tract.
The impact of oral or rectal formulations of peptides is not simply and issue of patient compliance. Data on intestinal absorption of peptidea suggests that the absolute bioavailability is in the order of 1%. Even if therapeutically relevant formulations can be prepared the implication is that approximately two orders of magnitude more drug will have to be used in GI tract formulations compared with an in.jection. This could have serious repercussions in two areas. Firstly. it is likely that GI tract formulations will be considerably more expensive than current therapies because of the need for large quantities of active ingredient. Secondly, if availability of the active ingredient is limited then companies providing formulations for delivery by the GI tract could be reducing the pool of patients that could be treated if in.jectable formulations were used.
ib not in question and would be significant in terms of patient compliance. One can envisage that multidisciplinary approaches by scientists in academia and within the pharmaceutical industry will lead to targeting of peptide and protein drugs to specific regions of the GI tract, such as the colon, and lead to both better compliance and therefore improved therapies. Moreover, the selection of the peptide and the disease indication is critical. The major opportunity rests with peptides that will need to be self-administered by the patient for disease indications where there is little competition from traditional pharmaceutical agents. The success of nasal salmon calcitonin showed the value of non-injectable formulations opening up markets. Market forces will continue to drive the need for GI tract formulations. It should be remembered that dDAVP. a small peptide, is already available as an oral formulation in some countries. This may represent the bridgehead that will expand as more therapeutic peptides and protein are identified and GI tract research makes breakthrough discoveries enabling safe and efficient delivery after oral or rectal administration.
Acknowledgements Many thanks to Natalie Schneiders ration of the references.
6. Challenges of colonic and rectal delivery peptides and protein drugs
for the prepa-
of
Achieving therapeutically relevant Gl tract deli\)ery of peptides and proteins is still a major challenge. The current state of the art is limited such that it is important to optimise the opportunity by careful selection of peptide, mode of delivery. and the site of absorption. Conventional wisdom suggests that the colon offers a window of opportunity. Attempts to achieve therapeutic levels of peptide and protein drugs in the vascular compartment following intracolonic administration have mostly relied on absorption enhancers. The use of such enhancers for chronic administration of drug is doubtful given the epithelial damage that many enhancers have been shown to cause. However, the need for more acceptable methods to achieve effective peptide absorption
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