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sible. A culture clash is inevitable: the best ionchannel assay is slow, and existing high-throughput assays do not capture the complexity of most ion channels. What is the solution to this impasse? The paper by Huang et al. describes a method to stimulate ion channelcontaining cells under computer control using external electric field changes while measuring the attendant changes in membrane potential using voltagesensitive dyes and high-speed optical recording. Changes in the external electric field alter the transmembrane potential transiently and thus alter the field experienced by the embedded ion channels. In this way, scientists can cycle ion channels through their various conformational states (Fig. 1) and allow drug candidates access to all these states7,8. Although membrane potential is a complicated nonlinear function of ion channel current, it was found nevertheless that conditions can be adjusted empirically to report the relative degree of inhibition of the channels1,9. Sodium channels, when open, depolarize the cells membrane potential; inhibition of sodium channels by a drug retards this depolarization. In the method of Huang et al., such changes in membrane potential are tracked with optical voltage-dependent dyes and correlated with channel inhibition. The authors show that the experimental conditions can be adapted (by changing the frequency of stimulation) to optimize characterization of particular drugs. Furthermore, because the method of interrogating membrane potential is optical, it can be carried out with very high throughput. The approach permits detection of different mechanisms of ion channel inhibition and can distinguish between use-dependent inhibitors7,8, such as lidocaine, and toxins such as tetrodotoxin from the Japanese puffer fish. This is important because drugs have different use-dependent kinetics (that is, different association/dissociation rates), and drugs that affect the channel in a use-dependent manner and have appropriate kinetics are believed to be safer than very slowly dissociating (non-use-dependent) toxins. This is quite exciting indeed. The method is not without limitations, however. First, it does not control membrane potential as in the voltage clamp method; it merely causes it to change. Second, the approach relies on the intrinsic excitability of cells; that is, the response of a cell will depend on the various combinations of ion channels in the membrane. This will differ with different cell types and cannot be fully controlled. Nevertheless, the approach of Huang et al. to retask an age-old approach in the service of drug discovery opens significant new opportunities for identifying ion channelmodulating drugs.
1. Huang, C.-J. et al. Nat. Biotechnol. 23, 389396 (2006). 2. Galvani L. De Bononiensi Scientiarum et Artium Instituto atque Academia Commentarii. 7:363-418 (1791). 3. Huxley, A.L. & Hodgkin, A.F. J. Physiol. 116, 424448 (1952a). 4. Neher, E. & Sakmann, B. Nature 260, 799802 (1976). 5. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Pflugers Arch. 391, 85100 (1981). 6. Doyle, D.A. et al. Science 280, 6977 (1998). 7. Hondeghem, L.M. & Katzung, B.G. Annu. Rev. Pharmacol. Toxicol. 24, 387423 (1984). 8. Hille, B. J. Gen. Physiol. 69, 497515 (1977). 9. Burnett, P. et al. J. Biomol. Screen 8, 660667 (2003).

2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

A peptide chaperone for transdermal drug delivery

Mark R Prausnitz
A peptide identified by in vivo phage display facilitates the transport of protein drugs.
Despite the increasing importance of protein therapeutics and vaccines, the need to deliver them by hypodermic injection remains a major limitation. Delivery of protein drugs through the skin is an attractive alternative to needles, but has proved elusive thus far. Findings reported by Chen et al.1 in this issue could change that. Using a novel high-throughput screen based on phage display, this study considered millions of peptides to find a chaperone that ferries proteins across the skin and found a unique sequence that dramatically increased transdermal delivery of insulin and human growth hormone in an animal model. The rewards for effective transdermal drug delivery are large. Drug delivery using skin patches has grown into a multibillion dollar industry, with multiple commercial and clinical successes for a variety of small drugs2. Patients like patches because of their convenience there is no need to remember to take frequent pills and no pain from hypodermic injections. Doctors like patches because of their efficacy transdermal delivery avoids the complications of poor absorption and enzymatic degradation associated with oral delivery and eliminates the peaks and valleys of drug concentration in the blood associated with bolus injections. And pharmaceutical companies like patches because of their profitabilitypatches are not only preferred by their customers but can often be used, in effect, to extend the patent life of a drug through new and improved delivery. These advantages have motivated the research community to overcome the primary challenge of transdermal delivery: the skins outer layer of stratum corneum is an extremely tough barrier that generally only permits entry of small, lipophilic drugs and uniformly excludes large, hydrophilic proteins (Fig. 1a). Various chemical enhancers, such as ethanol and surfactants, have been used to increase skin permeability to small molecules but, with few exceptions, have been ineffective in delivering proteins3. Physical approaches, which breach the skins barrier more aggressively, have recently met with some success for proteins and other compounds. Electric fields4, ultrasound5 and jet injectors6 have received US Food and Drug Administration approval for transdermal applications, and microneedles7 and thermal ablation8 are being studied in clinical trials. Although these methods show promise, they may be limited by the need for devices that could be large, costly and cumbersome. Chen et al. have taken a biological approach to increase skin permeability, in contrast to the chemical and physical approaches previously investigated. Application of a mixture of insulin and their peptide enhancer to the skin of a rat increased plasma insulin levels and reduced blood glucose levels for hours. Transdermal delivery of human growth hormone was similarly enhanced, suggesting that the approach may be broadly applicable. Thus, their new method has the potential to combine the simplicity of currently available patches that use chemical enhancers and the efficacy of physical enhancer devices under development. Chen et al. began with the hypothesis that appropriately selected peptide sequences could interact with skin to increase its permeability

Mark R. Prausnitz is at the School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0100, USA. e-mail: prausnitz@gatech.edu

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to proteins. To identify such peptides, they applied a phage display library to the skin of nude mice; phage that penetrated into the bloodstream were recovered and amplified (Fig. 1b). After a second round of screening, the most successful phage were found to have a consensus nucleotide sequence that coded for a common 9-mer peptide. The peptide was stabilized by adding an amino acid to each end, yielding the optimized peptide ACSSSPSKHCG. The peptides mechanism of action is not fully clear, but it is highly specific. Changes of just a single amino acid to the optimized peptide reduced its effectiveness, with some amino acids having greater effects than others. The mechanism also appears to involve an interaction between the peptide and the skin, rather than the peptide and the protein. Enzymelinked immunosorbent assay, dynamic light scattering and other binding assays did not show an association between the peptide and insulin. Moreover, transdermal delivery of both the hexamer and dimer forms of insulin was enhanced by the peptide equally well. Microscopy studies of fluorescently tagged compounds showed that both the peptide and insulin were present in hair follicles at especially high concentration, suggesting a transfollicular route across the skin. Although the findings of this study are exciting, many questions remain. In the absence of deeper mechanistic understanding, it is unclear whether this peptide enhancer will be broadly applicable to proteins or other macromolecules. Additional information is also needed to determine whether doses relevant to humans can be achieved, given that much less insulin is needed to modulate glucose level in a diabetic rat. Another question about scale-up to human use concerns the apparent transport pathway through hair follicles, because rats have a hairfollicle density more than 25 times greater than that of humans. Finally, although no adverse effects were mentioned in this study, a detailed assessment of safety is needed. As these questions are addressed in future studies, the prospect of a simple patch containing a peptide enhancer for protein delivery is tantalizing. Proteins make up a significant and growing fraction of recently approved therapeutics. Almost without exception, these proteins are administered by injection, which reduces patient compliance, complicates home use and poses serious safety threats in developing countries where needles are often reused. Protein delivery from a patch with equal efficacy and similar cost would almost certainly make hypodermic injection of many proteins

a
Stratum corneum Viable epidermis

ii

iii

2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

Dermis

Dermis

1st generation phage library

Screening round 1

Amplify

2nd generation phage library Sequence

Screening round 2

+
Increase serum insulin Decrease blood glucose
Bob Crimi

Peptide

Insulin

Figure 1 Phage-display screening to discover peptides that overcome skins barrier. (a) Transdermal delivery is limited largely by skins outer layer of stratum corneum. (i) Drugs from conventional patches follow a tortuous path, diffusing around cells within the lipid-rich extracellular matrix of stratum corneum. (ii) Transport directly across stratum corneum does not usually occur in intact skin. (iii) Diffusion via hair follicles is thought to be insignificant under normal circumstances. However, the peptide-enhanced insulin delivery demonstrated by Chen et al. appears to follow this pathway. (b) To find novel peptide enhancers, Chen et al. placed a phage display peptide library on the skin of nude mice. Phage that crossed the skin were harvested from the blood and amplified for a second round of in vivo selection. Sequencing of phage collected after the second screening yielded an optimal peptide that, when coadministered with insulin, increased serum insulin and decreased blood glucose levels in diabetic rats.

obsolete. This prospect is what makes finding a peptide chaperone for transdermal delivery such an exciting advance.
1. Chen, Y. et al. Nat. Biotechnol. 23, 405410 (2006). 2. Prausnitz, M.R., Mitragotri, S. & Langer, R. Nat. Rev. Drug Discov. 3, 115124 (2004). 3. Karande, P., Jain, A. & Mitragotri, S. Nat. Biotechnol. 22, 192197 (2004).

4. Kalia, Y.N., Naik, A., Garrison, J. & Guy, R.H. Adv. Drug Deliv. Rev. 56, 619658 (2004). 5. Lavon, I. & Kost, J. Drug Discov. Today 9, 670676 (2004). 6. Dean, H.J. Expert Opin. Drug Deliv. 2, 227236 (2005). 7. Prausnitz, M.R. Adv. Drug Deliv. Rev. 56, 581587 (2004). 8. Bramson, J. et al. Gene Ther. 10, 251260 (2003).

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