Cell-Penetrating Peptide

Cell-penetrating peptides in drug development: enabling intracellular targets

L. Chen, S.D. Harrison


A large body of literature has shown that CPPs (cell-penetrating peptides) are capable of carrying macromolecules across the plasma membrane. CPPs can penetrate a wide variety of tissue types and enable modulation of intracellular targets with molecules that, by themselves, are incapable of penetrating cells. As a result, CPPs are recognized for their potential value in validating intracellular targets that could lead to drug discovery programmes [Dietz and Bahr (2004) Mol. Cell Neurosci. 27, 85–131]. The potential for CPP–drug conjugates to be used as human therapeutic agents has not been extensively explored and there is limited knowledge regarding the characteristics of CPPs which are necessary for drug development. A better understanding of the properties of CPPs relating to in vivo pharmacology, pharmacokinetics, pharmacodynamics and safety will continue to inform and encourage novel drug development efforts using CPPs as therapeutics. Here we will discuss areas of interest for drug development of CPP-conjugated compounds.

  • cell-penetrating peptide
  • cell penetration
  • drug development
  • intracellular target
  • pharmacokinetics
  • toxicity


Over the last two decades, a number of CPPs (cell-penetrating peptides) have been identified and reported in the literature. The source from which these peptides have been derived from varies from viral proteins, such as VP22 [1,2] and HIV-Tat (transactivator of transcription) [3,4], to a homeobox protein in Drosophila, Antennapedia [5,6], and novel chemically designed peptides such as poly-arginine [7,8]. Despite the variability in their origins, these compounds are all capable of crossing biological membranes and carrying cargo with them into cells. The initial discovery of this class of peptides led to the idea that CPPs would play a significant role in novel drug discovery and development. CPPs effectively opened the door for cell-impermeable compounds to become intracellular targets. Classes of compounds that were inconceivable as therapeutic candidates in the past were now worthy of consideration for drug development. Compounds with poor uptake into tissues or bioavailability could now be redesigned with CPPs resulting in improved pharmacology. Existing therapies could be reconfigured for alternative, more effective or safer, routes of administration. The tremendous potential of CPPs in drug discovery and development has even led to the formation of a number of drug delivery companies with proprietary cell-penetrating technologies.

Despite the tremendous potential for use of CPPs in drug discovery and development, the majority of the research to date has been limited to drug discovery and not drug development. Notable drug development efforts include PsorBan®, a cyclosporin–poly-arginine conjugate formulated as a topical treatment for psoriasis [9], and KAI-9803, a PKC (protein kinase C) δ peptide inhibitor–Tat conjugate for the treatment of acute MI (myocardial infarction) [1012]. The overall lack of drug development with CPPs is likely to be a reflection of the uncertain technical challenges and costs of introducing an additional element to the mechanism and pharmacology of either a potential drug candidate or an approved drug when redesigning its use. In both of these circumstances, the increase in risk caused by introducing a CPP in drug development is high and will continue to impede the use of these peptides in drug development efforts until the risks are mitigated by an increase in the understanding of the pharmacological characteristics of CPPs relevant to drug development. To date, these studies are largely under-represented in the literature.

The majority of published literature is focused on a few distinct areas of investigation: identification of novel CPPs, mechanism of action of cell penetration and exploration of novel compounds or intracellular targets in cell-based systems [13,14]. These studies are critical for expanding our current understanding of CPPs in general and exemplify the predominant use of this technology for early research programmes. However, the risks associated with drug development are not readily addressed in these areas of research. Although identification of novel CPPs is useful in diversifying the pool of potential technologies, the existing peptides have been shown to be quite effective. The distinguishing features of different CPPs relevant for drug development are unclear because of the lack of preclinical and pharmacological data on these compounds. Further exploration into the basic mechanism of cell penetration is important in defining drug exposure and limitations of intracellular delivery. Yet these efforts do not directly mitigate the risks of safety and dosing selection for drug development. The use of CPPs to validate intracellular targets and demonstrate feasibility of use with novel compounds is critical for drug discovery. However, target validation is typically addressed through genetic and pharmacological approaches using reagents that penetrate cell membranes. However, CPP-conjugated novel compounds that demonstrate the potential for drug candidacy in target validation studies are seldom advanced to in vivo preclinical studies.

Traditional preclinical drug development relies on research in pharmacology, safety, pharmacokinetics/pharmacodynamics and chemistry manufacturing and controls. CPPs can alter the properties of a given compound in each of these areas. Further characterization of the features of CPPs will undoubtedly help to reduce the risks associated with these novel domains and encourage additional development efforts with CPPs.


A number of studies have used CPPs with cargoes of interest to demonstrate pharmacological activity in cell-based assays [13,14]. Fewer reports of in vivo pharmacology have been shown, although the number of publications is increasing. Not all reported studies of CPPs in animal models have focused on pharmacology. The use of peptides in traditional in vivo pharmacological models is challenging because of the metabolic instability of peptides in blood and plasma. Despite the hurdles of peptide degradation, numerous studies have shown efficacy and, in some cases, remarkable potency of CPP-conjugated compounds using in vivo animal models.

Studies using CPP-conjugated compounds in pharmacological models of oncology have been reported [13,14]. Gusarova et al. [15] fused nine N-terminal D-arginine residues to a tumour-suppressive peptide derived from the ARF (alternative reading frame) protein and administered this peptide by i.p. (intraperitoneal) injection to hepatocellular carcinoma-bearing mice. Mice which were treated daily with 5 mg/kg i.p. injections for 4 or 8 weeks showed a reduction in tumour size, proliferation and angiogenesis compared with control animals. Similarly, Harada et al. [16] generated a Tat ODD (oxygen-dependent domain)–caspase 3 fusion protein. Hypoxic tumour-bearing mice were treated with 2 or 20 mg/kg caspase 3 fusion protein or a control fusion protein by i.p. injection. Tumour-bearing mice treated with the caspase 3 fusion protein showed significantly blunted tumour growth compared with control animals.

Pharmacological studies of CPP-conjugated compounds have also been reported in animal models of ischaemic injury of both the brain and heart [11,12,1719]. Cao et al. [19] reported that i.p. injection (3–9 mg/kg) of a Tat–Bcl-XL fusion protein reduced infarct size of the brain and improved neurological scores compared with controls in a mouse model of ischaemic stroke. Bright et al. [18] reported that administration of a PKCδ peptide inhibitor conjugated to Tat as either an intra-carotid administration (60 μg/kg) or i.p. administration (200 μg/kg) was also effective in reducing infarct size in a rat model of ischaemic stroke. Other reports using a Tat-conjugated peptide inhibitor of JNK (Janus kinase), D-JNK11, demonstrated reduction of neuronal damage in in vivo stroke and optic nerve crush models [2023]. These studies showed that either i.p. [20,23] or intracerebrovascular administration [20,21] of D-JNK11 resulted in a reduction of neuronal damage. Inagaki and co-workers have reported that a PKCδ inhibitor peptide conjugated to Tat administered as an intracoronary injection was effective in reducing cardiac infarction in an in vivo porcine model of acute MI at a dose of 250 ng/kg which has led to the development of KAI-9803 for the treatment of acute MI patients [11,12]. The preclinical pharmacology demonstrating the function of this compound used in a clinically relevant route of administration was critical in both the regulatory and operational implementation of this compound.

Although these studies demonstrate the potential for drug development with CPP-conjugated compounds, most of these pharmacological reports do not demonstrate efficacy using clinically relevant routes of administration. Administration by i.p. is commonly used in murine models, but is not typically acceptable in the clinic. There are a few reports that demonstrate efficacy of CPP-conjugated compounds by i.v. (intravenous) [24] or subcutaneous injection [25], both of which would be more clinically acceptable. Previous studies that have produced preclinical data that mimicked clinical applications were done by Rothbard et al. [9] and Inagaki and co-workers [11,12]. Rothbard et al. [9] showed efficacy of poly-arginine-conjugated cyclosporine topically administered in a mouse model of contact dermatitis. This compound was later tested as a topical administration of cyclosporine for psoriasis patients in clinical trials [26]. Inagaki and co-workers demonstrated the efficacy of a Tat-conjugated PKCδ inhibitor against acute MI by intracoronary injection [11,12], which has recently been studied clinically as an intracoronary administration in acute MI patients [10].

The potency and efficacy of CPP-conjugated compounds in pharmacological models is critical in supporting drug development efforts with these compounds. CPPs have been shown to be quite effective in delivering therapeutic proteins and peptides, which suffer from metabolic plasma instability [2729]. Although the potency of CPP-conjugated compounds will largely depend on the cargo itself, the properties of the CPP can play a significant role in exposure and tissue distribution in order to improve potency. Optimization of CPPs to improve the in vivo potency of CPP-conjugated compounds can be directed towards tissue-specific delivery [3032], metabolic stabilization [29], increasing cellular uptake [33,34] or directed subcellular localization of cargo [33,35]. Alternative routes of administration to localize drug delivery in pharmacological models would provide alternative methods to improve the potency of CPP-conjugated compounds.

CPPs are often used to deliver compounds in cell-based assays in order to identify targets and study signalling mechanisms [13]. Studies have expanded the use of CPP-conjugated compounds to in vivo animal models demonstrating the feasibility of using CPP-conjugated compounds as potential drug candidates. Additional investigation of CPP-conjugated compounds in disease models will validate further the potential of CPP-conjugated compounds as drug candidates. These studies should consider using clinically relevant routes of administration and dose response to demonstrate the potency and efficacy of the compounds of interest.

Pharmacokinetics and ADME

A critical component of many clinical development programmes is dose-selection as it relates to efficacy and safety. For most drug development programmes, drug pharmacokinetics and ADME (absorption, distribution, metabolism and elimination) are used to improve the pharmacological properties of lead compounds and to plan dosing schedules in clinical trials. For CPPs conjugated to novel compounds that do not cross biological membranes by themselves, the CPP domain will be a significant factor in dose–efficacy and dose–safety relationships. Despite the significance of these types of studies for drug development, few studies have focused on the pharmacokinetics and ADME of CPP-conjugated compounds.

Metabolic instability of peptides in plasma and serum is a hurdle to peptide-based therapeutics. Design of protecting groups for metabolically labile sites, PEGylation of compounds and use of D-isomers or retro-inverso peptides where applicable are a few of the approaches that have been applied to overcome this problem. However, many of these approaches were designed for peptides with extracellular targets. Increasing the plasma or serum half-life of these peptides effectively increases exposure to the target of interest and potentially increases the potency, efficacy and duration of the pharmacological effect. Compounds conjugated to CPPs focus on intracellular targets and therefore may face additional challenges to achieve the same improvements in pharmacological efficiency.

Reducing the plasma instability of CPP-conjugated compounds by increasing the levels of active compound prior to cellular delivery may be an effective means of improving potency and efficacy. Some of the traditional approaches to improving the plasma stability of peptides do appear to improve plasma stability significantly. D-Isomers of some CPPs are as effective as L-isomers in cell penetration [34,36], suggesting that simple modifications may be sufficient to stabilize CPPs in plasma without having an impact on cell-penetrating activity. However, other methods of peptide stabilization such as PEGylation may not be a desirable approach to improving plasma stability. It is uncertain whether or not PEGylation would alter the cell-penetration capabilities of a CPP or if accumulation of PEG [poly(ethylene glycol)] inside the cell has potential safety implications [37].

While metabolic instability is a known problem with peptide-based therapeutics, there are many unknown characteristics of the pharmacology of CPPs. Clearance, metabolism and elimination mechanisms are generally well understood for small molecules; and peptide or protein therapeutics that do not cross biological membranes are thought to be cleared by proteases. In contrast, the mechanism of clearance, elimination and metabolism of CPPs has not been fully elucidated. Polyakov et al. [38] used technetium (Tc)-labelled Tat to investigate the disposition and possible clearance mechanism of Tat following i.v. bolus administration. The authors report rapid whole-body distribution of the labelled peptide with patterns indicative of renal and hepatobiliary excretion. Lee et al. [39] used radiolabelled Tat conjugates to examine the pharmacokinetics and plasma and tissue clearance of the carrier. The authors report that Tat conjugation increased membrane permeation and resulted in rapid plasma clearance of Tat-conjugated compounds. Furthermore, organ clearance of Tat-conjugated compounds was also reported to be greater than that of unconjugated controls.

Tissue distribution plays an important role in determining the potential safety risks and clearance mechanisms of a given compound. A few studies have investigated the tissue distribution of CPPs [3841]. Cai et al. [41] used a Tat–β-galactosidase fusion protein to investigate the tissue distribution and bioavailability of a Tat-conjugated protein when given as an i.v., i.p., i.p.v. (intra-portal vein) or oral administration. The authors demonstrate peak activity and protein levels 15 min after i.v., i.p. and i.p.v. administration in most tissues. Tissue distribution was observed in the liver, kidney, spleen, lung, bowel and brain. Interestingly, the authors report that the i.p.v. injection resulted in prolonged tissue activity and an increase in the liver distribution of the Tat-conjugated protein, suggesting that the liver may serve as a depot for the fusion protein. The finding of Tat distribution to the brain in this study was in contrast with other reports that show limited Tat distribution to the brain [39,40].

Additional studies of tissue distribution, clearance metabolism and pharmacokinetics of CPP-conjugated compounds will better characterize the pharmacological properties of this class of compounds. Determining the pharmacokinetics and ADME of different CPPs may reveal important distinguishing pharmacological properties of these peptides. A better understanding of the kinetics and mechanism of clearance will be informative with respect to where CPP-conjugated compounds may have the best clinical use and determine how CPPs can be optimized for improved pharmacological properties. Investigation of metabolic liabilities may enable maximization of potency, efficacy and possibly the duration of pharmacological effects. Exploration of pharmacokinetics and ADME by different routes of administration may also be important to broaden the potential clinical application of these compounds. Finally, research with a diverse set of cargoes may reveal cargo-specific differences in pharmacokinetics and ADME that would broaden the feasibility of using different CPPs across multiple therapeutic approaches.


Safety of CPPs is critical to developing a wider use of these domains for drug development. A few studies have examined the cellular cytotoxicity of CPPs [13,14,42]. Fewer studies have reported in vivo toxicity with CPPs [43,44]. Two studies in particular have reported an absence of toxicity when mice were treated with CPPs at high doses. Begley et al. [43] reported that daily i.p. injections of a PKCε activator peptide conjugated to Tat did not show any evidence of organ pathology at a dose of 20 nmol (estimated 60 μg per mouse). Toro et al. [44] reported that increasing doses up to 8 units/g (estimated 80 mg/kg) of PNP (purine nucleoside phosphorylase) conjugated to Tat was not toxic when i.p. injected into mice over a 4 week period. Although this suggests that Tat exerts no toxicity in these two studies, there is still a great deal to learn about toxicity of CPPs in vivo.

There are no general reports of in vivo toxicology of CPPs by clinically relevant routes of administration, which is of particular interest to regulatory authorities [45]. Both PsorBan® and the clinical study of our peptide inhibitor (DELTA MI) have reported excellent tolerability to Tat-conjugated compounds by topical and intracoronary administration respectively [10,26]. Toxicology studies by i.v. or subcutaneous injection would play a significant role in characterizing the safety of CPPs. Although there are a few reports of safety studies with Tat, there are no comparable examples with other CPPs. Safety with different peptides may be a critical factor in distinguishing the benefit of one CPP over another. The current reports have examined tissue pathology [43] and blood-based markers of liver or kidney function [44] as a means of determining safety. Other acute safety parameters should be included such as blood pressure, heart rate, respiratory function and animal behaviour. These endpoints are commonly assessed when determining the safety of novel compounds. Mutagenicity is also commonly assessed early in the development of novel compounds [46]. Finally, immunogenicity and immunotoxicity are of particular concern with peptides and biological agents [47]. Although it is unlikely that CPPs, with a short plasma half-life and rapid tissue distribution, will generate an immune response by i.v. administration, CPPs may have differential effects on the immune system when administered as a subcutaneous administration which may drain directly to the lymphatic system [48].

In the process of drug development, drug leads are often tested in in vitro kinase and receptor screens to identify non-specific interactions that may be indicative of potential safety concerns in vivo. Although these assays are not predictive of in vivo toxicology, they are often requested by regulatory authorities, particularly in the absence of in vivo safety data. As an active domain of potential drug leads, CPPs should be tested in these traditional screens to characterize activity, if any, of CPPs in these assays. Although it may seem unlikely that CPPs would have activity in these assays, it is not entirely unreasonable. Arginine-rich domains have been implicated in receptor-mediated interactions; in particular, it appears that arginine-rich peptides can serve as calcium mimetics against the calcium-sensing receptor in vitro [49]. Determination of the activity of CPPs in these assays may suggest toxicology signals to be expected with these peptides. Translation of findings from in vitro studies to in vivo safety data will also be important in mitigating risks associated with developing drugs with CPP domains.

To date, there is an absence of in vivo toxicology information with CPPs. Current reports have determined cellular toxicity in cell lines or examined safety when administered as i.p. injections. Further characterization of in vivo safety of CPPs will encourage the use of these domains for drug development and possibly distinguish the benefits and risks associated with different peptides. It should be noted that demonstration of an absence of toxicology with CPPs would not necessarily encourage drug development. Establishing a full understanding of the safety signals to be expected, and how these effects can be monitored and mitigated, is critical to developing safe drugs. An absence of this knowledge is often a risk of uncertainty that is equally challenging and a greater obstacle in drug development.


CPPs have established a role in the drug discovery process for compounds with intracellular targets. Many publications show the value of these peptides for target validation, cell-based efficacy with novel compounds and a greater understanding of biological mechanisms. Despite the prominence of CPPs in drug discovery, the potential of using CPPs in drug development has yet to be realized. An expansion of the efforts to demonstrate the feasibility and mitigate potential risks associated with developing CPP-conjugated compounds is required to move this field on to the next stage of development. Focused studies on in vivo pharmacology, pharmacokinetics, tissue distribution, metabolism and safety of CPPs will provide tremendous value and understanding of the use of these compounds for novel therapeutics. The comparison of different CPPs in the context of drug development criteria may be critical in distinguishing the benefit of some CPPs compared with other CPPs.


  • Cell-Penetrating Peptides: A Biochemical Society Focused Meeting held at University of Wolverhampton, Telford, U.K., 9–11 May 2007. Organized and Edited by B. Austen (St. George's, University of London, U.K.), J. Howl (Wolverhampton, U.K.), S. Jones (Wolverhampton, U.K.) and B. Lebleu (Montpellier, France).

Abbreviations: ADME, absorption, distribution, metabolism and elimination; CPP, cell-penetrating peptide; i.p., intraperitoneal; i.p.v., intra-portal vein; i.v., intravenous; MI, myocardial infarction; PKC, protein kinase C; Tat, transactivator of transcription


View Abstract