Enteric-coated capsules or tablets with additional excipients enable intestinal delivery.
Oral delivery of therapeutic peptide hormones offers the promise of greater patient compliance when compared to injectable formulations. Success is difficult to achieve because the hormones are relatively large (compared to digestible amino acids) and susceptible to proteolytic degradation in the gastro-intestinal tract. We have developed an enteric-coated solid dosage form with excipients that modulate intestinal proteolytic activity and enhance peptide transport.
Enteric-coated capsules or tablets with additional excipients enable intestinal delivery.
This article describes the elements of a novel oral delivery formulation, the subsequent tests with several peptide hormones in animals, and tests with salmon calcitonin in humans. The relative bioavailability of intact peptide hormones delivered using this formulation ranges from 1 to 10%, depending on the size, charge, and proteolytic susceptibility of the peptide.
The low bioavailability associated with oral delivery necessitates a large-scale, cost-effective production technology. Part II of this article will be published in an upcoming issue and will describe a direct-expression, recombinant peptide-production technology that is readily scalable to economically produce hundreds of kg/yr.
Alternate routes of delivery for macro-molecule drugs have been a topic of intense R&D to improve patient acceptance and adherence to chronic dosing regimens. Many companies and research groups have developed alternate delivery methods, including nasal, pulmonary, buccal, and transdermal administration and depot injection. However, oral delivery formulations in capsules or tablets offer the greatest convenience and patient compliance.
Therapeutic peptide hormones represent a major challenge for oral delivery. Developers of commercially viable oral peptide products face four obstacles:
Woodley1 and Lee2 have reviewed these obstacles and other information regarding proteolytic enzymes in the gastrointestinal (GI) tract. The relatively large size and hydrophilic nature of peptides severely limit the absorption of these molecules in the GI tract. In addition, peptides are susceptible to degradation by enzymes. Pepsin in the stomach can cleave peptide bonds and inactivate peptides. Peptides that survive passage through the stomach are susceptible to cleavage by intestinal proteases that are secreted from the pancreas or localized on the brush-border membranes of intestinal epithelial cells. Another impediment to oral absorption is the mucus layer in the GI tract that binds charged molecules, such as peptides, and prevents their absorption through the lumen of the intestine. Consequently, the bioavailability of peptides of a molecular size greater than two or three amino acids is extremely poor.
The rational design of orally active peptide formulations should be based on one or more of the following strategies:
In recent years, several technologies have emerged that fulfill at least some of the goals for an oral formulation. Animal and human studies have been conducted that demonstrate the bioavailability and biological activity of several orally delivered peptides. For example, peptide drugs can be conjugated to a polyethylene glycol- based amphiphilic oligomer. According to Soltero and Eckuribe, the resulting drug-polymer conjugate is absorbed more efficiently across the gastrointestinal wall and its resistance to proteases is increased.
However, a conjugated peptide drug constitutes a new chemical entity (NCE), compared to the peptide alone, and it may necessitate additional toxicology testing during clinical development.
In another example of a published oral technology, macromolecule drugs are mixed with proprietary delivery agents or carriers that bind to the drug and enhance passive transcellular transport. Once in circulation, the carrier dissociates from the drug. This technology necessitates selecting the appropriate carrier molecule for each peptide from a library of these compounds and adding a large excess of the carrier to the formulation to enhance bioavailability. A study of this technology in the oral delivery of salmon calcitonin (sCT) in human volunteers has been reported.4
The oral delivery of biologically active insulin has been demonstrated in diabetic dogs using enteric-coated microcapsules containing sodium cholate and soybean trypsin inhibitor as excipients.5 Though this study validates the concept of increasing bioavailability with the use of permeation enhancers and protease inhibition, because soybean trypsin inhibitor is a protein, it may not be practical for human therapeutics. It also may present regulatory problems.
Unigene Laboratories' proprietary technology,
which meets all the goals of orally active formulations, involves the preparation of a solid dosage form that contains the peptide along with other excipients in enteric-coated capsules or tablets. The excipients consist of up to three groups of compounds, depending on the peptide to be delivered. The Group A additive is a general inhibitor or modulator of intestinal protease activity. The Group B additive is a detergent that improves the solubility of the peptide, decreases interactions with intestinal mucus, and enhances paracellular transport. The Group C additive is a specific inhibitor of the primary enzymes that degrade the peptide. The Group C additives increase the stability of the molecule in the GI tract.
The enteric coating makes the capsule or tablet stable in acidic pH and allows it to pass through the stomach intact. As the pH in the intestine increases above 5.5, the coating dissolves and releases the peptide and excipients into a localized area of the intestine, where proteases with neutral to alkaline pH optima are inhibited.
This technology is applicable to a variety of peptides and small proteins, and it can achieve relative bioavailabilities ranging from 1 to 10%, depending on the size, charge, and structure of the peptide. No chemical modification or derivatization of the peptide is necessary to achieve peptide absorption. This is particularly advantageous when there is already an alternate approved formulation (for example, injectable or nasal) because the peptide in the oral formulation will not constitute an NCE. Also, all of the excipients used are generally regarded as safe or naturally occurring compounds.
The components of the formulation used for the oral delivery of sCT, a 32-amino acid amidated peptide used for the treatment of postmenopausal osteoporosis, other bone disorders, and hypercalcemia of malignancy, are shown in Table 1. The organic acid in the formulation reduces the pH in the local environment of the intestine where the capsule opens and releases its contents. Since the majority of intestinal and pancreatic proteolytic enzymes have neutral to alkaline pH optima, their activities are reduced or inhibited in the acidic area where the peptide is released from the solid dosage form. The acylcarnitine in the formulation facilitates the paracellular transport of sCT across the epithelial layer into the peripheral circulation.
Table 1. Solid Dosage Formulation Components for the Oral Delivery of sCT
Tablets containing sCT in combination with Group A and Group B excipients also have been evaluated in a 28-day toxicology study in rats and dogs, and no significant treatment-related effects were seen. The data are being held for future release.
Unigene prepared capsules and tablets with two or more of the three groups of excipients, depending on the peptide to be orally delivered, and obtained pharmacokinetic (PK) data in rats and dogs (and humans for sCT) to demonstrate the absorption of intact peptide in the peripheral circulation.
A single-dose clinical study in ten human volunteers was performed using the formulation given in Table 1 and size 00 capsules. Each subject received a single formulated capsule containing 500 mg sCT after an overnight fast. The PK profile of sCT was obtained by using an sCT-specific radioimmunoassay. Blood was collected prior to dosing and then, starting at 30 min post-dosing, every 15 min up to 200 min. Between 200 and 300 min, it was collected less frequently. The blood samples were then fractionated to obtain plasma, and sCT was quantified by radioimmunoassay. The PK profile for each subject is shown in Figure 1, left side. Nine of the ten subjects showed significant plasma levels of sCT, with peak plasma concentrations (Cmax) ranging from 142 to 599 pg/mL. The time required to reach Cmax (Tmax ) for each subject was variable, ranging from 90 to 180 min, and reflects the varying times for gastric emptying of the enteric-coated capsules in each subject. The mean PK profile for all nine subjects after normalization to a common Tmax is shown in Figure 1, right side. A fairly acute PK profile, with a mean Cmax of approximately 320 pg/mL, was observed.
Figure 1. Plasma Levels Measured in Humans Following Ingestion of a 500 mg sCT Capsule.
Previous studies in humans using this technology have shown that sCT in the circulation was biologically active, since a significant transient drop in serum calcium levels was observed (data not shown). Several oral studies with sCT in animal models, where plasma sCT was quantified with a two-site sandwich enzyme linked immunosorbent assay (ELISA), have shown that sCT delivered with Unigene's technology is intact, since this ELISA does not recognize small fragments of sCT.9 One of the antibodies is specific to the N-terminal region of the peptide and the other to the C- terminal region of the peptide. Thus only intact molecules of sCT in circulation that are recognized by both antibodies are measured by the assay.
The mean Cmax for sCT after oral administration was two-fold greater than the Cmax obtained by a subcutaneous dose of 100 International Units (IU), or 15 µg of sCT. It should be noted that this standard 100 IU injectable dose delivers sCT plasma levels that probably exceed those required to achieve a therapeutic response. Hence, a 200 mg dose of oral sCT delivered using Unigene's technology may be sufficient for therapeutic efficacy.
Several peptides, including luteinizing hormone-releasing hormone, leuprolide, desmopressin, parathyroid hormone (PTH) analogs, insulin, glucagon-like peptide 1, and other glucose regulatory peptides have been delivered using this oral technology to achieve therapeutically relevant plasma levels in animal studies. It has been found that the bioavailability of a given peptide depends not only on the size and charge of the peptide, but also on the presence of structural features that render the peptide more protease resistant, such as blocked
For example, equivalent formulations of arg-vasopressin, a naturally occurring peptide, and desmopressin, an analog of arg-vasopressin with blocked N- and C- termini and D- arginine in position 2, were each given to groups of eight dogs in oral capsules. The area under the curve (AUC0-240min) of the analog, as calculated by the trapezoidal rule, was 3.1-fold higher than that of the parent compound. The Cmax of the unformulated arg-vasopressin analog was less than one-thirtieth that of the formulated, enteric-coated capsules, showing that oral bioavailability is dependent on the enteric coating and the excipients in the formulation. Also, the bioavailability of the analog, when delivered with Unigene's oral technology, was higher than that of a currently marketed desmopressin tablet, with a 3.4 fold higher Cmax value and an AUC0-240min that was 7.9-fold higher (Table 2).
Table 2. Effect of Oral Administration of Desmopressin to Dogs
We tested oral delivery in Beagle dogs to determine if the dog is a good model for preclinical PK studies. Using sCT as a model peptide, the correlation between plasma levels in Beagles and humans was established in five separate studies, as shown in Figure 2. In each study, capsules with the identical formulation and the same amount of sCT peptide were given to dogs and humans. We plotted the mean
levels to species for each capsule lot, and the data show a linear correlation between the plasma levels, with an
of 0.918. The absolute values in dogs and humans are obviously different due to the difference in body weight.
Figure 2. sCT Values for Dogs and Humans Showing a Linear Correlation
Significant plasma levels of several peptides of various sizes and charges have been detected using this technology in dogs. Dose-ranging studies with a proprietary glucose regulatory peptide have demonstrated that there is a dose-dependent increase in plasma levels of the peptide (Figure 3). At doses ranging from 0.4 mg to 4.5 mg per capsule (n = 8 for each dose), with the identical formulation, the mean plasma levels of the peptide increased linearly from a Cmax of approximately 200 pg/mL to greater than 3,000 pg/mL, with an R2 of 0.98.
Figure 3. Cmax in Dog Plasma Varies Linearly with Administered Dose. Eight dogs were used in each of the five studies and they were matched to 10-15 human volunteers.
The studies described in Figures 2 and 3 demonstrate that the dog is a good model for preclinical PK studies with this formulation and that plasma levels in dogs increase linearly with the increase in dose and are predictive of the levels expected in humans.
Despite the recent advances in oral delivery technology, the bioavailability of peptides with any of the current technologies is much lower than that obtained by injection. Thus, for peptides that are used for chronic therapeutic indications having large markets, or for peptides where the required dose is high (for example, PTH and insulin), oral delivery places tremendous cost and scale-up burdens on the API manufacturer. Various recombinant protein production technologies have been developed that can deliver kilograms or even metric tons of API. However, the production of peptides, and particularly of peptide hormones that require
-terminal amidation for biological activity, remains a significant challenge.
A direct-expression technology for the efficient and cost-effective production of peptides, along with a technology for in vitro amidation, will be described in the second part of this article, which will appear in an upcoming issue. We believe this recombinant peptide-production technology is readily scalable to production levels of hundreds of kg/yr.
Unigene's oral delivery technology was primarily developed by William Stern, with significant contributions by Jim Gilligan. Amy Sturmer and other scientists have contributed to the immunoassays for quantifying the various peptides evaluated in the oral program.
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