PREFORMULATION STUDIES OF PEPTIDES KTTKS AND PAL-KTTKS 309 In the second heating runs, weight losses were less than 1% for both peptides. After both heating processes in TGA, the pans containing peptides were inspected visually there were no macroscopic changes in the appearance of peptide powders. In agreement with the present results, some researchers (16) showed that a DSC transition of around 63.1°C in a lyophilized synthetic trapezoid-inspired peptide fragment disappeared in the second DSC heating run. It was concluded by investigators to be due to the presence of bound water and subsequent water loss in the second run. S TABILITY STUDIES Ensuring the stability of permeants during the skin permeation experiment is crucial. The concentration of KTTKS solution was 100 μg/mL at the beginning of the stability study. After 48 h, the concentration of KTTKS solution reached 99.3 ± 1.7 μg/mL. Thus, it could be concluded that KTTKS remains stable at 32°C at least for 48 h. Therefore, there is no concern about the instability of this permeant during the skin permeation studies for at least 48 h. DISC USSION KTTK S and its derivative are popular in the cosmetic industry because of their anti- wrinkle properties, but many of their physicochemical properties remain unknown. This study is an attempt to defi ne the physicochemical properties of these peptides and to in- vestigate the effects of covalent attachment of a fatty acid on peptide properties. In t he case of UV absorbance, λmax of both peptides was at less than 200 nm. KTTKS and Pal-KTTKS have no aromatic amino acids in their structure, and the UV absorbance of them is due to the peptide bonds. The photons are absorbed by peptide bond at the maximum wavelength of below 210 nm (17). Note that the wavelengths below 200 nm belong to vacuum UV (18). The UV radiation is forcefully absorbed by atmospheric oxygen in the vacuum UV range (19). Therefore, a free oxygen environment is required to accu- rately assay KTTKS and Pal-KTTKS if UV spectroscopy at the maximum wavelength is to be used. Because KTTKS and Pal-KTTKS possess a broad absorption peak, it might be said that it is possible to measure the absorption at the longer wavelength, e.g., at 205 nm, but we noted that at this wavelength, the UV radiation is absorbed by many compounds used in buffers (20), which are mainly applied in skin permeation studies. In X- ray studies, both peptides showed a broad peak (hump) at around 2θ = 19° (see Figure 4), which was absent in palmitic acid. Such a broad peak at a wide angle region, which has also been reported for other peptides (21), might indicate that these peptides contain either some amorphous structures or some disordered crystalline structures. Both peptides also show peaks at 2θ  10 that indicate some long-range orders. The presence of some degrees of orders in the structure was also confi rmed by showing birefringence in the cross-polarized light. The presence of new peaks in the XRD pattern of Pal-KTTKS, which is not present in the pattern of KTTKS, means that the structures of these peptides are not the same therefore, some possible changes in the physicochemical properties of peptides, such as solubility, dissolution rate, and fl owability, are expected and should be considered by formulators.

JOURNAL OF COSMETIC SCIENCE 310 Palmitic a cid molecules form dimers because of OH….O hydrogen bonds and these di- mers pack into bilayers (22,23). Palmitic acid has a small polar head group (–COOH). Changing the head group to bulky KTTKS probably imposes some degree of disorder into the structure. Besides this, the molecules become longer with higher d-spacing: 34.6 Å for palmitic acid versus 44.2 Å for Pal-KTTKS. Pal-KTTKS also shows a higher d-spacing than KTTKS that are defi nitely due to the presence of long chain (C16) in the structure. Based on the results of partitioning and solubility tests, KTTKS has a highly hydrophilic nature (logP 0 and high water solubility). Because higher lipophilicity is required for good permeation through the skin (logP 0–3), KTTKS, like many other peptides such as Ala–Ala–Pro–Val (24), tetragastrin (25), and TRH (26), is not a good candidate for skin delivery. The results of the in vitro skin permeation study of KTTKS, which showed KTTKS did not permeate across the skin (27), is in good agreement with the present fi nding. KTTKS is a p entapeptide without a hydrophobic tail, but Pal-KTTKS has a 16-carbon chain (palmitic acid) as the hydrophobic tail, so Pal-KTTKS is a peptide amphiphile. The peptide amphiphiles are capable of forming a diversity of aggregates, such as micelles, cylindrical fibrils, sheets, and vesicles (28). Here, the results of tensiometry indicate that Pal-KTTKS has surface activity and reduces the surface tension of water to 50.3 ± 0.4 mN/m. The CMC of Pal-KTTKS in water was also determined by the ring method and found to be 0.024 ± 0.004 mM. There are no reports on the CMC value of Pal-KTTKS in aqueous solutions using the ring method. This CMC value resembles polysorbate 80 (0.02–0.03 mM) (29), which is structurally similar to Pal-KTTKS. The CMC value could be considered as the solubility of the individual molecules with surface activity because above this concen- tration, the molecules are not dissolved in the monomeric forms and aggregate as micelles (30). As said, this value for Pal-KTTKS was 0.024 ± 0.004 mM or 19.25 ± 2.9 mg/L thus, considering only individual molecules, it could be said this peptide amphiphile is practically insoluble in water. Cycled DSC and TG A results show that both peptide powders are hygroscopic. The hy- groscopic nature of some peptides, especially when present as porous lyophilized pow- ders, is a well-known property. On the other hand, it has been argued that peptides containing charged amino acids (such as arginine, aspartic acid, glutamic acid, histidine, and lysine) are hygroscopic (31). KTTKS and its derivative have two lysine amino acids, and therefore, it is possible that they absorb water. Hence, two water-related transitions (T1 KTTKS and T1 Pal-KTTKS ) in their DSC thermograms disappear on reheating, which is in agreement with weight losses observed in TGA thermograms. Finally, as far a s formulation is concerned, techniques such as UV, XRD, polarized light microscopy, SEM, and thermal analysis can be used to monitor changes during pharmaceu- tical processing, stability studies, and storage by the formulator. Some of these techniques, e.g., DSC, can be used to determine drug–excipients interactions. During pharmaceutical processing and storage, a formulator should be aware of the polymorphism and the pos- sibility of change of a polymorphic form into another due to processing (such as mixing and heating) and storage. For example, if a formulator uses these substances in the sus- pension dosage form (the presence of solid particles in the formulation), the stability during storage can be monitored using XRD or polarized light microscopy, as described earlier. The same applications apply to the SEM morphologies. SEM and polarized light