Treatment for chemotherapy‐induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain

Abstract

Parathyroid hormone (PTH) agonists and antagonists have been shown to improve hair growth after chemotherapy; however, rapid clearance and systemic side-effects complicate their usage. To facilitate delivery and retention to skin, we fused PTH agonists and antagonists to the collagen binding domain (CBD) of Clostridium histolyticum collagenase. in-vitro studies showed that the agonist fusion protein, PTH-CBD, bound collagen and activated the PTH/parathyroid hormone-related peptide receptor in SaOS-2 cells. The antagonist fusion proteins, PTH(7–33)-CBD and PTH([−1]–33)-CBD, also bound collagen and antagonized PTH(1–34) effect in SaOS-2 cells; however, PTH(7–33)-CBD had lower intrinsic activity. Distribution studies confirmed uptake of PTH-CBD to the skin at 1 and 12 hr after subcutaneous injection. We assessed in vivo efficacy of PTH-CBD and PTH(7–33)-CBD in C57BL/6J mice. Animals were depilated to synchronize the hair follicles; treated on Day 7 with agonist, antagonist, or vehicle; treated on Day 9 with cyclophosphamide (150 mg/kg i.p.) or vehicle; and sacrificed on Day 39. Normal mice (no chemo and no treatment) showed rapid regrowth of hair and normal histology. Chemo + Vehicle mice showed reduced hair regrowth and decreased pigmentation; histology revealed reduced number and dystrophic anagen/catagen follicles. Chemo + Antagonist mice were grossly and histologically indistinguishable from Chemo + Vehicle mice. Chemo + Agonist mice showed more rapid regrowth and repigmentation of hair; histologically, there was a normal number of hair follicles, most of which were in the anagen phase. Overall, the agonist PTH-CBD had prominent effects in reducing chemotherapy-induced damage of hair follicles and may show promise as a therapy for chemotherapy-induced alopecia.

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

“…Meanwhile, either a ␤-branched residue or Leu is found at the position equivalent to Thr957 in most of the CBDs. Six other residues identified by 15 N-HSQC-NMR titration to interact with minicollagen are not well conserved. Since divergent CBDs (s3 and s3b) adopted a similar cleft-shaped binding pocket, other CBDs may also adopt a similar collagen-binding structure.…”

“…The s3b binds to collagenous peptides with triple-helical conformation but not to the similar peptides lacking triple-helical conformation or to gelatin (denatured collagen), suggesting that the CBD-collagen interaction is conformation specific (4,7). The most recent solution studies of s3b in complex with collagenous peptides by smallangle X-ray scattering (SAXS) and heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) titration of various spin-labeled collagenous peptides to 15 N-labeled s3b showed that s3b binds unidirectionally to the C terminus of collagenous peptides (10).…”

“…It was uncertain as to how Ser896 in s3 could construct the dual calciumbinding site in place of the bidentate Asp. To address its structureand-function relationship applicable for various drug delivery applications (14)(15)(16)(17)(18), structural work on s3 was initiated. The structures of dissimilar CBD molecules in the metallopeptidase family M9B enabled us to compare and contrast their Ca 2ϩ sites and collagen-binding mechanism.…”

The Effect of Parathyroid Hormones on Hair Follicle Physiology: Implications for Treatment of Chemotherapy-Induced Alopecia

“…The PTH(7-34)-CBD and PTH-CBD subcutaneous injection in mice initially resulted in whole-animal distribution and, later, was followed by the redistribution of applied peptides to collagen-rich tissues such as bone and skin. No symptoms of hyperparathyroidism or calcium serum level alterations were found in the study [88]. The majority of hairs in the histopathological samples of treated mice stayed long in anagen VI, confirming the very potent role of the peptides used.…”

“…A single dose of the peptide PTH(1-33) caused a hair regrowth in mice within 30 days and an increase in the number of anagen phase VI hair follicles. Surprisingly, PTH-CBD antagonists have no significant effect on hair growth [88]. In another study, PTH agonists and antagonists were CBD-linked and applied subcutaneously to mice treated with cyclophosphamide.…”