Cyclic Dipeptide Shuttles as a Novel Skin Penetration Enhancement Approach: Preliminary Evaluation with Diclofenac

Abstract

This study demonstrates the effectiveness of a peptide shuttle in delivering diclofenac into and through human epidermis. Diclofenac was conjugated to a novel phenylalanyl-N-methyl-naphthalenylalanine-derived diketopiperazine (DKP) shuttle and to TAT (a classical cell penetrating peptide), and topically applied to human epidermis in vitro. DKP and TAT effectively permeated into and through human epidermis. When conjugated to diclofenac, both DKP and TAT enhanced delivery into and through human epidermis, though DKP was significantly more effective. Penetration of diclofenac through human epidermis (to receptor) was increased by conjugation to the peptide shuttle and cell penetrating peptide with enhancement of 6x by DKP-diclofenac and 3x by TAT-diclofenac. In addition, the amount of diclofenac retained within the epidermis was significantly increased by peptide conjugation. COX-2 inhibition activity of diclofenac was retained when conjugated to DKP. Our study suggests that the peptide shuttle approach may offer a new strategy for targeted delivery of small therapeutic and diagnostic molecules to the skin.

Fig 1. Synthesis of DKP Phe-N-MeNal.

Fig 2. Synthesis of DKP Phe(Diclofenac)-N-MeNal conjugate.

Fig 3. Comparison of human skin permeation profiles of DKP and TAT alone.

Indicated values are mean ± (sem) of n = 7, *P<0.05.

Fig 4. Human skin permeation of diclofenac alone, diclofenac as admixture with DKP, diclofenac as DKP conjugate and diclofenac as TAT conjugate.

The data is represented as mean ± sem (n = 5), *P<0.05.

Fig 5. Amount of DKP, diclofenac, DKP-diclofenac conjugate, TAT-diclofenac conjugate and TAT in the skin after permeation experiments (donor amount of 150 μg).

Data is represented as mean ± sem (n = 4), * P<0.05.

Fig 6. Percent inhibition of COX-2 by diclofenac,DKP-diclofenac conjugate and DKP alone over the concentration range of 0.00015–0.31 mM.

The indicated values are means ± SD (n = 3). The COX-2 inhibition caused by diclofenac was tested for significance against DKP alone and DKP-diclofenac conjugate (* p<0.05).