Peptide Progress

Refined manufacturing and delivery promises more advanced drug offerings

eptides are among the most biologically relevant molecules and comprise one of the most productive areas of pharmaceutical research and development. According to Frost and Sullivan, more than 40 peptide drugs are on the market, and close to 300 are in clinical testing.

Among the approved agents are natural peptides such as insulin, oxytocin, exendin-4, parathyroid hormone, and calcitonin. Several synthetic or derived peptides or peptide analogs have also been developed into successful products, including Fuzeon, Integrilin, DDAVP, Sandostatin, Lupron, and Symlin. One form of insulin, Lilly's Humalog, enjoyed sales of $1.1 billion in 2004.

What does it take to introduce a new peptide drug? In addition to discovery, development, and success in the clinic, developers need to address the problem of scale-up in manufacturing and, eventually, delivery. Most approved peptide drugs, including the blockbuster insulin, are administered by injection or infusion. At one time, the well-known drawbacks of injectable-dosage forms held back the development of peptide drugs. That no longer needs to be the case.

CORNUCOPIA FOR DISCOVERY

The pipelines of nearly every pharmaceutical company today include peptides or peptide-like drugs. Some, like Novo Nordisk, have shifted their discovery and development efforts almost entirely to peptides.

As Edwards, et al., wrote in 1999: "We are on the brink of a therapeutic revolution" based on peptide drugs. ¹

Peptides, or proteins, regulate most of human physiology through the endocrine/paracrine systems. They serve as hormones, neurotransmitters, growth factors, enzymes, and structural components of cells.

Insulin was the first peptide introduced into human clinical practice. Discovered by Banting in 1921, this 51-amino acid peptide with a molecular weight of 5,808 daltons quickly revolutionized the treatment of diabetes. Despite the introduction of numerous peptide drugs since the 1920s, insulin remains the leading peptide therapeutic-a perennial blockbuster in both its native and chemically modified forms.

One strategy for "discovering" new peptide therapies involves chemical modification of known active peptides. Esterification, oligomerization, or amino acid substitution, for example, can in some cases enhance a peptide's activity while conferring on it desirable pharmacokinetic properties such as extended plasma half-life.

Insulin has been introduced in several formats and delivery vehicles, including buccal, rectal, sublingual, and, more recently, inhaled and intranasal forms. Patients with diabetes now enjoy slow-acting insulin products that enable them to maintain basal levels of the drug, as well as fast-acting forms taken at meal times.

Although many pharmacologically relevant peptides are already known, completion of the human genome project is expected to reveal dozens-if not hundreds-of additional peptides with regulatory and disease-modulating activity. Within a few years, we expect the discovery of naturally occurring peptides to rival in importance the recent discovery of several classes of small regulatory RNAs. The analogy here is striking, because as recently as 2000 no one knew small regulatory RNAs existed, much less understood their far-reaching influence on human health.

Discovering peptide drugs will require significant advances in how researchers identify, quantify, and analyze peptides as small as three amino acids in length. Given the number and concentrations of confounding molecules in biological samples, that won't be easy. The broad dynamic range for biological molecules means that traditional analytical methods like high-performance liquid chromatography mass spectrometry (HPLC-MS) will require highly selective and innovative sample preparation methods to identify peptides whose existence in blood may be fleeting.

Unigene Laboratories, Inc. (Fairfield, N.J.) has developed a new enzyme-based assay for the discovery of amidated peptides that should, ultimately, identify these low-concentration peptide hormones and that will complement LC and LC/MS methods-thereby fueling the discovery of many more biologically active peptides suitable for pharmaceutical development.

DELIVERY OBSTACLES

This figure shows results from a clinical study in which 10 human volunteers received a single dose of 500 ?g of oral salmon calcitonin (sCT) after an overnight fast. Nine of the 10 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 ranged from 90 to 180 minutes, reflecting varying times for gastric emptying. When the pharmacokinetic (PK) profile from all of the volunteers was normalized to a common Tmax to eliminate the variability of gastric emptying, the resulting mean PK profile showed a sharp peak. The mean Cmax value was approximately 325 pg/mL. This indicates that the desired therapeutic exposure of sCT, which is approximately 50 pg/ml, can be achieved with a dose of approximately 100 ?g.

SOURCE: UNIGENE LABORATORIES, INC.

Orally delivered peptides and proteins face a hostile gastric environment: proteolytic enzymes in the stomach and intestine and the intestinal permeability barrier. These obstacles result in relatively low bioavailability of orally delivered formulations, which places a burden on the manufacturing of peptides due to the large doses needed.

In addition to the fact that the large size and hydrophilicity of peptides severely limit their absorption through the gastrointestinal (GI) tract, they are also susceptible to degradation by enzymes in the stomach-primarily pepsin-and by the stomach's acidic environment. Peptides that survive the stomach are susceptible to cleavage by intestinal proteases secreted from the pancreas or localized on the brush border membranes of intestinal epithelia. The mucus layer of the GI tract, which binds polar molecules, serves as a further barrier to absorption through the lumen of the intestine. Consequently, the bioavailability of peptides of more than two to three amino acids is extremely poor. ²

The rational design of orally active peptide pharmaceuticals should strive to inhibit or modulate proteolysis, enhance absorption in the stomach or intestine (by facilitating paracellular or transcellular transport and/or by increasing penetration through the mucus barrier), and increase the circulating half-life of the peptide in situations requiring sustained presence for therapeutic efficacy.

Several technologies that fulfill some or all of these goals have been tested in both animals and humans. ^3-6 Unigene's oral delivery technology, which requires no chemical modification of the peptide, uses up to four groups of excipients, depending on the peptide to be delivered. ^7-8 Excipient groups comprise organic acids that:

• Serve as general protease inhibitors or modulators;

• Enhance paracellular transport;

• Act as detergents for improving peptide solubility and transport while reducing interaction with mucus; and

• Are protease-specific inhibitors for enhancing circulating half-life.

The enteric coating confers stability to a capsule or tablet in an acidic pH, allowing it to pass through the stomach intact. As the pH in the intestine rises above 5.5, the coating dissolves and releases peptide and excipients into a localized area of the intestine.

Unigene has prepared capsules and tablets and obtained human pharmacokinetic data that demonstrate absorption of intact peptide into the systemic circulation. The oral delivery of salmon calcitonin (sCT), an amidated 32-amino acid peptide for treating postmenopausal osteoporosis and hypercalcemia of malignancy, is shown in Figure 1 (see below).

Unigene has also demonstrated delivery in animals for lute-inizing hormone-releasing hormone, leuprolide, desmopressin, parathyroid hormone (PTH) analogs, insulin, glucogen-like peptide-1, and other glucose regulatory peptides. Researchers have determined that bioavailability 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 N- and C-termini or the incorporation of D-amino acids.

MANUFACTURING

Because most peptides are highly potent, the low bioavail-ability of orally delivered peptides, which ranges from 1% to 10%, should not be problematic from a therapeutic standpoint. It carries special significance for manufacturing cost and scale, however, particularly for peptide drugs with high dosing requirements (e.g., Fuzeon and insulin).

Although chemical synthesis has meant great strides in reducing costs and improving scalability for peptide production, recombinant technology has become the method of choice for the large-scale manufacture of larger peptides (25 amino acids or more). Recombinant expression of foreign proteins in micro-organisms and cells provides the best combination of cost-effectiveness, scalability, and environmental safety.

The manufacture of peptides in recombinant organisms began in the early 1980s. Since then, many different host cells and organism types have been used to produce peptides.

Microbial fermentation, particularly in Escherichia coli, has several significant advantages over mammalian cell culture. E. coli fermentations are rapid, predictable, free of downstream contaminants associated with cells, and less costly than cell culture.

Conventional bacterial fermentation systems are not without their limitations, however. For example, the relatively small size and lack of tertiary structure of most peptides makes them susceptible to rapid degradation in the cytoplasm of expressing bacteria and yeast. This drawback may be mitigated by expressing the product with a much larger protein fusion partner; this generally protects the peptide from proteolysis.

Liberation of the product from the fusion partner requires chemical or enzymatic cleavage, however, which adds at least two processing steps (cleavage and purification) and results in significantly reduced peptide yield. Also, lysing the bacterial cell to release the peptide product causes release of all the bacterial proteins, as well as DNA and bacterial endotoxins.

Purifying these process-related contaminants away from the peptide of interest further increases the number of purification steps required. An ideal expression system would be one that allowed for the production of peptides without a fusion partner and secreted the expressed peptide from the cell into the growth medium-leaving the bacterial cells intact. Unfortunately, obtaining excreted products from E. coli is difficult because the organisms do not normally excrete peptide or protein products. Further, they produce pro-teases that break down foreign proteins intracellularly.

A further complication in producing peptide hormones in bacteria or yeast is the frequent requirement that these products be amidated at the C-terminus of the hormone for full biological activity. Prokaryotes lack peptidylglycine-amidating monooxygenase (PAM), the enzyme that carries out this post-translational amidation. Peptides produced in E. coli are not C-terminally amidated.

To address these issues, Unigene has developed a manufacturing platform that efficiently produces amidated peptide hormones through the use of two recombinant cell lines. The glycine-extended precursor of the desired peptide is first produced in recombinant E. coli using a proprietary "direct expression" technology. ⁹ The expression construct incorporates an upstream signal sequence that causes the peptide to translocate from the cytoplasm to the periplasm, at which point the signal sequence is cleaved.

Further innovations in the growth conditions and in the components of the growth medium allow the peptide to be excreted into the growth medium. The E. coli host cell is a proprietary protease minus cell that allows for the accumulation of the peptide in the growth medium without significant degradation. Because E. coli does not excrete appreciable quantities of endogenous proteins, the peptide product in the conditioned medium provides a relatively enriched starting material for purification, thus reducing the number of purification steps and increasing the yields from purification.

After purification, the peptide is treated in vitro with PAM, separately produced from recombinant Chinese Hamster Ovary (CHO) cells. PAM quantitatively converts a variety of C-terminally glycine-extended peptides to the corresponding peptide amides at a mass ratio of enzyme to substrate of 1:1000 or greater, depending on the glycine residue's immediate neighbor. The quantity of PAM needed is, therefore, a small fraction of the amount of peptide to be produced. The higher cost of production of PAM in CHO cells does not add appreciably to the overall cost of the process. One or more chromatography steps then separate amidated product from precursor and other minor contaminants. After purification and amidation, the peptide is typically more than 98% pure.

The direct expression process is readily scalable up to 1,000 liters with no loss of productivity. Yields will vary depending on the peptide, but products like sCT, PTH analogs, glucose regulatory peptide analogs, secretin, and growth hormone releasing factor have been expressed at up to 1 g/liter of intact peptide. In instances in which peptide degradation occurred in the growth medium, changes in the nutrient feed significantly reduced it.

NOT WITHOUT RISKS

Although the future looks bright for peptide drugs, the usual risks remain-as Pfizer recently learned through its experience with Exubera, its inhaled insulin product.

It was widely reported that Pfizer would take a $2.8 billion charge related to the failure of Exubera, its inhaled insulin product; sales were only $12 million in the nine months after its introduction. Since that time, programs to develop inhaled insulin at Novo Nordisk and Eli Lilly have been terminated.

Exubera failed less because of insulin's shortcomings than because of the way the product was formulated. Exubera's delivery device was cumbersome, difficult to use, and did not allow for dose modulation the way injection does. Physicians may also have been wary of the dosage form, which used insulin-coated particles rather than a mist or nebulized dose.

Further, the prescribing information for Exubera described concerns about decreased lung function-an issue not uncommon when particulate matter enters the lungs. Exubera demonstrated that, even with insulin's established position in diabetes treatment and the backing of a top-five pharmaceutical company, failure is always a possibility.

Discovering peptide drugs will require significant advances in how researchers identify, quantify, and analyze peptides as small as three amino acids in length. Given the number and concentrations of confounding molecules in biological samples, that won't be easy.

Nevertheless, there has never been a more exciting time to be involved in peptide pharmaceutical development. Oral delivery methods have changed the paradigm for peptide drugs irrevocably-and for the better. The results of human genome research should provide peptide drug candidates for years to come.

A robust manufacturing platform can support the right delivery vehicle for today's-and tomorrow's-peptide drugs. These technologies may enable the introduction of several oral peptide hormone drugs for administration in chronic osteoporosis and diabetes. While certain individual development projects are early stage, the trend is positive and the possibilities are plentiful.?

Dr. Levy is president and CEO of Unigene Laboratories, Inc. Reach him at

REFERENCES

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2. Woodley JF. Enzymatic barriers for GI peptide and protein delivery. Crit Rev Ther Drug Carrier Syst. 1994;11(2-3):61-95.

3. Martins S, Sarmento B, Ferreira DC, et al. Lipid-based colloidal carriers for peptide and protein delivery-liposomes versus lipid nanoparticles. Int J Nanomedicine. 2007;2(4):595-607.

4. Buclin T, Cosma Rochat M, Burckhardt P, et al. Bioavailability and biological efficacy of a new oral formulation of salmon calcitonin in healthy volunteers. J Bone Miner Res. 2002;17(8):1478-1485.

5. Deutel B, Greindl M, Thaurer M, et al. Novel insulin thiomer nanoparticles: in vivo evaluation of an oral drug delivery system [published online ahead of print December 27, 2007]. Biomacromolecules. 2008;9(1):278-285.

6. Owusu-Ababio G, Karami K, Maher J, et al. Utility of a novel oral delivery agent in promoting heparin absorption across different sites of rat gastrointestinal tissues. Paper presented at: AAPS Annual Meeting; October 2002; Toronto, Canada.

7. Stern W, Gilligan JP, inventors; Unigene Laboratories, Inc., assignee. Oral salmon calcitonin pharmaceutical products. US patent 5 912 014. June 15, 1999.

8. Stern W, Gilligan JP, inventors; Unigene Laboratories, Inc., assignee. Oral peptide pharmaceutical products. US patent 6 086 918. July 11, 2000.

9. Ray MV, Meenan CP, Consalvo AP, et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli. Protein Expr Purif. 2002;26(2):249-259.