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Oral peptide delivery: Translational challenges due to physiological effects
Accepted Manuscript Oral peptide delivery: physiological effects
Translational
challenges
due
to
Puneet Tyagi, Sergei Pechenov, J. Anand Subramony PII: DOI: Reference:
S0168-3659(18)30498-X doi:10.1016/j.jconrel.2018.08.032 COREL 9441
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
26 June 2018 21 August 2018 22 August 2018
Please cite this article as: Puneet Tyagi, Sergei Pechenov, J. Anand Subramony , Oral peptide delivery: Translational challenges due to physiological effects. Corel (2018), doi:10.1016/j.jconrel.2018.08.032
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ACCEPTED MANUSCRIPT Oral Peptide Delivery: Translational Challenges due to Physiological Effects
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Puneet Tyagi, Sergei Pechenov, and J Anand Subramony* [email protected] MedImmune Inc, United States
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Corresponding author at: One MedImmune Way, Gaithersburg, MD 20878, United States.
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Abstract
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Oral delivery of peptide therapeutics as a convenient alternate to injections has been an area of research for the pharmaceutical scientific community for the last several decades. However, systemic delivery of therapeutic peptides via the oral route has been a
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daunting task due to the low pH denaturation of the peptides in the stomach, enzymatic instability, and poor transport across the tight
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junctions resulting in very low bioavailability. The low bioavailability is accompanied by large intra- and inter-subject variability leading to translational issues, preventing the development of successful peptide therapeutics. The inter-subject variability leads to large differences in pharmacologic responses in individuals and thus the dose required to produce therapeutic effect could vary between individuals making the development of drug product a very difficult task. A substantial amount of research has been (and continues to be) performed with a focus on getting acceptable absorption and reproducible results. Nonetheless, the high variability and low bioavailability during oral administration of peptides is still a work in progress and under-explored in a systematic way. 1
ACCEPTED MANUSCRIPT While there are several review articles and scattered publications that discuss potential technologies for oral peptide delivery, a detailed look into the physiological challenges and absorption barriers which are a hindrance to successful clinical translation, is lacking. Herein, we have analyzed the physiological barriers within the gastrointestinal (GI) tract that are the root causes for the low bioavailability and high variability of oral delivery of peptides in humans. In particular, we have taken a detailed look at the key
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influencing factors such as the nature of various GI tract parameters, components of the GI tract that influences the uptake, site of
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absorption, pH of the gastric and intestinal compartments, food effect, and role of peptidases in affecting oral peptide absorption. Lack of in vitro - in vivo correlations and variability in animal models have also been highlighted as key impediments in understanding the challenges.
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The unique perspective presented herein for overcoming the physiological absorption barriers, will offer better
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developability approaches and will positively impact clinical translation of future oral peptide therapeutics. A deep understanding of these effects are vital, given the emergence of microbiome and oral biologic drug delivery that are fast emerging as the next wave of
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personalized patient centric therapies.
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1. Introduction
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Therapeutic peptides have come a long way since insulin was discovered and used as a therapy for diabetes. After insulin,
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oxytocin was chemically synthesized in 1950 and was approved in 1980 for the treatment of uterine contraction [1]. Since then the therapeutic peptide market expanded exponentially and currently over 60 peptide products are available in the market. Market
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research predicts that the peptide therapeutic market is expected to reach ~ $ 46 billion by 2025 with a highly impressive 9.1% CAGR from 2016 mainly due to the utility of peptide therapeutics in the area of oncology and metabolic disorders [2]. Most of the peptides are administered to patients through injections via the parenteral route. However, the injectable route is not the most convenient, and has issues with needle phobia and non-patient compliance. Consequently, there is a need for development of 2
ACCEPTED MANUSCRIPT alternate delivery routes for administration of peptides. Oral delivery can improve patient compliance and provide the needed convenience and therefore is of great interest for the development of peptide therapeutics [3, 4]. Oral dosage forms are also easy to scale up and convenient in terms of packaging and handling compared to liquid injections. The last two decades have seen several
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efforts for the development of oral peptide therapeutics (Table 1). However, peptides are not designed by nature to be systemically
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bioavailable for oral administration and the reasons are manifold. Oral bioavailability of peptides is very low due to peptide
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degradation in the GI tract (due to protease and acidic pH) and their poor permeability across the epithelial barrier because of high
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molecular weight, large size [5], and amphiphilicity. Figure 1 summarizes the published data on oral bioavailability in humans and interestingly it follows an inverse trend with MW. Most peptides have <1% absolute bioavailability with high variability when
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delivered via oral route [6, 7]. For example, bioavailability of desmopressin acetate oral tablets in humans ranges from ~ 0.08 to
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0.16% [11, 12]. Despite these low numbers, Minirin (desmopressin acetate) tablet has become a viable product as it is convenient to
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administer it orally for the indication it is used for (to control nighttime bedwetting in children) and also due to its wide therapeutic
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window. However, for larger peptides like insulin, it is essential to have low variability because of its low therapeutic window and the need for accurate onset and efficacy for the needs of glycemic control for diabetic patients.
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Table 1. Clinically advanced technologies for oral peptide delivery (source: PharmaCircle™). Interested readers are encourage d to refer to clinical trials database (clinicaltrials.gov) for most recent updates.
Company
Peptide
Technology
Development stage
Biocon Ltd.
Insulin
HIM2; Absorption enhancers and enzyme
Phase I
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Chiasma Inc.
Octreotide
Enteris BioPharma, Inc.
Leuprolide
Emisphere Technologies Inc /Novo Nordisk Merrion Pharmaceuticals /Novo Nordisk
Insulin GLP-1 analogue
Oramed Pharmaceuticals
Insulin
Cara Therapeutics
Pain indication
Insulin
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GSK
Transient Permeability Enhancer (TPE®) technology; oil based suspension Peptelligence™; absorption enhancers Eligen®; absorption enhancers GIPET™; absorption enhancers POD™; Absorption enhancers and enzyme inhibitors
PTH
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Phase III
Phase II Phase I Phase III Phase I Phase II Phase II
Unigene/Enteris
Phase I/Terminated
Unigene/Enteris
Phase II/Terminated
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Figure 1. Oral bioavailability of small molecule drugs and peptides in comparison to their molecular weights [8-12]. Inset graph shows the bioavailability of peptide drugs. The trend follows an inverse relationship between MW and oral bioavailability (BAV) in humans. Figure does not show the variability for BAV.
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ACCEPTED MANUSCRIPT To understand the reasons for the low systemic bioavailability and pharmacokinetic (PK) variability of peptides after oral administration, a close look at the transport properties of the peptide and its molecular dynamics during its residence within the GI tract are important. In the stomach and along the GI tract, peptide molecules are susceptible to peptidase and protease
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action, working on breaking the peptide down to single amino acids [4]. The amount of survived
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peptide is proportional to its protease and acid stability and reversibly proportional to the amount
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of time it is exposed to the low pH of stomach, and the amount and activity of the respective proteases. After transiting through the stomach, the peptide goes into intestines, where the GI
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tract lining is protected by a layer of mucin. Peptide molecules need to transport and cross the
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mucin layer before it can enter into the layer of epithelial cells. These cells efficiently transport amino acids and small polypeptides, but prevent larger hydrophilic, potentially bioactive
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molecules, from entering systemic circulation [13]. Yet some of the molecules can reach the
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circulation via passive route, by passing between the cells, the so called tight junctions, or via active cellular transport way aided by a transporter ligand that is present on the peptide [14].
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Hence peptide properties, namely size and amphiphilicity, exposure time in the GI tract, amount
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of peptide present at the site of absorption, are some of the key factors that can influence bioavailability. Physiological attributes, such as amount of proteases and peptidases present, pH
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microenvironment, gastric and intestinal surface area, thickness of mucus layer, presence of accompanying food, permeability, or leakiness of GI tract lining, are some of the other intrinsic barriers that determine the bioavailability. The transport across GI tract is quite variable since it is a dynamic system, whereby its function depends on the age, gender, fed state, time of the day, dietary preferences, and GI health status resulting in great intra- and inter- subject PK variability [15]. Particularly the GI tract attributes are critical if the compound has a very narrow absorption
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ACCEPTED MANUSCRIPT window. The above-mentioned parameters are some of the key reasons why peptides have extremely low bioavailability on their own. In order to increase bioavailability for oral peptide delivery, several approaches including use of technologies have been tested in preclinical models and in humans. However, due to reasons
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outlined above, high inter-subject variability is seen in many of these studies, and therefore a
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systematic approach is required to further understand the root causes for poor bioavailability and
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high inter-subject variability. For example, in an oral insulin clinical trial report published by Kapitza et. al.[16] in 2010, the bioavailability varied by more than 100% in the first hour. In
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another study, oral administration of the Gonadotropin-releasing hormone (GnRH) antagonist
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acyline in a tablet dosage form resulted in highly variable pharmacokinetic data. The variability among subjects was so high that the clinical outcomes between different doses were also not
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significantly different [17]. For peptides with wide therapeutic window, such as GLP-1, high PK
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variability may be less of an obstacle, but nausea due to high Cmax could be a problem. Nevertheless, nausea can be managed by dose titration and by using controlled release
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technologies. For peptides with a narrow therapeutic window, such as insulin, high PK
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variability may result in either lack of therapeutic effect or fatal hypoglycemia which are very critical to patients.
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As high variability and low bioavailability continue to act as hurdles for oral peptide delivery, the purpose of this review is to understand and highlight the physiological factors that impact the development of oral peptide therapeutics in preclinical models and in human subjects during clinical translation. For broader reviews covering oral delivery of peptides, readers are directed to relevant published literature [4, 18-22].
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ACCEPTED MANUSCRIPT 2. Variability in GI tract parameters Parameters of the digestive tract, such as surface area, motility and permeability, can affect the absorption of peptides. Additionally, variations in gastric volume, vascularity of the intestinal lining that impacts the blood flow, thickness of the mucus layer, and site of absorption can also
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alter the absorption and hence the bioavailability of the peptides. In a recent publication,
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Sugihara, et al systematically compared 113 human bioequivalence studies of 74 small molecule
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actives [23]. Their analysis suggested that factors such as luminal fluid volume, transit time, and presence of metabolizing enzymes play a significant role in intra and inter subject variability of
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drug absorption. Though small molecules are different than peptides, we can roughly infer from
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the data that these factors can influence the absorption of peptides as well. Let us look at some of these GI parameters in the following paragraphs.
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Surface area: The surface of the stomach has a small area relative to the small intestine. The
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small intestine has folds, villi, and microvilli that enlarge the surface are of the intestine. A range of 520-1536 cm2 has been reported for human stomach mucosal surface area [24].
More
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recently, Helander et al. [25] used morphometry to assess the stomach area in healthy volunteers
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and reported that the mucosal surface area of the stomach is ~ 500 cm2 in alignment with the previously reported range. Variability in the small intestine length has been reported to range
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from 160 - 430 cm in people with no abdominal disorder (measured using radiography) [26] and a range of 385 - 538 for patients who have undergone laparotomy [27]. As the primary role of small intestine is the uptake of nutrients such as peptides, variations in length and surface area can appreciably alter the bioavailability of peptides. Motility and resulting exposure: Variations in the small intestine motility can result in significant variations and poor bioavailability of peptides by altering the site of absorption and/or
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ACCEPTED MANUSCRIPT duration of exposure. In healthy volunteers [28], speed of phase III contractions in duodenumjejenum area varies from 2.4 - 10 cm/minutes. The variation in duration of phase III contractions (ranges from 3.6-5.7 minutes) in a healthy individual is not as variable as speed of contraction. In a diseased state, such as patients suffering from Crohn’s disease, the variation in speed of phase
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III contractions can go from 2.1-22.5 cm/minute. Another significant observation in diseased
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patients is the lack of phase III contractions in ~30% of the patients. High speed of contraction
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can cause poor peptide absorption in some patients. Further, the absence of contraction can cause large gradient of peptide deposition within the small intestine by which there will be localization
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of peptide dose in one area and a more concentrated exposure in another area, leading to
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variability.
Intestinal permeability: Region dependent absorption has been observed for many
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therapeutic peptides including calcitonin [29]. Modifications and inter-subject variability in
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intestinal permeability has been assessed by measuring urinary excretion of non-metabolized sugars (lactulose and mannitol) administered orally [30]. Several diseases such as irritable bowel
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syndrome (IBS) are associated with modification in intestinal permeability marked by diarrhea
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and weight loss. In a study to measure intestinal permeability in healthy volunteers vs. patients with IBS [31], subjects were dosed with 5 g of lactulose and 2 g of mannitol dissolved in 100 ml
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of water. Urine from the subjects were collected at different time intervals and analyzed to obtain a measure of permeability. The results indicated that in healthy volunteers, the intestinal permeability varied from 0.01 - 0.06 lactulose/mannitol excretion ratio. Furthermore, for patients suffering from IBS, the ratio varied from 0.01 - 0.24.
As a standard, a lactulose/mannitol
excretion ratio ≥ 0.07 is taken to indicate the presence of an increase in permeability.
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ACCEPTED MANUSCRIPT Site of absorption: Permeability of peptides can vary from the site of absorption in the intestine. Langguth et al. calculated wall permeability of metkephamid, a synthetic opioid pentapeptide, and observed that the permeability was highest in the ileum, followed by jejunum. The bioavailability of metkephamid increased from 0.22% (following oral administration) to
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1.79% when the peptide was administered into the ileum [32]. Similar to the results by Langguth
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linear polypeptide, than in the duodenum and colon in dogs [29].
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et al., Sinko et al. also observed higher ileal absorption of salmon calcitonin, a 32-amino acid
Region dependent studies using a lipopeptide, LY303366, wherein duodenal, jejunal, and
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colonic bolus administration of the same dose and volume were conducted in Beagle dogs [33].
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It was observed that LY303366 had significantly lower drug plasma levels following administration through the colon and jejunum (Cmax of 0.3 and 0.6 µg/ml, respectively) as
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compared to oral and duodenal administration (Cmax of 1.6 and 1.5µg/ml, respectively). It is
form.
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really important to carry out a site of absorption study before designing the oral peptide dosage Formulations technologies such as immediate release and delayed release as well as
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enteric coating can be engineered in the dosage form based on the site of absorption studies.
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Similarly, for top GI and stomach absorption, gastro retention would be a suitable approach. Absorption site transition time: Following gastric emptying, an enteric dosage form will
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disintegrate in small intestine. Depending on the enteric coating, disintegration can occur in either of the intestinal segments (duodenum, jejunum, or ileum). If the dosage form disintegrates within the first 5 minutes following gastric emptying, it is considered to happen in the duodenum. Disintegration happening from 5 to 60 minutes and from 60 to 75 minutes is considered to happen in jejunum and ileum, respectively. The average intestinal transit time in
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ACCEPTED MANUSCRIPT fasted dogs and humans has been reported to range from 111 ± 17 minutes [34] and 170 ± 30 minutes, respectively [35]. Lee et al. reported the intestinal transit time for tablet formulations containing calcitonin and observed that disintegration of two formulations occurred in the jejunum (transit times of ≤ 50
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minutes) and one formulation had transit times from 20 – 90 minutes [36], indicating absorption
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from the ileum. In addition to the small intestine, the transit time in human large intestine can
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vary in the range of 8 – 72 hours [37]. This indicates that variability in transit times can result in dosage form being in an undesirable site, and eventually leading to poor bioavailability and huge
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variability between patients.
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Gastric volume: The volume of liquid being secreted in the stomach can affect the solubility of the active molecule and might also alter the rate of degradation depending upon the pH of the
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contents. Following overnight fasting, healthy volunteers were observed to have a mean gastric
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volume of 59.6 ± 4.2 (males) and 46.3 ± 4.2 ml (females), with a range of 0 – 225 ml [38]. Similar results of mean gastric volume of 50 ml with a range of 0 – 180 ml have also been
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reported by Leech et al. [39]. Furthermore, in diseased state (such as of ulcers) the gastric
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volumes following an overnight fasting can go up to > 80 ml. Following collection of gastric secretion after overnight fasting in healthy men, Iijima et al. observed that the pH of the gastric
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secretion was 4.4 ± 1.2 mEq/10 min. Iijima et al. measured the H+ concentration of the gastric juice by titration and reported the output in a 10-min period [13]. Gastric mucus: Gastric mucus is divided into an adherent layer (adhering to the gastric mucosa) and a soluble layer (mostly freshly secreted). Iijima et al. quantified the soluble mucin concentration to range from 93.6 ± 37.8 μg hexose/ml in healthy volunteers. In the same study, the amount of reported mucus (determined by multiplying the mucin concentration by the
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ACCEPTED MANUSCRIPT volume of gastric juice collected in the 10-min period) varied from 3.2 ± 1.2 mg hexose/10 minutes in humans[13]. In a study consisting of 10 patients, Huh et al., acquired data for mucous thickness in stomach to vary from 1.03 mm - 1.64 mm [40]. Variation in mucus secretion rate and output can alter the clearance of active before it is exposed to the epithelial cell layer for
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activity. In addition, mucosal blood flow also plays an important role in the production of mucus
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and bicarbonates. Karzai et al. demonstrated the gastric mucosal microcirculatory blood flow to
Effect of gastric and intestinal pH
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3.
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be 61 ± 33 units [41].
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Gastric pH is an important factor contributing to the stability of the peptide. It has been reported that the gastric pH is fairly stable and within a narrow range in young individuals [42].
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A median fasting gastric pH of 1.7 (range 1.4-2.1) and median fasting pH in mid duodenum of
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6.1 (range 5.8-6.5) was found in 24 young healthy men and women [42]. During meals, pH in the stomach might increase to 6, but quickly returns to normal within a few minutes. However, with
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age, elderly people tend to have more pH variation. A study conducted on healthy volunteers 65
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or older, it was found that 10% of the people had a high fasting gastric pH of ~6 [43]. Furthermore, many individuals took many hours before the pH came back to normal value of 1.7.
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Some individuals were found to have achlorhydria and did not see a decrease in gastric pH for up to 6 hours [43]. Such high variations can have a significant impact on the presence of peptidases in the stomach and eventually induce variations in the extent of peptide absorption if the solubility factors of the peptide for better absorption have a narrow pH window dependence. In addition to age, disease states can also affect the pH. People suffering from cystic fibrosis tend to produce a lot of mucus, which can block the secretions from the pancreas. An acid-base
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ACCEPTED MANUSCRIPT imbalance is the result, which eventually causes lower and variable intestinal pH in cystic fibrosis patients [44]. In the study by Youngberg et al., [44], it was noticed that the pH in the duodenum for 25% of cystic fibrosis patients was below 5.5, up to 2 hours after meals. A change in the GI tract pH, as seen in cystic fibrosis patients, poses serious problems in oral
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delivery of peptides as it can affect the solubility of the peptide. A variation in pH can also serve
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as a major factor in peptide stability. Furthermore, as many tablets are enteric coated for delivery
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in the intestine, a low pH can lead to improper disintegration of tablet. Lee et al. [36] have performed a series of experiments with calcitonin (a 32-amino acid linear polypeptide) which
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highlighted the variation in disintegration of enteric coated tablets. Lee et al. showed that,
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following oral administration of enteric coated tablets containing citric acid, the time to reach intestinal pH trough varied from 35 to 90 minutes. Therefore, a careful choice of enteric coating
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material is a key factor in oral peptide delivery.
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Another important aspect of pH variation lies in the location where the enteric coated tablet is disintegrating. An oral enteric coated tablet formulation containing 565 mg of citric acid and
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1.2 mg of calcitonin was found to give high plasma concentration of calcitonin in dogs when the
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tablet disintegrates in the late jejunum or ileum. The authors suggested that this was because the pH recovery in the late jejunum or ileum is slow (takes up to ~140 minutes). In comparison,
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when the tablet disintegration occurs in the duodenum, the plasma concentration of calcitonin was low and the pH recovery was fast (takes up to ~15 minutes) [45]. In the absence of citric acid in the formulation, no calcitonin was observed in the plasma and no decrease in intestinal pH was observed [36]. Therefore, a decrease in intestinal pH seems to be necessary for the absorption of calcitonin. This again confirms that understanding the site of absorption is beneficial for optimum delivery of peptides, and the dosage form can be designed to target the
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ACCEPTED MANUSCRIPT optimal site of absorption using controlled release technologies and or by creating local micro pH environment to maintain appropriate solubility and super saturation.
4. Effect of gastric emptying
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It is widely considered that peptide absorption from the stomach is very slow whereas the
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absorption from the small intestine is much fast and rapid. This is believed to be due to the large
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surface area of the small intestine. Gastric emptying is likely to be a rate limiting step in the absorption of peptides and can therefore account for much of the observed variation (in time of
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onset of action) amongst patients. Gastric emptying can be influenced by many factors including
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posture, pH, presence of other drugs, and hormonal activity. For example, Steingotter et al. observed that gastric peristalsis was slower in patients in upside down position in comparison to
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patients in seated position. Also, intra gastric distribution was significantly different in the two
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volunteer groups [46]. Common drugs such as clonidine (used to treat hypertension) and Sumatriptan (used to treat migraine headaches) have also been shown to modulate gastric
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emptying significantly [47].
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Gastric emptying during fasted state happens when migrating complex, a band of regular contractions, periodically moves indigestible solids from the stomach to the distal small intestine.
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Studies have confirmed that non-disintegrating units (such as enteric coated tablets) are emptied out from the stomach in the third phase of the cyclic motility pattern. Thus, the intestinal absorption of peptides delivered as an enteric coated formulation largely depends upon the gastric emptying. Variation in gastric emptying can occur if the dosage form linger in the less muscular body of the stomach and not be emptied out to the small intestine [48], thus delaying gastric emptying. Lee et al. observed gastric emptying time ranging from 30 - 150 minutes in
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ACCEPTED MANUSCRIPT beagle dogs dosed with exactly the same formulations of calcitonin [36]. As the gastric emptying in fasted state is dependent on a variable migrating complex, we might observe improper disintegration and dissolution of the dosage form. In comparison, in the fed state, the formulation is expected to have a prolonged gastric residence, thereby ensuring proper dissolution [49]. Fed
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state can also be characterized by elevated pH, which in achlorhydria patients can lead to
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dissolution of enteric coated tablets in stomach with resulting lower bioavailability and dilution
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with food components. In addition to inter subject variations, diseases can also alter the gastric emptying time. Achlorhydria has been shown to go together with delayed gastric emptying [50].
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In a study comprising of elderly achlorhydria patients, it was observed that, on an average, the
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patients took 40 minutes to empty 50% of a 300 ml of consumed orange juice. In comparison, healthy volunteers cleared the same amount in 18 minutes [50]. Gastric emptying has also been
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reported to be delayed in gastric ulcers and gastric carcinoma, whereas an accelerated gastric
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emptying was seen in celiac disease and duodenal ulcers [51].
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5. Effect of meal and water intake
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Studies have indicated that bioavailability of small molecule drugs are affected by meal timings. However, peptides and proteins have not been evaluated extensively for effect of food
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effect on oral delivery. Furthermore, vast structural differences in small molecules and peptide/proteins makes it difficult to extrapolate any learnings from the small molecule area. In a study conducted by Karsdal et al., the effect of meal and water uptake on oral salmon calcitonin was observed in 56 healthy women [52]. The formulation [53] contained 0.8 mg of calcitonin and 200 mg of 5-CNAC, [8-(N-2- hydroxy-5-chloro-benzoyl)-amino-caprylic acid)], a permeation enhancer. The study evaluated the pharmacokinetic and pharmacodynamics of
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ACCEPTED MANUSCRIPT salmon calcitonin when taken with different volumes of water (50 or 200 ml) and at different times before a standard meal (10, 30 or 60 minutes). It was observed that the level of maximum concentration and AUC of the plasma levels was significantly dependent upon the amount of water intake, suggesting an influence of water intake on peptide absorption. Administration of
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the peptide with 50 ml of water achieved two- to three-fold higher absorption in comparison to
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the administration with 200 ml water. The effect of water intake was significantly higher
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irrespective of the time post meal was taken. A better efficacy was also observed when the dose was administered with 50 ml of water. More evaluation is needed to confirm if higher
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bioavailability is a result of higher concentration of peptide and permeation enhancer.
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In another study using the oral calcitonin formulation (0.8 mg calcitonin and 200mg 5CNAC), the effect of predose meal was evaluated on the oral bioavailability [54]. The predose
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meal at 4 and 2 hours before dosing significantly decreased relative oral bioavailability of oral
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calcitonin to 26% compared with that of the dose in the fasting state. Meals taken 10 minutes after dosing also resulted in decrease in bioavailability to 59%. This hints upon the potential
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influence of gastric retention and food interaction on bioavailability of the orally administered
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peptide. However, many such studies are needed to properly verify the effect of food. Li et al. evaluated the effect of meal composition on the systemic availability of LY303366, a
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lipopeptide being developed as an antifungal agent [33]. The study evaluated the effect of different types of foods (proteins, lipid, and carbohydrate), and osmolality on the plasma concentrations of LY303366. Of the food types tested, protein (125 kcal diet) and lipid (250 kcal diet) decreased the plasma level of LY303366 in comparison to a fasted plasma concentration. The effect of a hyperosmolar solution (480 mOsm/kg) on the plasma concentrations was not significant. However, a significant decrease in drug plasma levels was observed with
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ACCEPTED MANUSCRIPT administration of the 250 kcal, hyperosmolar liquid nutrient meal of mixed caloric composition. The authors hypothesized that the possible cause of a negative effect of meals might be due to the influence of meal viscosity and/or binding of the peptide to the meal ingredients. Li et al. suggested that these effects can make a major impact on the peptide bioavailability if significant
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portion of the drug is absorbed in the upper small intestine. As the peptide LY303366 contains
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lipid, the chances of the peptide binding to lipid diet is highly plausible. A similar reduction in
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bioavailability was observed when EP01572, a novel, synthetic ghrelin agonist was dosed with food [55]. In comparison to dosing without food, the Cmax and AUC values decreased by half
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when the dose was given with food. Tuvia et al. also observed lower plasma octreotide levels
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when oral octreotide formulations were administered with a high-fat, high-calorie meal [56].
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6. Effect of peptidases
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Many proteases and peptidases degrades the peptides in the GI tract, leading to poor bioavailability. However, there is only limited data available for the variation in the activity of
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peptidases, as it is hard to quantify. Tor Lindberg [57] observed that the specific activity of
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dipeptidases (attacking hydrophobic amino acids) in the small intestine mucosa of adult humans ranges from 23.7 - 125.2 units/mg nitrogen where one unit is defined as the peptidase activity
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hydrolyzing 1 µmole of dipeptide per min at 40 ºC. However, the study was performed in diseased patients.
Wang et al. [58] have compiled a good summary list of peptide stability in human gastric fluid in the presence of pepsin (after 2 hours) and human small intestine fluid in presence of pancreatin (after 30 minutes). From the list of 17 peptides or so they studied, it is interesting to note that only 3 of them had any percentage peptide left in small intestinal fluid containing
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ACCEPTED MANUSCRIPT pancreatin (namely cyclosporin 99%, desmopressin 25%, and octreotide 22%) and only those 3 peptides have made it as oral peptide therapeutic products (Neoral™, Minrin™, and
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Octreolin™).
Potential tools to overcome the physiological barrier/methodology
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Parameters that impact peptide bioavailability
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Variability in GI tract -
Intestinal permeability
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Site of absorption
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Absorption site residence time
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Gastric mucus
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Can be tackled by a combination of fine tuning physico-chemical properties of permeation enhancers complementing the properties of peptide (such as LogP, pH dependent solubility).
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Perform site of absorption studies and deliver peptide to that location via controlled release.
Mucolytic agents; mucus penetrating particles
Dosing in fasted state Peptidase inhibitors; peptide modification to enhance stability against peptidases
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Effect of peptidases
pH modifiers/buffering agents to modulate the immediate peptide environment.
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Effect of gastric and intestinal pH Effect of meal uptake
Mucoadhesives can be used to retain the formulation at the absorption site to maximize absorption.
Rodents are not ideal due to issues with dosing oral formulations; pigs and dogs are widely preferred due to anatomical similarities to humans. Use of higher samples (n value) to make bioavailability values statistically significant.
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Variability in preclinical models
Table 2. List of relevant parameters that can led to low peptide bioavailability and potential tools to overcome the physiological barrier or methodology.
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ACCEPTED MANUSCRIPT 7. Variability in preclinical models used to evaluate oral delivery systems A reliable prediction of the oral bioavailability is crucial for the development of delivery systems. To that end, different animal models are utilized which can mimic conditions across the GI tract[59]. Rats, pigs, dogs, and non-human primates are the common models used to evaluate
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oral delivery systems. Inter- and intra-species variability exists in the gastric physiology and can
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greatly affect the outcome in the animal models (Table 3). Also, it is difficult to dose solid oral
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dosages in rats and preliminary work has relied on administering by direct intraduodenal injections. Gastric emptying can vary from 0.7 - 2.1 hours in rats depending upon the dosage
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form density and size [60]. In comparison, the gastric emptying of solids in humans takes 68
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minutes. Pigs have a highly variable gastric emptying time, ranging from 1.5 - 6 hours. In addition to inherent variability in gastric emptying time, the size of dosage form can also affect
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the gastric emptying time. Sagawa et al. reported the gastric emptying of a capsule with 6 x 5 x
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25 mm3 size to be 1.4 hours in fasted Beagle dogs [61]. In contrast, the gastric emptying of a standard 10 g meal and 200 g meal was 9.4 and 20 hours, respectively. In a study conducted with
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telemetric capsules (7 x 20 mm), gastric emptying of the capsules was similar in humans and
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dogs (1.2 ± 0.4 hours) [62]. In another study, 1 mm particles emptied out within 2 hours from both humans and dogs [63]. Gastric motility is an additional complicating factor and can induce
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significant variability in drug absorption. Dogs and humans were found to have similar phase III activity, lasting for 18.6 and 19 minutes, respectively [64]. Phase III activity in the stomach is responsible for moving food into the small intestine. The periodicity of the phase III cycles was also similar in humans and dogs, with cycle being seen every 106 minutes in dogs and 128 minutes in humans [65]. However, dogs have highly permeable intestinal wall, which can demonstrate higher drug absorption in comparison to humans. Furthermore, in dogs, the gastric
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ACCEPTED MANUSCRIPT emptying time is ~10 to 20 times that of humans while the gastric retention time and the GI transit time (in the fasted state) is nearly one half of that in humans. The thickness of mucus layer varies considerably in dogs (425 ± 7 µm) [66] and humans (144 µm) [67]. In comparison, rats have a mucus thickness ranging from a mean of 31.3 – 69.4 µm [68]. Also, the length of the
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GI tract ranges from 17 cm for rats to 177 cm in humans. The length of GI tract in pigs is closest
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to humans at 125 cm [69]. Basal gastric acid output is highly variable in dogs with a range of 0.3
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– 1.5 ml/minute. In comparison, pigs and humans have a gastric acid output at approximately 1 ml/minute [70].
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Thus, the choice of preclinical model should be carefully made if the absorption is in the
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stomach or top GI and for compounds with a very narrow absorption window [71]. The length of the GI components (colon, caecum, small intestine and stomach) matches very well between pigs
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and humans and therefore using a pig model might be appropriate for confirmatory studies once
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preliminary bioavailability is determined from the dog model [71]. Also, it is better to repeat the studies in a given animal model for consistency in pharmacokinetic parameters before a dosage
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form is advanced to the next stage.
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ACCEPTED MANUSCRIPT Table 3. Comparison of key GI tract parameters across different pre-clinical and species and humans commonly used for pharmacokinetic and efficacy evaluations [66, 71-74].
Gastric emptying time Gastric retention time Small intestine transit time Absolute water content (g/cm gut length) pH of the stomach (fed) pH of small intestine Length of gut Stomach fundus mucus thickness Average stomach fluid capacity
Humans
Rats
Dogs
Pigs
1 hour
0.7-2.1 hours
3.9 – 5.3 hours
1.5-6 hours
46 minutes
29 minutes
23 minutes
224 – 252 minutes
21.4 – 25.1 hours (fed state)
96 – 128 minutes (fed state)
0.58
0.06
2 – 4.5
3
5.0 – 7.0
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144 ± 52 µm
C A
3 – 4 hours
0.62
3–5
2.2 – 4.3
6.2 – 7.5
6.0 – 7.5
17 cm
90 cm
125 cm
31.3 ± 11.4 µm
425 µm
190.7 ± 80.7 µm
3.4 ml
4.33 L
8.0 L
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1 – 1.6 L
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48 minutes
0.86
6.5 – 7.1
177 cm
C S
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T P
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ACCEPTED MANUSCRIPT 8. Gastric simulation and modelling of the GI tract Various high throughput in vitro systems have been developed to simulate drug absorption and transport. A popular in vitro system is the Caco-2 tight junction cell layer derived from the human carcinoma [75]. The Caco-2 cells have served as a qualitative tool for permeability
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coefficient calculation of small molecular weight compounds [76]. However, the predictive value
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of Caco-2 cells is stalled by their inability to fully mimic in vivo phenomenon such as peristaltic
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movements. Furthermore, Caco-2 cells solely consist of enterocytes and do not reflect the in vivo physiology of intestinal epithelium. In addition to the above factors, lack of mucus layer in Caco-
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2 cells makes them less optimal to understand the absorption of peptides. Human ex vivo
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intestinal tissues have served as a better model to understand preclinical human intestinal permeability. Small pieces of human intestinal tissue are obtained from patients and permeability
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studies using human small intestinal or colon tissue have been performed [77]. The FDA also
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recommends the use of excised intestinal tissues to assess permeability [78]. However, availability of sufficient healthy human tissues and maintenance of their viability is critical and
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can hinders this type of research. Furthermore, differences in eating habits, environment, and
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medications can affect the tissue morphology and result in inter subject variability. In addition to human ex vivo tissues, porcine intestinal tissue is another option and has the advantage of ease of
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tissue availability. Gastrointestinal morphology in pigs appears to be comparable to humans as opposed to other preclinical animal species such as monkeys and dogs [79]. Several computer controlled models aim to simulate the luminal settings of the stomach and small intestine [80]. Parameters such as pH, gastric emptying, and addition of gastric and intestinal juices have been included in such models. TIM-1 (TNO gastro-Intestinal Model 1), a multi-compartmental model, designed to realistically simulate GI tract conditions, is composed
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ACCEPTED MANUSCRIPT of the stomach and the three parts of the small intestine, the duodenum, jejunum, and ileum [81]. TIM-1 allows close modeling of luminal dynamic procedures occurring within the human body. Parameters such as gastric and small intestinal transit, flow rates and composition of gastric and intestinal fluids, pH values, and removal of water and metabolites, transit time and peristalti c In the InTESTine™ system ex vivo
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mixing have been included in the simulation protocols.
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intestinal segments are used for prediction of permeability at a medium thoroughput, wherein
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upto 96 segments can be evaluated in one experiment [82]. Prediction of intestinal permeability using porcine intestinal tissue in the InTESTine™ system provides a better alternative in
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comparison to Ussing chambers. Furthermore, a unique integrated approach has been developed
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in recent times[83] that combines the TIM-1 model with InTESTine™ and in silico modeling to predict drug absorption in humans.
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The Dynamic Gastric Model (DGM) [84] is an in vitro model that simulates the physical,
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mechanical and biochemical milieus of the human stomach. The DGM includes addition of dynamically controlled gastric juices and enzymes. The rate of addition of the juices is
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dependent upon the pH of the meal (which is detected by a pH electrode). In addition, the system
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also provides the food bolus with simulated peristaltic movements, which in turn help the food to move further. In a direct comparison of DGM with a USP Type II dissolution apparatus, it was
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observed that DGM took into account the shear forces of the gastric walls during disintegration and dissolution [85]. In comparison, USP Type II apparatus did not exhibit high shear forces despite increase in shear rates. In Vivo conditions are such that the shear rates are relatively low but the shear forces are greater [86]. This indicates that the DGM model is more apt at mimicking the in vivo conditions in comparison to the USP Type II apparatus.
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ACCEPTED MANUSCRIPT In silico models have also been employed to integrate data from in vitro assays towards formulation screening in order to predict in vivo performance. Early models were not extensive and did not take into account some integral factors such as pH dependence and surface area. The GastroPlus™ software (Simulation Plus, CA) included some of these factors and accounts for
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release, dissolution, metabolism, and absorption through different GI tract compartments
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(stomach, small intestine, and colon) [87].
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Despite the availability of multiple models, a key concern for in vitro models is providing an accurate estimation of the in vivo state. Due to the complexity of the human GI tract, none of the
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in vitro simulation and modeling can accurately replace in vivo experiments. The complexity is
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further multiplied when the drug molecules are larger in size such as peptides and proteins when compared to small molecules.
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9. Methodologies to address GI variability and to increase bioavailability of peptides
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9a. Traditional Approaches
Intestinal absorption enhancers: Formulations based on permeation enhancers promote
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transport of peptides across tight junctions. Fatty acids (eg. sodium caprate [88]), bile salts (eg.
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sodium taurodeoxycholate [89]), anionic polymers (eg. poly acrylic acid [90]), cationic polymers (eg. chitosan and trimethyl chitosan [91]) and surfactants (eg. sodium dodecyl sulphate) are
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commonly used drug permeation enhancers. While some success has been observed with absorption enhancers, they do not necessarily work upto expectation and warrant more evaluations. Peptidase inhibitors:
Co-administration of peptidase inhibitors with peptides can
significantly improve oral bioavailability. Puromycin has shown to increase absorption of metkephamid by ~ 7-fold, increasing from 0.5% in absence of inhibitor to 3.5% in presence of
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ACCEPTED MANUSCRIPT puromycin. This was ascribed to the inhibition of aminopeptidase N by puromycin [92]. Despite the significant improvement that peptidase inhibitors provide, long term use is still questionable. Long term inhibition of peptidases can lead to improper digestion of nutritive proteins and peptides and can also result in stimulation of peptidases as an endogenous regulatory mechanism.
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pH modifiers: As mentioned earlier, most of these peptidases have activity at pH 7 to 8.
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Enteric-coated tablet formulations containing a pH modifier such as citric acid have been used to
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alter the intestinal pH so that it is sub-optimal for enzyme activity [93]. In addition to using pH modifying excipients, peptide modifications can be made that make the peptides resistant to
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degradation in the GI tract. For example, proline- and hydroxyproline-containing peptides have
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been found to be resistant to degradation by digestive enzymes. Furthermore, presence of C terminal proline has been reported to make peptides resistant to some peptidases [94].
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Dosing in fasted state: Limited studies are available for the effect of food and water intake on
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peptide bioavailability [52, 56]. Further evaluation with different peptides is needed before any substantial conclusions can be drawn regarding the effect of food on peptide absorption and
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bioavailability.
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Inhibitors of CYP3A and P-gp: The results presented have demonstrated the significant impact that CYP3A and P-gp may have on the bioavailability of peptides. Inhibition of CYP3A
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and/or P-gp can be expected to improve the bioavailability and lower the variability observed in systemic drug concentrations of orally delivered peptides. D-α-tocopheryl-poly (ethylene glycol) 1000 succinate (TPGS) is an excipient that has been found to improve the absorption of cyclosporine in volunteers [95] apparently by inhibition of P-gp. Various juices such as grape fruit juice have been shown to inhibit CYP3A. Furocoumarin derivatives have been isolated from grape fruit juice which are considered to be responsible for CYP3A inhibition.
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ACCEPTED MANUSCRIPT Peptide properties and their modifications: In an attempt to improve the intestinal permeability of peptides, various research groups have suggested modifications to the peptide structure. Borchardt et al. pointed out that a positive and neutral charge enhances permeation across intestinal mucosa [96]. Permeability was found to depend on the size too, suggesting
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paracellular transport. In another paper by the Borchardt et al., it was shown that hydrogen
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bonding is a major factor in determining the intestinal permeability of peptides [97]. A role of β-
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turn structure has also been suggested by Borchardt group in determining permeability across CaCo-2 cells [98]. In a separate study by Trier et al., it was shown that acylation of glucagon like
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peptides enhances cell membrane binding [99]. However, there is much uncertainty in how these
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modifications to peptide structure might alter PK and affect biological activity. Varying the molecular mass can also play a role in peptide absorption. The potency of the orally administered
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peptides decreases as the chain length increases. In a study by Roberts et al., thyrotropin
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releasing hormone (MW ~362 Da), luteinizing hormone-releasing hormone (MW ~1182 Da), and insulin (MW ~5800 Da) showed an inverse relationship between molecular weight and
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potency [100].
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Dosage form design: Gastroretentive formulations have been adopted to enhance the retention of the dosage form in the stomach [101, 102]. Polymers such as chitosan and trimethyl
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chitosan are advantageous for bioadhesive systems for oral peptide delivery as they act as permeation enhancers as well as adhesives. Lueβen et al. [103] reported an enhancement in the intestinal absorption of buserelin, a nonapeptide, in presence of chitosan, a mucoadhesive polymer. It was assumed that chitosan increases peptide absorption by opening the intercellular tight junctions. Thanou et al.[91] reported a 34- to 121-fold increase in octreotide absorption when administered with a chitosan derivative, trimethyl chitosan at 1.5% polymer solution.
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ACCEPTED MANUSCRIPT As mucus has a high turnover rate, nonspecific mucoadhesive systems are not very effective in vivo. Cell specific adhesion has been attained by targeting cell surface glycans using lectins and found to improve absorption. Lectin conjugated nanoparticles showed 23% absorption when compared to < 0.5% with nanoparticles without surface lectins [104]. However, lectins have
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some toxicity related concerns [105].
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Particulate delivery systems such as microparticles and nanoparticles have also been used to
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enhance absorption following oral delivery[106, 107]. Nanoparticles are expected to pass through the intestinal mucosa intact. Different polymers such as chitosan, cellulose, and poly
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(lactide-co-glycolide) have been used for oral delivery of peptides such as insulin. In one such
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study, insulin loaded chitosan nanoparticles showed a significant improvement in efficacy to reduce glucose levels following oral administration when compared to insulin chitosan solution
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[108].
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Site specific delivery has also been proposed to enhance oral bioavailability of peptides. Colon specific delivery is anticipated to be beneficial for peptides as the proteolytic activity is to
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a minimum in the colon [109]. Saffran et al. developed a colon targeted system, wherein peptide
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drugs were coated with azo-polymers that do not degrade in the stomach and small intestine
drug.
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[110]. Once the system reaches the colon, microflora degrades the azo-polymer and release the
Lipids have been known to enhance bioavailability by reduction of gastric emptying. A relation between chain length of fatty acids and the slowing of gastric emptying has been shown by Hunt and Knox [111]. 9b. Novel technology based approaches for overcoming the oral peptide bioavailability
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ACCEPTED MANUSCRIPT Chuang et al. used self-assembling bubble carriers [112] to deliver insulin. This study in diabetic rats used a foaming agent (sodium bicarbonate) that can generate CO 2 bubbles upon exposure to a diethylene triamine pentaacetic acid (DTPA) dianhydride created acidic aqueous environment. The relative insulin bioavailability was 21.7 ± 1.7 when the foaming agent was
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used in comparison to 0.5 ± 0.3 for free insulin. In addition to using insulin as a model peptide, a
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surfactant (SDS, sodium dodecyl sulfate) was also added in the formulation. The authors propose
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that improved oral bioavailability was due to DTPA (please mention DTPA was added in the formulation and expansion of the same) and SDS, which serve as protease inhibitors and
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absorption enhancers. they improve the oral bioavailability of insulin and induce hypoglycemic
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effects.
In another novel approach, GLP-1 mimetic exenatide was successfully encapsulated in
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nanoparticles made of a mixture of albumin and dextran, and further cross-linked using sodium
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trimetaphosphate [113]. Apparent increase in lymphatic uptake, due to the presence of dextran, resulted in improvement of the relative oral bioavailability of exenatide in rats compared to
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subcutaneous injection. Exenatide has also been modified to include albumin binding domai ns
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[114]. These long acting analogs of exenatide displayed better pharmacokinetics and pharmacological effects compared to native exenatide in diabetic mice.
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Many technologies are being developed and are in the clinic to improve bioavailability or oral peptides (Table 3). The Eligen® technology contains a small carrier, such as sodium N-[8(2-hydroxybenzoyl) Amino] Caprylate (SNAC) [115], which is attached non-covalently with biomolecules. The drug-carrier complex is able to cross the epithelial membrane much more efficiently than the drug alone. Davies et al. recently reported a significant reduction in body weight with oral semaglutide formulation prepared using SNAC [116]. The Peptelligence™
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ACCEPTED MANUSCRIPT technology constitutes citric acid (pH modifier), an acylcarnitine, an antioxidant, and other functional excipients such as disintegrants and binder [93]. Oral delivery of octreotide (via capsules) has been testing Using another novel platform called TPE® (Transient Permeability Enhancer). The GIPET technology combines enteric coating with permeation enhancers
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(medium chain fatty acids) to facilitate peptide’s absorption across the intestinal epithelium [17,
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117, 118]. Novel hydrophilic aromatic alcohols are also being used as permeation enhancers
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[119], towards development of oral calcitonin and insulin formulations [120]. Aguirre et al. [121] provide a good overview of the technologies that are either commercially available, or are in
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preclinical/clinical development. However, many of these technologies suffer from very low
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bioavailability and variability challenges mentioned before.
In another recent advancement, Rani therapeutics has reported a new approach for oral
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delivery of large molecules using a pill which according to the inventors is a “mini swallowable
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auto-injector that delivers drug directly into the highly vascular intestinal wall allowing the drug to be absorbed quickly into the blood stream” (https://www.medgadget.com/2017/07/pill-
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replace-needles-interview-mir-imran-chairman-ceo-rani-therapeutics.html).
The mechanism of
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action of the pill depends on gaseous reactions that provides momentum to make the microneedle containing the drug inject into the tissues. In another work, Traverso et al. [122] has
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demonstrated a microneedle based oral delivery system that has comparable the blood-glucose response following insulin administration via the GI tract vs subcutaneous route. The safety aspects of these technologies and widespread reproducibility in various animal models is yet to be seen.
10. Conclusions
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ACCEPTED MANUSCRIPT Oral delivery of therapeutic peptides requires deeper understanding of the physiological barriers and disruptive approaches to overcome the same in an integrated fashion. We have highlighted that the physiological challenges are manifold and must be accounted for in addition to overcoming protease inhibition and acid stability issues that are commonly mentioned for this The life of a peptide during oral delivery involves transition through the
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area of research.
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stomach where it is denatured by acids and degraded by peptidases/proteases. The remaining
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peptide will face permeability barriers in the GI tract and a fraction of it will be absorbed, whereas the rest is excreted out unabsorbed. Figure 2 summarizes the various root causes for the
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variability encountered in translational studies.
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Overcoming these challenges by utilizing learnings from small molecule oral dosage form development and a systematic approach to address GI physiological issues is a must if we have
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to develop oral peptide therapeutics. While technology and formulation based approaches
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routinely follow the use of intestinal absorption enhancers [88, 91], peptidase inhibitors [92], pH modifiers [93, 94] and modifications of the physicochemical properties of the peptide [97, 99] ,
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they address only half of the problems. In addition, peptide size, lipophilicity, and systemic
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circulation time to compensate for the effects of first pass must be modified and tested to increase bioavailability. Few novel technology based approaches have been reported in the last
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couple of years such as bubble carriers [112], use of nanoparticles [113], and an ingestible microneedle pill [122]. However, coupling the utility of a technology with overcoming the appropriate physiological barrier effect is a must. A key understanding of various endogenous factors affecting the absorption and transport of the peptide across the site of absorption is a good step to start with. Enabling technologies for oral peptide delivery should also address increasing
30
ACCEPTED MANUSCRIPT the residence time of the peptide at the site of absorption and tuning the microenvironment of the
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dosage form to the GI tract environment for acceptable solubility and absorption.
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ACCEPTED MANUSCRIPT
T P
I R
C S
U N
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T P
E C
C A
Figure 2. Schematic representation of the factors impacting variability, and approaches to address them during oral administration of peptides. Gastroretention can potentially be beneficial for peptides that are absorbed through upper GI tract. Successful colonic delivery suffers from disadvantages such as variable transit time and heterogenous microflora, a challenge that can be addres sed in the near future.
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ACCEPTED MANUSCRIPT Chronic diseases such as type 2 diabetes can benefit from oral delivery as it can overcome the need for frequent injections. Improving the half-life of the orally bioavailable peptide is another important aspect that can provide momentum to oral peptide delivery by way of offering less frequent doses (QD vs BID or TID) to patients. Weight based dosing and administering
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peptide in a fasted state, and with the right amount of fluid can help with improving variability.
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Peptide drug delivery systems that can tackle the variabilities can offer promise to creating better
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drug products.
Oral peptide delivery is an exciting area of research due to the scalability of tablets and
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capsules as drug products and the convenience it offers to patients, and hence will continue to
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attract the attention of scientists. The demand for efficient oral peptide therapeutics and dosage forms will continue to increase due to the prevalence of cancer and the need to have non-
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injection based treatment methodologies. Several cancer therapeutic development programs are
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already utilizing small molecule oral therapy compared to IV injections and this is an area where peptides can further add utility particularly in the area of immune-peptide therapeutics for
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oncology [123]. While technologies and approaches are being improved, attention needs to be
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given to the physiological parameters highlighted in this article, which may further enhance development of oral peptide therapeutics in a holistic way. The immediate next steps should be
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to understand and overcome the GI physiological aspects for overcoming absorption barriers, as “gut filling” and the contents of the GI tract will truly call the shots on oral peptide delivery.
Acknowledgement The authors would like to thank Ben Ambalong of MedImmune for his assistance with Figure 2.
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Oral Peptide Delivery: Translational Challenges due to Physiological Effects
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Puneet Tyagi, Sergei Pechenov, and J Anand Subramony*
Corresponding author: J Anand Subramony
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MedImmune Inc,
One MedImmune Way,
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Gaithersburg, MD 20878
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Phone: 301-398-1418, E-mail: [email protected]
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ACCEPTED MANUSCRIPT TABLES Table 1. Clinically advanced technologies for oral peptide delivery (source: PharmaCircle™). Interested readers are encouraged to refer to clinical trials database (clinicaltrials.gov) for most recent updates.
Peptide
Technology
Insulin
Chiasma Inc.
Octreotide
Enteris BioPharma, Inc.
Leuprolide
Emisphere Technologies Inc /Novo Nordisk Merrion Pharmaceuticals /Novo Nordisk
Insulin GLP-1 analogue
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AN
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Insulin
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HIM2; Absorption enhancers and enzyme inhibitors Transient Permeability Enhancer (TPE®) technology; oil based suspension Peptelligence™; absorption enhancers Eligen®; absorption enhancers GIPET™; absorption enhancers POD™; Absorption enhancers and enzyme inhibitors
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Biocon Ltd.
Oramed Pharmaceuticals
Dev
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Company
Insulin
Pain indication
Unigene/Enteris
Ph
GSK
PTH
Unigene/Enteris
Ph
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Cara Therapeutics
Table 2. List of relevant parameters that can led to low peptide bioavailability and potential tools to overcome the physiological barrier or methodology. Parameters that impact peptide bioavailability Variability in GI tract - Intestinal permeability
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Potential tools to overcome the physiological barrier/methodology
Can be tackled by a combination of fine tuning physico-chemical properties of permeation
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Site of absorption
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Absorption site residence time
Effect of meal uptake Effect of peptidases
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Variability in preclinical models
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- Gastric mucus Effect of gastric and intestinal pH
enhancers complementing the properties of peptide (such as LogP, pH dependent solubility). Perform site of absorption studies and deliver peptide to that location via controlled release. Mucoadhesives can be used to retain the formulation at the absorption site to maximize absorption. Mucolytic agents; mucus penetrating particles pH modifiers/buffering agents to modulate the immediate peptide environment. Dosing in fasted state Peptidase inhibitors; peptide modification to enhance stability against peptidases Rodents are not ideal due to issues with dosing oral formulations; pigs and dogs are widely preferred due to anatomical similarities to humans. Use of higher samples (n value) to mak bioavailability values statistically significant.
Humans
Rats
Dogs
Pigs
1 hour
0.7-2.1 hours
3.9 – 5.3 hours
1.5-6 hours
46 minutes
29 minutes
23 minutes
48 minutes
224 – 252 minutes
21.4 – 25.1 hours (fed state)
96 – 128 minutes (fed state)
3 – 4 hours
0.58
0.06
0.86
0.62
2 – 4.5
3
3–5
2.2 – 4.3
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Gastric emptying time Gastric retention time Small intestine transit time Absolute water content (g/cm gut length) pH of the stomach (fed)
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Table 3. Comparison of key GI tract parameters across different pre-clinical and species and humans commonly used for pharmacokinetic and efficacy evaluations [66, 71-74].
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ACCEPTED MANUSCRIPT 6.5 – 7.1
6.2 – 7.5
6.0 – 7.5
177 cm
17 cm
90 cm
125 cm
144 ± 52 µm
31.3 ± 11.4 µm
425 µm
190.7 ± 80.7 µm
1 – 1.6 L
3.4 ml
4.33 L
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pH of small intestine Length of gut Stomach fundus mucus thickness Average stomach fluid capacity
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Translational
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Puneet Tyagi, Sergei Pechenov, J. Anand Subramony PII: DOI: Reference:
S0168-3659(18)30498-X doi:10.1016/j.jconrel.2018.08.032 COREL 9441
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
26 June 2018 21 August 2018 22 August 2018
Please cite this article as: Puneet Tyagi, Sergei Pechenov, J. Anand Subramony , Oral peptide delivery: Translational challenges due to physiological effects. Corel (2018), doi:10.1016/j.jconrel.2018.08.032
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ACCEPTED MANUSCRIPT Oral Peptide Delivery: Translational Challenges due to Physiological Effects
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Puneet Tyagi, Sergei Pechenov, and J Anand Subramony* [email protected] MedImmune Inc, United States
*
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Corresponding author at: One MedImmune Way, Gaithersburg, MD 20878, United States.
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Abstract
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Oral delivery of peptide therapeutics as a convenient alternate to injections has been an area of research for the pharmaceutical scientific community for the last several decades. However, systemic delivery of therapeutic peptides via the oral route has been a
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daunting task due to the low pH denaturation of the peptides in the stomach, enzymatic instability, and poor transport across the tight
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junctions resulting in very low bioavailability. The low bioavailability is accompanied by large intra- and inter-subject variability leading to translational issues, preventing the development of successful peptide therapeutics. The inter-subject variability leads to large differences in pharmacologic responses in individuals and thus the dose required to produce therapeutic effect could vary between individuals making the development of drug product a very difficult task. A substantial amount of research has been (and continues to be) performed with a focus on getting acceptable absorption and reproducible results. Nonetheless, the high variability and low bioavailability during oral administration of peptides is still a work in progress and under-explored in a systematic way. 1
ACCEPTED MANUSCRIPT While there are several review articles and scattered publications that discuss potential technologies for oral peptide delivery, a detailed look into the physiological challenges and absorption barriers which are a hindrance to successful clinical translation, is lacking. Herein, we have analyzed the physiological barriers within the gastrointestinal (GI) tract that are the root causes for the low bioavailability and high variability of oral delivery of peptides in humans. In particular, we have taken a detailed look at the key
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influencing factors such as the nature of various GI tract parameters, components of the GI tract that influences the uptake, site of
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absorption, pH of the gastric and intestinal compartments, food effect, and role of peptidases in affecting oral peptide absorption. Lack of in vitro - in vivo correlations and variability in animal models have also been highlighted as key impediments in understanding the challenges.
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The unique perspective presented herein for overcoming the physiological absorption barriers, will offer better
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developability approaches and will positively impact clinical translation of future oral peptide therapeutics. A deep understanding of these effects are vital, given the emergence of microbiome and oral biologic drug delivery that are fast emerging as the next wave of
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personalized patient centric therapies.
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1. Introduction
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Therapeutic peptides have come a long way since insulin was discovered and used as a therapy for diabetes. After insulin,
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oxytocin was chemically synthesized in 1950 and was approved in 1980 for the treatment of uterine contraction [1]. Since then the therapeutic peptide market expanded exponentially and currently over 60 peptide products are available in the market. Market
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research predicts that the peptide therapeutic market is expected to reach ~ $ 46 billion by 2025 with a highly impressive 9.1% CAGR from 2016 mainly due to the utility of peptide therapeutics in the area of oncology and metabolic disorders [2]. Most of the peptides are administered to patients through injections via the parenteral route. However, the injectable route is not the most convenient, and has issues with needle phobia and non-patient compliance. Consequently, there is a need for development of 2
ACCEPTED MANUSCRIPT alternate delivery routes for administration of peptides. Oral delivery can improve patient compliance and provide the needed convenience and therefore is of great interest for the development of peptide therapeutics [3, 4]. Oral dosage forms are also easy to scale up and convenient in terms of packaging and handling compared to liquid injections. The last two decades have seen several
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efforts for the development of oral peptide therapeutics (Table 1). However, peptides are not designed by nature to be systemically
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bioavailable for oral administration and the reasons are manifold. Oral bioavailability of peptides is very low due to peptide
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degradation in the GI tract (due to protease and acidic pH) and their poor permeability across the epithelial barrier because of high
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molecular weight, large size [5], and amphiphilicity. Figure 1 summarizes the published data on oral bioavailability in humans and interestingly it follows an inverse trend with MW. Most peptides have <1% absolute bioavailability with high variability when
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delivered via oral route [6, 7]. For example, bioavailability of desmopressin acetate oral tablets in humans ranges from ~ 0.08 to
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0.16% [11, 12]. Despite these low numbers, Minirin (desmopressin acetate) tablet has become a viable product as it is convenient to
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administer it orally for the indication it is used for (to control nighttime bedwetting in children) and also due to its wide therapeutic
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window. However, for larger peptides like insulin, it is essential to have low variability because of its low therapeutic window and the need for accurate onset and efficacy for the needs of glycemic control for diabetic patients.
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Table 1. Clinically advanced technologies for oral peptide delivery (source: PharmaCircle™). Interested readers are encourage d to refer to clinical trials database (clinicaltrials.gov) for most recent updates.
Company
Peptide
Technology
Development stage
Biocon Ltd.
Insulin
HIM2; Absorption enhancers and enzyme
Phase I
3
ACCEPTED MANUSCRIPT inhibitors
Chiasma Inc.
Octreotide
Enteris BioPharma, Inc.
Leuprolide
Emisphere Technologies Inc /Novo Nordisk Merrion Pharmaceuticals /Novo Nordisk
Insulin GLP-1 analogue
Oramed Pharmaceuticals
Insulin
Cara Therapeutics
Pain indication
Insulin
D E
T P
I R
C S
U N
A
M
T P
E C
GSK
Transient Permeability Enhancer (TPE®) technology; oil based suspension Peptelligence™; absorption enhancers Eligen®; absorption enhancers GIPET™; absorption enhancers POD™; Absorption enhancers and enzyme inhibitors
PTH
C A
4
Phase III
Phase II Phase I Phase III Phase I Phase II Phase II
Unigene/Enteris
Phase I/Terminated
Unigene/Enteris
Phase II/Terminated
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Figure 1. Oral bioavailability of small molecule drugs and peptides in comparison to their molecular weights [8-12]. Inset graph shows the bioavailability of peptide drugs. The trend follows an inverse relationship between MW and oral bioavailability (BAV) in humans. Figure does not show the variability for BAV.
5
ACCEPTED MANUSCRIPT To understand the reasons for the low systemic bioavailability and pharmacokinetic (PK) variability of peptides after oral administration, a close look at the transport properties of the peptide and its molecular dynamics during its residence within the GI tract are important. In the stomach and along the GI tract, peptide molecules are susceptible to peptidase and protease
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action, working on breaking the peptide down to single amino acids [4]. The amount of survived
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peptide is proportional to its protease and acid stability and reversibly proportional to the amount
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of time it is exposed to the low pH of stomach, and the amount and activity of the respective proteases. After transiting through the stomach, the peptide goes into intestines, where the GI
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tract lining is protected by a layer of mucin. Peptide molecules need to transport and cross the
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mucin layer before it can enter into the layer of epithelial cells. These cells efficiently transport amino acids and small polypeptides, but prevent larger hydrophilic, potentially bioactive
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molecules, from entering systemic circulation [13]. Yet some of the molecules can reach the
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circulation via passive route, by passing between the cells, the so called tight junctions, or via active cellular transport way aided by a transporter ligand that is present on the peptide [14].
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Hence peptide properties, namely size and amphiphilicity, exposure time in the GI tract, amount
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of peptide present at the site of absorption, are some of the key factors that can influence bioavailability. Physiological attributes, such as amount of proteases and peptidases present, pH
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microenvironment, gastric and intestinal surface area, thickness of mucus layer, presence of accompanying food, permeability, or leakiness of GI tract lining, are some of the other intrinsic barriers that determine the bioavailability. The transport across GI tract is quite variable since it is a dynamic system, whereby its function depends on the age, gender, fed state, time of the day, dietary preferences, and GI health status resulting in great intra- and inter- subject PK variability [15]. Particularly the GI tract attributes are critical if the compound has a very narrow absorption
6
ACCEPTED MANUSCRIPT window. The above-mentioned parameters are some of the key reasons why peptides have extremely low bioavailability on their own. In order to increase bioavailability for oral peptide delivery, several approaches including use of technologies have been tested in preclinical models and in humans. However, due to reasons
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outlined above, high inter-subject variability is seen in many of these studies, and therefore a
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systematic approach is required to further understand the root causes for poor bioavailability and
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high inter-subject variability. For example, in an oral insulin clinical trial report published by Kapitza et. al.[16] in 2010, the bioavailability varied by more than 100% in the first hour. In
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another study, oral administration of the Gonadotropin-releasing hormone (GnRH) antagonist
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acyline in a tablet dosage form resulted in highly variable pharmacokinetic data. The variability among subjects was so high that the clinical outcomes between different doses were also not
M
significantly different [17]. For peptides with wide therapeutic window, such as GLP-1, high PK
ED
variability may be less of an obstacle, but nausea due to high Cmax could be a problem. Nevertheless, nausea can be managed by dose titration and by using controlled release
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technologies. For peptides with a narrow therapeutic window, such as insulin, high PK
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variability may result in either lack of therapeutic effect or fatal hypoglycemia which are very critical to patients.
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As high variability and low bioavailability continue to act as hurdles for oral peptide delivery, the purpose of this review is to understand and highlight the physiological factors that impact the development of oral peptide therapeutics in preclinical models and in human subjects during clinical translation. For broader reviews covering oral delivery of peptides, readers are directed to relevant published literature [4, 18-22].
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ACCEPTED MANUSCRIPT 2. Variability in GI tract parameters Parameters of the digestive tract, such as surface area, motility and permeability, can affect the absorption of peptides. Additionally, variations in gastric volume, vascularity of the intestinal lining that impacts the blood flow, thickness of the mucus layer, and site of absorption can also
T
alter the absorption and hence the bioavailability of the peptides. In a recent publication,
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Sugihara, et al systematically compared 113 human bioequivalence studies of 74 small molecule
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actives [23]. Their analysis suggested that factors such as luminal fluid volume, transit time, and presence of metabolizing enzymes play a significant role in intra and inter subject variability of
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drug absorption. Though small molecules are different than peptides, we can roughly infer from
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the data that these factors can influence the absorption of peptides as well. Let us look at some of these GI parameters in the following paragraphs.
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Surface area: The surface of the stomach has a small area relative to the small intestine. The
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small intestine has folds, villi, and microvilli that enlarge the surface are of the intestine. A range of 520-1536 cm2 has been reported for human stomach mucosal surface area [24].
More
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recently, Helander et al. [25] used morphometry to assess the stomach area in healthy volunteers
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and reported that the mucosal surface area of the stomach is ~ 500 cm2 in alignment with the previously reported range. Variability in the small intestine length has been reported to range
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from 160 - 430 cm in people with no abdominal disorder (measured using radiography) [26] and a range of 385 - 538 for patients who have undergone laparotomy [27]. As the primary role of small intestine is the uptake of nutrients such as peptides, variations in length and surface area can appreciably alter the bioavailability of peptides. Motility and resulting exposure: Variations in the small intestine motility can result in significant variations and poor bioavailability of peptides by altering the site of absorption and/or
8
ACCEPTED MANUSCRIPT duration of exposure. In healthy volunteers [28], speed of phase III contractions in duodenumjejenum area varies from 2.4 - 10 cm/minutes. The variation in duration of phase III contractions (ranges from 3.6-5.7 minutes) in a healthy individual is not as variable as speed of contraction. In a diseased state, such as patients suffering from Crohn’s disease, the variation in speed of phase
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III contractions can go from 2.1-22.5 cm/minute. Another significant observation in diseased
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patients is the lack of phase III contractions in ~30% of the patients. High speed of contraction
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can cause poor peptide absorption in some patients. Further, the absence of contraction can cause large gradient of peptide deposition within the small intestine by which there will be localization
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of peptide dose in one area and a more concentrated exposure in another area, leading to
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variability.
Intestinal permeability: Region dependent absorption has been observed for many
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therapeutic peptides including calcitonin [29]. Modifications and inter-subject variability in
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intestinal permeability has been assessed by measuring urinary excretion of non-metabolized sugars (lactulose and mannitol) administered orally [30]. Several diseases such as irritable bowel
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syndrome (IBS) are associated with modification in intestinal permeability marked by diarrhea
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and weight loss. In a study to measure intestinal permeability in healthy volunteers vs. patients with IBS [31], subjects were dosed with 5 g of lactulose and 2 g of mannitol dissolved in 100 ml
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of water. Urine from the subjects were collected at different time intervals and analyzed to obtain a measure of permeability. The results indicated that in healthy volunteers, the intestinal permeability varied from 0.01 - 0.06 lactulose/mannitol excretion ratio. Furthermore, for patients suffering from IBS, the ratio varied from 0.01 - 0.24.
As a standard, a lactulose/mannitol
excretion ratio ≥ 0.07 is taken to indicate the presence of an increase in permeability.
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ACCEPTED MANUSCRIPT Site of absorption: Permeability of peptides can vary from the site of absorption in the intestine. Langguth et al. calculated wall permeability of metkephamid, a synthetic opioid pentapeptide, and observed that the permeability was highest in the ileum, followed by jejunum. The bioavailability of metkephamid increased from 0.22% (following oral administration) to
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1.79% when the peptide was administered into the ileum [32]. Similar to the results by Langguth
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linear polypeptide, than in the duodenum and colon in dogs [29].
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et al., Sinko et al. also observed higher ileal absorption of salmon calcitonin, a 32-amino acid
Region dependent studies using a lipopeptide, LY303366, wherein duodenal, jejunal, and
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colonic bolus administration of the same dose and volume were conducted in Beagle dogs [33].
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It was observed that LY303366 had significantly lower drug plasma levels following administration through the colon and jejunum (Cmax of 0.3 and 0.6 µg/ml, respectively) as
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compared to oral and duodenal administration (Cmax of 1.6 and 1.5µg/ml, respectively). It is
form.
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really important to carry out a site of absorption study before designing the oral peptide dosage Formulations technologies such as immediate release and delayed release as well as
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enteric coating can be engineered in the dosage form based on the site of absorption studies.
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Similarly, for top GI and stomach absorption, gastro retention would be a suitable approach. Absorption site transition time: Following gastric emptying, an enteric dosage form will
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disintegrate in small intestine. Depending on the enteric coating, disintegration can occur in either of the intestinal segments (duodenum, jejunum, or ileum). If the dosage form disintegrates within the first 5 minutes following gastric emptying, it is considered to happen in the duodenum. Disintegration happening from 5 to 60 minutes and from 60 to 75 minutes is considered to happen in jejunum and ileum, respectively. The average intestinal transit time in
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ACCEPTED MANUSCRIPT fasted dogs and humans has been reported to range from 111 ± 17 minutes [34] and 170 ± 30 minutes, respectively [35]. Lee et al. reported the intestinal transit time for tablet formulations containing calcitonin and observed that disintegration of two formulations occurred in the jejunum (transit times of ≤ 50
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minutes) and one formulation had transit times from 20 – 90 minutes [36], indicating absorption
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from the ileum. In addition to the small intestine, the transit time in human large intestine can
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vary in the range of 8 – 72 hours [37]. This indicates that variability in transit times can result in dosage form being in an undesirable site, and eventually leading to poor bioavailability and huge
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variability between patients.
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Gastric volume: The volume of liquid being secreted in the stomach can affect the solubility of the active molecule and might also alter the rate of degradation depending upon the pH of the
M
contents. Following overnight fasting, healthy volunteers were observed to have a mean gastric
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volume of 59.6 ± 4.2 (males) and 46.3 ± 4.2 ml (females), with a range of 0 – 225 ml [38]. Similar results of mean gastric volume of 50 ml with a range of 0 – 180 ml have also been
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reported by Leech et al. [39]. Furthermore, in diseased state (such as of ulcers) the gastric
CE
volumes following an overnight fasting can go up to > 80 ml. Following collection of gastric secretion after overnight fasting in healthy men, Iijima et al. observed that the pH of the gastric
AC
secretion was 4.4 ± 1.2 mEq/10 min. Iijima et al. measured the H+ concentration of the gastric juice by titration and reported the output in a 10-min period [13]. Gastric mucus: Gastric mucus is divided into an adherent layer (adhering to the gastric mucosa) and a soluble layer (mostly freshly secreted). Iijima et al. quantified the soluble mucin concentration to range from 93.6 ± 37.8 μg hexose/ml in healthy volunteers. In the same study, the amount of reported mucus (determined by multiplying the mucin concentration by the
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ACCEPTED MANUSCRIPT volume of gastric juice collected in the 10-min period) varied from 3.2 ± 1.2 mg hexose/10 minutes in humans[13]. In a study consisting of 10 patients, Huh et al., acquired data for mucous thickness in stomach to vary from 1.03 mm - 1.64 mm [40]. Variation in mucus secretion rate and output can alter the clearance of active before it is exposed to the epithelial cell layer for
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activity. In addition, mucosal blood flow also plays an important role in the production of mucus
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and bicarbonates. Karzai et al. demonstrated the gastric mucosal microcirculatory blood flow to
Effect of gastric and intestinal pH
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3.
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be 61 ± 33 units [41].
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Gastric pH is an important factor contributing to the stability of the peptide. It has been reported that the gastric pH is fairly stable and within a narrow range in young individuals [42].
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A median fasting gastric pH of 1.7 (range 1.4-2.1) and median fasting pH in mid duodenum of
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6.1 (range 5.8-6.5) was found in 24 young healthy men and women [42]. During meals, pH in the stomach might increase to 6, but quickly returns to normal within a few minutes. However, with
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age, elderly people tend to have more pH variation. A study conducted on healthy volunteers 65
CE
or older, it was found that 10% of the people had a high fasting gastric pH of ~6 [43]. Furthermore, many individuals took many hours before the pH came back to normal value of 1.7.
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Some individuals were found to have achlorhydria and did not see a decrease in gastric pH for up to 6 hours [43]. Such high variations can have a significant impact on the presence of peptidases in the stomach and eventually induce variations in the extent of peptide absorption if the solubility factors of the peptide for better absorption have a narrow pH window dependence. In addition to age, disease states can also affect the pH. People suffering from cystic fibrosis tend to produce a lot of mucus, which can block the secretions from the pancreas. An acid-base
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ACCEPTED MANUSCRIPT imbalance is the result, which eventually causes lower and variable intestinal pH in cystic fibrosis patients [44]. In the study by Youngberg et al., [44], it was noticed that the pH in the duodenum for 25% of cystic fibrosis patients was below 5.5, up to 2 hours after meals. A change in the GI tract pH, as seen in cystic fibrosis patients, poses serious problems in oral
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delivery of peptides as it can affect the solubility of the peptide. A variation in pH can also serve
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as a major factor in peptide stability. Furthermore, as many tablets are enteric coated for delivery
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in the intestine, a low pH can lead to improper disintegration of tablet. Lee et al. [36] have performed a series of experiments with calcitonin (a 32-amino acid linear polypeptide) which
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highlighted the variation in disintegration of enteric coated tablets. Lee et al. showed that,
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following oral administration of enteric coated tablets containing citric acid, the time to reach intestinal pH trough varied from 35 to 90 minutes. Therefore, a careful choice of enteric coating
M
material is a key factor in oral peptide delivery.
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Another important aspect of pH variation lies in the location where the enteric coated tablet is disintegrating. An oral enteric coated tablet formulation containing 565 mg of citric acid and
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1.2 mg of calcitonin was found to give high plasma concentration of calcitonin in dogs when the
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tablet disintegrates in the late jejunum or ileum. The authors suggested that this was because the pH recovery in the late jejunum or ileum is slow (takes up to ~140 minutes). In comparison,
AC
when the tablet disintegration occurs in the duodenum, the plasma concentration of calcitonin was low and the pH recovery was fast (takes up to ~15 minutes) [45]. In the absence of citric acid in the formulation, no calcitonin was observed in the plasma and no decrease in intestinal pH was observed [36]. Therefore, a decrease in intestinal pH seems to be necessary for the absorption of calcitonin. This again confirms that understanding the site of absorption is beneficial for optimum delivery of peptides, and the dosage form can be designed to target the
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ACCEPTED MANUSCRIPT optimal site of absorption using controlled release technologies and or by creating local micro pH environment to maintain appropriate solubility and super saturation.
4. Effect of gastric emptying
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It is widely considered that peptide absorption from the stomach is very slow whereas the
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absorption from the small intestine is much fast and rapid. This is believed to be due to the large
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surface area of the small intestine. Gastric emptying is likely to be a rate limiting step in the absorption of peptides and can therefore account for much of the observed variation (in time of
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onset of action) amongst patients. Gastric emptying can be influenced by many factors including
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posture, pH, presence of other drugs, and hormonal activity. For example, Steingotter et al. observed that gastric peristalsis was slower in patients in upside down position in comparison to
M
patients in seated position. Also, intra gastric distribution was significantly different in the two
ED
volunteer groups [46]. Common drugs such as clonidine (used to treat hypertension) and Sumatriptan (used to treat migraine headaches) have also been shown to modulate gastric
PT
emptying significantly [47].
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Gastric emptying during fasted state happens when migrating complex, a band of regular contractions, periodically moves indigestible solids from the stomach to the distal small intestine.
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Studies have confirmed that non-disintegrating units (such as enteric coated tablets) are emptied out from the stomach in the third phase of the cyclic motility pattern. Thus, the intestinal absorption of peptides delivered as an enteric coated formulation largely depends upon the gastric emptying. Variation in gastric emptying can occur if the dosage form linger in the less muscular body of the stomach and not be emptied out to the small intestine [48], thus delaying gastric emptying. Lee et al. observed gastric emptying time ranging from 30 - 150 minutes in
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ACCEPTED MANUSCRIPT beagle dogs dosed with exactly the same formulations of calcitonin [36]. As the gastric emptying in fasted state is dependent on a variable migrating complex, we might observe improper disintegration and dissolution of the dosage form. In comparison, in the fed state, the formulation is expected to have a prolonged gastric residence, thereby ensuring proper dissolution [49]. Fed
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state can also be characterized by elevated pH, which in achlorhydria patients can lead to
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dissolution of enteric coated tablets in stomach with resulting lower bioavailability and dilution
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with food components. In addition to inter subject variations, diseases can also alter the gastric emptying time. Achlorhydria has been shown to go together with delayed gastric emptying [50].
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In a study comprising of elderly achlorhydria patients, it was observed that, on an average, the
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patients took 40 minutes to empty 50% of a 300 ml of consumed orange juice. In comparison, healthy volunteers cleared the same amount in 18 minutes [50]. Gastric emptying has also been
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reported to be delayed in gastric ulcers and gastric carcinoma, whereas an accelerated gastric
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emptying was seen in celiac disease and duodenal ulcers [51].
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5. Effect of meal and water intake
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Studies have indicated that bioavailability of small molecule drugs are affected by meal timings. However, peptides and proteins have not been evaluated extensively for effect of food
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effect on oral delivery. Furthermore, vast structural differences in small molecules and peptide/proteins makes it difficult to extrapolate any learnings from the small molecule area. In a study conducted by Karsdal et al., the effect of meal and water uptake on oral salmon calcitonin was observed in 56 healthy women [52]. The formulation [53] contained 0.8 mg of calcitonin and 200 mg of 5-CNAC, [8-(N-2- hydroxy-5-chloro-benzoyl)-amino-caprylic acid)], a permeation enhancer. The study evaluated the pharmacokinetic and pharmacodynamics of
15
ACCEPTED MANUSCRIPT salmon calcitonin when taken with different volumes of water (50 or 200 ml) and at different times before a standard meal (10, 30 or 60 minutes). It was observed that the level of maximum concentration and AUC of the plasma levels was significantly dependent upon the amount of water intake, suggesting an influence of water intake on peptide absorption. Administration of
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the peptide with 50 ml of water achieved two- to three-fold higher absorption in comparison to
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the administration with 200 ml water. The effect of water intake was significantly higher
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irrespective of the time post meal was taken. A better efficacy was also observed when the dose was administered with 50 ml of water. More evaluation is needed to confirm if higher
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bioavailability is a result of higher concentration of peptide and permeation enhancer.
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In another study using the oral calcitonin formulation (0.8 mg calcitonin and 200mg 5CNAC), the effect of predose meal was evaluated on the oral bioavailability [54]. The predose
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meal at 4 and 2 hours before dosing significantly decreased relative oral bioavailability of oral
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calcitonin to 26% compared with that of the dose in the fasting state. Meals taken 10 minutes after dosing also resulted in decrease in bioavailability to 59%. This hints upon the potential
PT
influence of gastric retention and food interaction on bioavailability of the orally administered
CE
peptide. However, many such studies are needed to properly verify the effect of food. Li et al. evaluated the effect of meal composition on the systemic availability of LY303366, a
AC
lipopeptide being developed as an antifungal agent [33]. The study evaluated the effect of different types of foods (proteins, lipid, and carbohydrate), and osmolality on the plasma concentrations of LY303366. Of the food types tested, protein (125 kcal diet) and lipid (250 kcal diet) decreased the plasma level of LY303366 in comparison to a fasted plasma concentration. The effect of a hyperosmolar solution (480 mOsm/kg) on the plasma concentrations was not significant. However, a significant decrease in drug plasma levels was observed with
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ACCEPTED MANUSCRIPT administration of the 250 kcal, hyperosmolar liquid nutrient meal of mixed caloric composition. The authors hypothesized that the possible cause of a negative effect of meals might be due to the influence of meal viscosity and/or binding of the peptide to the meal ingredients. Li et al. suggested that these effects can make a major impact on the peptide bioavailability if significant
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portion of the drug is absorbed in the upper small intestine. As the peptide LY303366 contains
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lipid, the chances of the peptide binding to lipid diet is highly plausible. A similar reduction in
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bioavailability was observed when EP01572, a novel, synthetic ghrelin agonist was dosed with food [55]. In comparison to dosing without food, the Cmax and AUC values decreased by half
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when the dose was given with food. Tuvia et al. also observed lower plasma octreotide levels
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when oral octreotide formulations were administered with a high-fat, high-calorie meal [56].
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6. Effect of peptidases
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Many proteases and peptidases degrades the peptides in the GI tract, leading to poor bioavailability. However, there is only limited data available for the variation in the activity of
PT
peptidases, as it is hard to quantify. Tor Lindberg [57] observed that the specific activity of
CE
dipeptidases (attacking hydrophobic amino acids) in the small intestine mucosa of adult humans ranges from 23.7 - 125.2 units/mg nitrogen where one unit is defined as the peptidase activity
AC
hydrolyzing 1 µmole of dipeptide per min at 40 ºC. However, the study was performed in diseased patients.
Wang et al. [58] have compiled a good summary list of peptide stability in human gastric fluid in the presence of pepsin (after 2 hours) and human small intestine fluid in presence of pancreatin (after 30 minutes). From the list of 17 peptides or so they studied, it is interesting to note that only 3 of them had any percentage peptide left in small intestinal fluid containing
17
ACCEPTED MANUSCRIPT pancreatin (namely cyclosporin 99%, desmopressin 25%, and octreotide 22%) and only those 3 peptides have made it as oral peptide therapeutic products (Neoral™, Minrin™, and
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Octreolin™).
Potential tools to overcome the physiological barrier/methodology
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Parameters that impact peptide bioavailability
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Variability in GI tract -
Intestinal permeability
-
Site of absorption
-
Absorption site residence time
-
Gastric mucus
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Can be tackled by a combination of fine tuning physico-chemical properties of permeation enhancers complementing the properties of peptide (such as LogP, pH dependent solubility).
ED
M
Perform site of absorption studies and deliver peptide to that location via controlled release.
Mucolytic agents; mucus penetrating particles
Dosing in fasted state Peptidase inhibitors; peptide modification to enhance stability against peptidases
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Effect of peptidases
pH modifiers/buffering agents to modulate the immediate peptide environment.
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Effect of gastric and intestinal pH Effect of meal uptake
Mucoadhesives can be used to retain the formulation at the absorption site to maximize absorption.
Rodents are not ideal due to issues with dosing oral formulations; pigs and dogs are widely preferred due to anatomical similarities to humans. Use of higher samples (n value) to make bioavailability values statistically significant.
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Variability in preclinical models
Table 2. List of relevant parameters that can led to low peptide bioavailability and potential tools to overcome the physiological barrier or methodology.
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ACCEPTED MANUSCRIPT 7. Variability in preclinical models used to evaluate oral delivery systems A reliable prediction of the oral bioavailability is crucial for the development of delivery systems. To that end, different animal models are utilized which can mimic conditions across the GI tract[59]. Rats, pigs, dogs, and non-human primates are the common models used to evaluate
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oral delivery systems. Inter- and intra-species variability exists in the gastric physiology and can
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greatly affect the outcome in the animal models (Table 3). Also, it is difficult to dose solid oral
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dosages in rats and preliminary work has relied on administering by direct intraduodenal injections. Gastric emptying can vary from 0.7 - 2.1 hours in rats depending upon the dosage
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form density and size [60]. In comparison, the gastric emptying of solids in humans takes 68
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minutes. Pigs have a highly variable gastric emptying time, ranging from 1.5 - 6 hours. In addition to inherent variability in gastric emptying time, the size of dosage form can also affect
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the gastric emptying time. Sagawa et al. reported the gastric emptying of a capsule with 6 x 5 x
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25 mm3 size to be 1.4 hours in fasted Beagle dogs [61]. In contrast, the gastric emptying of a standard 10 g meal and 200 g meal was 9.4 and 20 hours, respectively. In a study conducted with
PT
telemetric capsules (7 x 20 mm), gastric emptying of the capsules was similar in humans and
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dogs (1.2 ± 0.4 hours) [62]. In another study, 1 mm particles emptied out within 2 hours from both humans and dogs [63]. Gastric motility is an additional complicating factor and can induce
AC
significant variability in drug absorption. Dogs and humans were found to have similar phase III activity, lasting for 18.6 and 19 minutes, respectively [64]. Phase III activity in the stomach is responsible for moving food into the small intestine. The periodicity of the phase III cycles was also similar in humans and dogs, with cycle being seen every 106 minutes in dogs and 128 minutes in humans [65]. However, dogs have highly permeable intestinal wall, which can demonstrate higher drug absorption in comparison to humans. Furthermore, in dogs, the gastric
19
ACCEPTED MANUSCRIPT emptying time is ~10 to 20 times that of humans while the gastric retention time and the GI transit time (in the fasted state) is nearly one half of that in humans. The thickness of mucus layer varies considerably in dogs (425 ± 7 µm) [66] and humans (144 µm) [67]. In comparison, rats have a mucus thickness ranging from a mean of 31.3 – 69.4 µm [68]. Also, the length of the
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GI tract ranges from 17 cm for rats to 177 cm in humans. The length of GI tract in pigs is closest
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to humans at 125 cm [69]. Basal gastric acid output is highly variable in dogs with a range of 0.3
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– 1.5 ml/minute. In comparison, pigs and humans have a gastric acid output at approximately 1 ml/minute [70].
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Thus, the choice of preclinical model should be carefully made if the absorption is in the
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stomach or top GI and for compounds with a very narrow absorption window [71]. The length of the GI components (colon, caecum, small intestine and stomach) matches very well between pigs
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and humans and therefore using a pig model might be appropriate for confirmatory studies once
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preliminary bioavailability is determined from the dog model [71]. Also, it is better to repeat the studies in a given animal model for consistency in pharmacokinetic parameters before a dosage
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form is advanced to the next stage.
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ACCEPTED MANUSCRIPT Table 3. Comparison of key GI tract parameters across different pre-clinical and species and humans commonly used for pharmacokinetic and efficacy evaluations [66, 71-74].
Gastric emptying time Gastric retention time Small intestine transit time Absolute water content (g/cm gut length) pH of the stomach (fed) pH of small intestine Length of gut Stomach fundus mucus thickness Average stomach fluid capacity
Humans
Rats
Dogs
Pigs
1 hour
0.7-2.1 hours
3.9 – 5.3 hours
1.5-6 hours
46 minutes
29 minutes
23 minutes
224 – 252 minutes
21.4 – 25.1 hours (fed state)
96 – 128 minutes (fed state)
0.58
0.06
2 – 4.5
3
5.0 – 7.0
D E
144 ± 52 µm
C A
3 – 4 hours
0.62
3–5
2.2 – 4.3
6.2 – 7.5
6.0 – 7.5
17 cm
90 cm
125 cm
31.3 ± 11.4 µm
425 µm
190.7 ± 80.7 µm
3.4 ml
4.33 L
8.0 L
T P
E C
1 – 1.6 L
U N
48 minutes
0.86
6.5 – 7.1
177 cm
C S
I R
T P
A
M
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ACCEPTED MANUSCRIPT 8. Gastric simulation and modelling of the GI tract Various high throughput in vitro systems have been developed to simulate drug absorption and transport. A popular in vitro system is the Caco-2 tight junction cell layer derived from the human carcinoma [75]. The Caco-2 cells have served as a qualitative tool for permeability
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coefficient calculation of small molecular weight compounds [76]. However, the predictive value
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of Caco-2 cells is stalled by their inability to fully mimic in vivo phenomenon such as peristaltic
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movements. Furthermore, Caco-2 cells solely consist of enterocytes and do not reflect the in vivo physiology of intestinal epithelium. In addition to the above factors, lack of mucus layer in Caco-
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2 cells makes them less optimal to understand the absorption of peptides. Human ex vivo
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intestinal tissues have served as a better model to understand preclinical human intestinal permeability. Small pieces of human intestinal tissue are obtained from patients and permeability
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studies using human small intestinal or colon tissue have been performed [77]. The FDA also
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recommends the use of excised intestinal tissues to assess permeability [78]. However, availability of sufficient healthy human tissues and maintenance of their viability is critical and
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can hinders this type of research. Furthermore, differences in eating habits, environment, and
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medications can affect the tissue morphology and result in inter subject variability. In addition to human ex vivo tissues, porcine intestinal tissue is another option and has the advantage of ease of
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tissue availability. Gastrointestinal morphology in pigs appears to be comparable to humans as opposed to other preclinical animal species such as monkeys and dogs [79]. Several computer controlled models aim to simulate the luminal settings of the stomach and small intestine [80]. Parameters such as pH, gastric emptying, and addition of gastric and intestinal juices have been included in such models. TIM-1 (TNO gastro-Intestinal Model 1), a multi-compartmental model, designed to realistically simulate GI tract conditions, is composed
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ACCEPTED MANUSCRIPT of the stomach and the three parts of the small intestine, the duodenum, jejunum, and ileum [81]. TIM-1 allows close modeling of luminal dynamic procedures occurring within the human body. Parameters such as gastric and small intestinal transit, flow rates and composition of gastric and intestinal fluids, pH values, and removal of water and metabolites, transit time and peristalti c In the InTESTine™ system ex vivo
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mixing have been included in the simulation protocols.
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intestinal segments are used for prediction of permeability at a medium thoroughput, wherein
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upto 96 segments can be evaluated in one experiment [82]. Prediction of intestinal permeability using porcine intestinal tissue in the InTESTine™ system provides a better alternative in
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comparison to Ussing chambers. Furthermore, a unique integrated approach has been developed
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in recent times[83] that combines the TIM-1 model with InTESTine™ and in silico modeling to predict drug absorption in humans.
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The Dynamic Gastric Model (DGM) [84] is an in vitro model that simulates the physical,
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mechanical and biochemical milieus of the human stomach. The DGM includes addition of dynamically controlled gastric juices and enzymes. The rate of addition of the juices is
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dependent upon the pH of the meal (which is detected by a pH electrode). In addition, the system
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also provides the food bolus with simulated peristaltic movements, which in turn help the food to move further. In a direct comparison of DGM with a USP Type II dissolution apparatus, it was
AC
observed that DGM took into account the shear forces of the gastric walls during disintegration and dissolution [85]. In comparison, USP Type II apparatus did not exhibit high shear forces despite increase in shear rates. In Vivo conditions are such that the shear rates are relatively low but the shear forces are greater [86]. This indicates that the DGM model is more apt at mimicking the in vivo conditions in comparison to the USP Type II apparatus.
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ACCEPTED MANUSCRIPT In silico models have also been employed to integrate data from in vitro assays towards formulation screening in order to predict in vivo performance. Early models were not extensive and did not take into account some integral factors such as pH dependence and surface area. The GastroPlus™ software (Simulation Plus, CA) included some of these factors and accounts for
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release, dissolution, metabolism, and absorption through different GI tract compartments
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(stomach, small intestine, and colon) [87].
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Despite the availability of multiple models, a key concern for in vitro models is providing an accurate estimation of the in vivo state. Due to the complexity of the human GI tract, none of the
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in vitro simulation and modeling can accurately replace in vivo experiments. The complexity is
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further multiplied when the drug molecules are larger in size such as peptides and proteins when compared to small molecules.
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9. Methodologies to address GI variability and to increase bioavailability of peptides
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9a. Traditional Approaches
Intestinal absorption enhancers: Formulations based on permeation enhancers promote
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transport of peptides across tight junctions. Fatty acids (eg. sodium caprate [88]), bile salts (eg.
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sodium taurodeoxycholate [89]), anionic polymers (eg. poly acrylic acid [90]), cationic polymers (eg. chitosan and trimethyl chitosan [91]) and surfactants (eg. sodium dodecyl sulphate) are
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commonly used drug permeation enhancers. While some success has been observed with absorption enhancers, they do not necessarily work upto expectation and warrant more evaluations. Peptidase inhibitors:
Co-administration of peptidase inhibitors with peptides can
significantly improve oral bioavailability. Puromycin has shown to increase absorption of metkephamid by ~ 7-fold, increasing from 0.5% in absence of inhibitor to 3.5% in presence of
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ACCEPTED MANUSCRIPT puromycin. This was ascribed to the inhibition of aminopeptidase N by puromycin [92]. Despite the significant improvement that peptidase inhibitors provide, long term use is still questionable. Long term inhibition of peptidases can lead to improper digestion of nutritive proteins and peptides and can also result in stimulation of peptidases as an endogenous regulatory mechanism.
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pH modifiers: As mentioned earlier, most of these peptidases have activity at pH 7 to 8.
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Enteric-coated tablet formulations containing a pH modifier such as citric acid have been used to
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alter the intestinal pH so that it is sub-optimal for enzyme activity [93]. In addition to using pH modifying excipients, peptide modifications can be made that make the peptides resistant to
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degradation in the GI tract. For example, proline- and hydroxyproline-containing peptides have
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been found to be resistant to degradation by digestive enzymes. Furthermore, presence of C terminal proline has been reported to make peptides resistant to some peptidases [94].
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Dosing in fasted state: Limited studies are available for the effect of food and water intake on
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peptide bioavailability [52, 56]. Further evaluation with different peptides is needed before any substantial conclusions can be drawn regarding the effect of food on peptide absorption and
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bioavailability.
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Inhibitors of CYP3A and P-gp: The results presented have demonstrated the significant impact that CYP3A and P-gp may have on the bioavailability of peptides. Inhibition of CYP3A
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and/or P-gp can be expected to improve the bioavailability and lower the variability observed in systemic drug concentrations of orally delivered peptides. D-α-tocopheryl-poly (ethylene glycol) 1000 succinate (TPGS) is an excipient that has been found to improve the absorption of cyclosporine in volunteers [95] apparently by inhibition of P-gp. Various juices such as grape fruit juice have been shown to inhibit CYP3A. Furocoumarin derivatives have been isolated from grape fruit juice which are considered to be responsible for CYP3A inhibition.
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ACCEPTED MANUSCRIPT Peptide properties and their modifications: In an attempt to improve the intestinal permeability of peptides, various research groups have suggested modifications to the peptide structure. Borchardt et al. pointed out that a positive and neutral charge enhances permeation across intestinal mucosa [96]. Permeability was found to depend on the size too, suggesting
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paracellular transport. In another paper by the Borchardt et al., it was shown that hydrogen
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bonding is a major factor in determining the intestinal permeability of peptides [97]. A role of β-
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turn structure has also been suggested by Borchardt group in determining permeability across CaCo-2 cells [98]. In a separate study by Trier et al., it was shown that acylation of glucagon like
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peptides enhances cell membrane binding [99]. However, there is much uncertainty in how these
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modifications to peptide structure might alter PK and affect biological activity. Varying the molecular mass can also play a role in peptide absorption. The potency of the orally administered
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peptides decreases as the chain length increases. In a study by Roberts et al., thyrotropin
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releasing hormone (MW ~362 Da), luteinizing hormone-releasing hormone (MW ~1182 Da), and insulin (MW ~5800 Da) showed an inverse relationship between molecular weight and
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potency [100].
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Dosage form design: Gastroretentive formulations have been adopted to enhance the retention of the dosage form in the stomach [101, 102]. Polymers such as chitosan and trimethyl
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chitosan are advantageous for bioadhesive systems for oral peptide delivery as they act as permeation enhancers as well as adhesives. Lueβen et al. [103] reported an enhancement in the intestinal absorption of buserelin, a nonapeptide, in presence of chitosan, a mucoadhesive polymer. It was assumed that chitosan increases peptide absorption by opening the intercellular tight junctions. Thanou et al.[91] reported a 34- to 121-fold increase in octreotide absorption when administered with a chitosan derivative, trimethyl chitosan at 1.5% polymer solution.
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ACCEPTED MANUSCRIPT As mucus has a high turnover rate, nonspecific mucoadhesive systems are not very effective in vivo. Cell specific adhesion has been attained by targeting cell surface glycans using lectins and found to improve absorption. Lectin conjugated nanoparticles showed 23% absorption when compared to < 0.5% with nanoparticles without surface lectins [104]. However, lectins have
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some toxicity related concerns [105].
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Particulate delivery systems such as microparticles and nanoparticles have also been used to
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enhance absorption following oral delivery[106, 107]. Nanoparticles are expected to pass through the intestinal mucosa intact. Different polymers such as chitosan, cellulose, and poly
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(lactide-co-glycolide) have been used for oral delivery of peptides such as insulin. In one such
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study, insulin loaded chitosan nanoparticles showed a significant improvement in efficacy to reduce glucose levels following oral administration when compared to insulin chitosan solution
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[108].
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Site specific delivery has also been proposed to enhance oral bioavailability of peptides. Colon specific delivery is anticipated to be beneficial for peptides as the proteolytic activity is to
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a minimum in the colon [109]. Saffran et al. developed a colon targeted system, wherein peptide
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drugs were coated with azo-polymers that do not degrade in the stomach and small intestine
drug.
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[110]. Once the system reaches the colon, microflora degrades the azo-polymer and release the
Lipids have been known to enhance bioavailability by reduction of gastric emptying. A relation between chain length of fatty acids and the slowing of gastric emptying has been shown by Hunt and Knox [111]. 9b. Novel technology based approaches for overcoming the oral peptide bioavailability
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ACCEPTED MANUSCRIPT Chuang et al. used self-assembling bubble carriers [112] to deliver insulin. This study in diabetic rats used a foaming agent (sodium bicarbonate) that can generate CO 2 bubbles upon exposure to a diethylene triamine pentaacetic acid (DTPA) dianhydride created acidic aqueous environment. The relative insulin bioavailability was 21.7 ± 1.7 when the foaming agent was
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used in comparison to 0.5 ± 0.3 for free insulin. In addition to using insulin as a model peptide, a
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surfactant (SDS, sodium dodecyl sulfate) was also added in the formulation. The authors propose
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that improved oral bioavailability was due to DTPA (please mention DTPA was added in the formulation and expansion of the same) and SDS, which serve as protease inhibitors and
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absorption enhancers. they improve the oral bioavailability of insulin and induce hypoglycemic
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effects.
In another novel approach, GLP-1 mimetic exenatide was successfully encapsulated in
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nanoparticles made of a mixture of albumin and dextran, and further cross-linked using sodium
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trimetaphosphate [113]. Apparent increase in lymphatic uptake, due to the presence of dextran, resulted in improvement of the relative oral bioavailability of exenatide in rats compared to
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subcutaneous injection. Exenatide has also been modified to include albumin binding domai ns
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[114]. These long acting analogs of exenatide displayed better pharmacokinetics and pharmacological effects compared to native exenatide in diabetic mice.
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Many technologies are being developed and are in the clinic to improve bioavailability or oral peptides (Table 3). The Eligen® technology contains a small carrier, such as sodium N-[8(2-hydroxybenzoyl) Amino] Caprylate (SNAC) [115], which is attached non-covalently with biomolecules. The drug-carrier complex is able to cross the epithelial membrane much more efficiently than the drug alone. Davies et al. recently reported a significant reduction in body weight with oral semaglutide formulation prepared using SNAC [116]. The Peptelligence™
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ACCEPTED MANUSCRIPT technology constitutes citric acid (pH modifier), an acylcarnitine, an antioxidant, and other functional excipients such as disintegrants and binder [93]. Oral delivery of octreotide (via capsules) has been testing Using another novel platform called TPE® (Transient Permeability Enhancer). The GIPET technology combines enteric coating with permeation enhancers
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(medium chain fatty acids) to facilitate peptide’s absorption across the intestinal epithelium [17,
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117, 118]. Novel hydrophilic aromatic alcohols are also being used as permeation enhancers
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[119], towards development of oral calcitonin and insulin formulations [120]. Aguirre et al. [121] provide a good overview of the technologies that are either commercially available, or are in
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preclinical/clinical development. However, many of these technologies suffer from very low
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bioavailability and variability challenges mentioned before.
In another recent advancement, Rani therapeutics has reported a new approach for oral
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delivery of large molecules using a pill which according to the inventors is a “mini swallowable
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auto-injector that delivers drug directly into the highly vascular intestinal wall allowing the drug to be absorbed quickly into the blood stream” (https://www.medgadget.com/2017/07/pill-
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replace-needles-interview-mir-imran-chairman-ceo-rani-therapeutics.html).
The mechanism of
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action of the pill depends on gaseous reactions that provides momentum to make the microneedle containing the drug inject into the tissues. In another work, Traverso et al. [122] has
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demonstrated a microneedle based oral delivery system that has comparable the blood-glucose response following insulin administration via the GI tract vs subcutaneous route. The safety aspects of these technologies and widespread reproducibility in various animal models is yet to be seen.
10. Conclusions
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ACCEPTED MANUSCRIPT Oral delivery of therapeutic peptides requires deeper understanding of the physiological barriers and disruptive approaches to overcome the same in an integrated fashion. We have highlighted that the physiological challenges are manifold and must be accounted for in addition to overcoming protease inhibition and acid stability issues that are commonly mentioned for this The life of a peptide during oral delivery involves transition through the
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area of research.
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stomach where it is denatured by acids and degraded by peptidases/proteases. The remaining
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peptide will face permeability barriers in the GI tract and a fraction of it will be absorbed, whereas the rest is excreted out unabsorbed. Figure 2 summarizes the various root causes for the
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variability encountered in translational studies.
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Overcoming these challenges by utilizing learnings from small molecule oral dosage form development and a systematic approach to address GI physiological issues is a must if we have
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to develop oral peptide therapeutics. While technology and formulation based approaches
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routinely follow the use of intestinal absorption enhancers [88, 91], peptidase inhibitors [92], pH modifiers [93, 94] and modifications of the physicochemical properties of the peptide [97, 99] ,
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they address only half of the problems. In addition, peptide size, lipophilicity, and systemic
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circulation time to compensate for the effects of first pass must be modified and tested to increase bioavailability. Few novel technology based approaches have been reported in the last
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couple of years such as bubble carriers [112], use of nanoparticles [113], and an ingestible microneedle pill [122]. However, coupling the utility of a technology with overcoming the appropriate physiological barrier effect is a must. A key understanding of various endogenous factors affecting the absorption and transport of the peptide across the site of absorption is a good step to start with. Enabling technologies for oral peptide delivery should also address increasing
30
ACCEPTED MANUSCRIPT the residence time of the peptide at the site of absorption and tuning the microenvironment of the
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dosage form to the GI tract environment for acceptable solubility and absorption.
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T P
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Figure 2. Schematic representation of the factors impacting variability, and approaches to address them during oral administration of peptides. Gastroretention can potentially be beneficial for peptides that are absorbed through upper GI tract. Successful colonic delivery suffers from disadvantages such as variable transit time and heterogenous microflora, a challenge that can be addres sed in the near future.
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ACCEPTED MANUSCRIPT Chronic diseases such as type 2 diabetes can benefit from oral delivery as it can overcome the need for frequent injections. Improving the half-life of the orally bioavailable peptide is another important aspect that can provide momentum to oral peptide delivery by way of offering less frequent doses (QD vs BID or TID) to patients. Weight based dosing and administering
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peptide in a fasted state, and with the right amount of fluid can help with improving variability.
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Peptide drug delivery systems that can tackle the variabilities can offer promise to creating better
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drug products.
Oral peptide delivery is an exciting area of research due to the scalability of tablets and
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capsules as drug products and the convenience it offers to patients, and hence will continue to
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attract the attention of scientists. The demand for efficient oral peptide therapeutics and dosage forms will continue to increase due to the prevalence of cancer and the need to have non-
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injection based treatment methodologies. Several cancer therapeutic development programs are
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already utilizing small molecule oral therapy compared to IV injections and this is an area where peptides can further add utility particularly in the area of immune-peptide therapeutics for
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oncology [123]. While technologies and approaches are being improved, attention needs to be
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given to the physiological parameters highlighted in this article, which may further enhance development of oral peptide therapeutics in a holistic way. The immediate next steps should be
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to understand and overcome the GI physiological aspects for overcoming absorption barriers, as “gut filling” and the contents of the GI tract will truly call the shots on oral peptide delivery.
Acknowledgement The authors would like to thank Ben Ambalong of MedImmune for his assistance with Figure 2.
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Oral Peptide Delivery: Translational Challenges due to Physiological Effects
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Puneet Tyagi, Sergei Pechenov, and J Anand Subramony*
Corresponding author: J Anand Subramony
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MedImmune Inc,
One MedImmune Way,
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Phone: 301-398-1418, E-mail: [email protected]
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ACCEPTED MANUSCRIPT TABLES Table 1. Clinically advanced technologies for oral peptide delivery (source: PharmaCircle™). Interested readers are encouraged to refer to clinical trials database (clinicaltrials.gov) for most recent updates.
Peptide
Technology
Insulin
Chiasma Inc.
Octreotide
Enteris BioPharma, Inc.
Leuprolide
Emisphere Technologies Inc /Novo Nordisk Merrion Pharmaceuticals /Novo Nordisk
Insulin GLP-1 analogue
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Insulin
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HIM2; Absorption enhancers and enzyme inhibitors Transient Permeability Enhancer (TPE®) technology; oil based suspension Peptelligence™; absorption enhancers Eligen®; absorption enhancers GIPET™; absorption enhancers POD™; Absorption enhancers and enzyme inhibitors
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Biocon Ltd.
Oramed Pharmaceuticals
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Insulin
Pain indication
Unigene/Enteris
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GSK
PTH
Unigene/Enteris
Ph
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Cara Therapeutics
Table 2. List of relevant parameters that can led to low peptide bioavailability and potential tools to overcome the physiological barrier or methodology. Parameters that impact peptide bioavailability Variability in GI tract - Intestinal permeability
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Potential tools to overcome the physiological barrier/methodology
Can be tackled by a combination of fine tuning physico-chemical properties of permeation
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Site of absorption
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Absorption site residence time
Effect of meal uptake Effect of peptidases
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Variability in preclinical models
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- Gastric mucus Effect of gastric and intestinal pH
enhancers complementing the properties of peptide (such as LogP, pH dependent solubility). Perform site of absorption studies and deliver peptide to that location via controlled release. Mucoadhesives can be used to retain the formulation at the absorption site to maximize absorption. Mucolytic agents; mucus penetrating particles pH modifiers/buffering agents to modulate the immediate peptide environment. Dosing in fasted state Peptidase inhibitors; peptide modification to enhance stability against peptidases Rodents are not ideal due to issues with dosing oral formulations; pigs and dogs are widely preferred due to anatomical similarities to humans. Use of higher samples (n value) to mak bioavailability values statistically significant.
Humans
Rats
Dogs
Pigs
1 hour
0.7-2.1 hours
3.9 – 5.3 hours
1.5-6 hours
46 minutes
29 minutes
23 minutes
48 minutes
224 – 252 minutes
21.4 – 25.1 hours (fed state)
96 – 128 minutes (fed state)
3 – 4 hours
0.58
0.06
0.86
0.62
2 – 4.5
3
3–5
2.2 – 4.3
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Gastric emptying time Gastric retention time Small intestine transit time Absolute water content (g/cm gut length) pH of the stomach (fed)
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Table 3. Comparison of key GI tract parameters across different pre-clinical and species and humans commonly used for pharmacokinetic and efficacy evaluations [66, 71-74].
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ACCEPTED MANUSCRIPT 6.5 – 7.1
6.2 – 7.5
6.0 – 7.5
177 cm
17 cm
90 cm
125 cm
144 ± 52 µm
31.3 ± 11.4 µm
425 µm
190.7 ± 80.7 µm
1 – 1.6 L
3.4 ml
4.33 L
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pH of small intestine Length of gut Stomach fundus mucus thickness Average stomach fluid capacity
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Graphical abstract
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8.0 L