Alopecia (hair loss) is a psychologically and emotionally distressing side effect of cancer chemotherapeutic drugs 1. Chemotherapy-induced alopecia (CIA) can result in anxiety, depression, a negative body image, lowered self-esteem, and a reduced sense of well-being 2,3. The incidence and severity of CIA are variable and related to the particular chemotherapeutic protocols 4. It is estimated that 65–85% of chemotherapy patients experience some degree of alopecia 5,6. Overall, 47–58% of female cancer patients considered hair loss to be the most traumatic aspect of chemotherapy and 8% would decline treatment for fear of hair loss 2,5.
The mechanism of CIA is unknown and probably specific for each chemotherapeutic agent 7. There appears to be a direct effect of at least some chemotherapeutics causing damage to the hair follicles 8,9. There are currently no effective therapies for CIA 10,11, nor are there any satisfactory methods of preventing CIA 12. The only currently approved prevention for CIA is scalp hypothermia during chemotherapeutic courses to reduce scalp blood flow and reduce delivery, and hence the damage, of the chemotherapeutics to the skin. Clinical reports and testimonials do provide support showing efficacy for this treatment 13–15. However, the therapy is very expensive (Penguin Cold Cap total cost $600 US/month plus shipping), and there are concerns that vasoconstriction in the scalp may promote the development of cutaneous scalp metastasis 16.
Parathyroid hormone (PTH) agonists and antagonists have been shown to effectively manipulate the hair follicle response to chemotherapy-induced damage in depilated mice 17. However, the regrowth of hair required multiple intraperitoneal injections, which may be the result of limited delivery and retention of the drug to the skin. To improve skin delivery and retention, we synthesized fusion proteins linking PTH agonists and antagonists to a collagen-binding domain (CBD) derived from ColH collagenase of Clostridium histolyticum. We showed that these compounds are concentrated and retained in the skin after intraperitoneal or subcutaneous administration 18,19. In a depilated mouse model of cyclophosphamide (CYP)-induced alopecia, a single dose of the agonist, PTH-CBD, restored hair growth over a 30-day period, increasing the number of anagen VI phase hair follicles; CBD-linked PTH antagonists had no evident effect 18. Although depilation has the advantage of synchronizing the hair follicles and maximizing the chemotherapy-induced damage, depilation itself causes injury to hair follicles as well, which may have affected our experimental results 20,21. In fact, we confirmed in a pilot study (results below) that PTH-CBD therapy does indeed alter the course of recovery following depilation injury without chemotherapy. During the course of conducting long-term experiments on the effects of PTH-CBD on chemotherapy-induced osteoporosis, we observed that providing three cycles of CYP chemotherapy 1–2 weeks apart, in the absence of depilation, results in long-term hair loss, presumably because spacing the cycles of chemotherapy captures a different set of hair follicles in the anagen phase each time 22. We therefore proceeded with a series of studies using this model to test the effects of PTH-CBD agonist in CIA in mice in the absence of depilation.
Healthy female mice (C57BL/6J), 3–5 weeks of age, weighing 13–15 g were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and housed in cages at the animal facility at Ochsner Clinic Foundation under standard living conditions, including a diet consisting of 18% protein purchased from Harlan Company (Barton, Illinois, USA and Madison, Wisconsin, USA). The mice were allowed to acclimatize for a 2-week period before the start of the experiment. Approval for these studies was obtained from the Institutional Animal Care and Use Committee (Ochsner Clinic Foundation, New Orleans, Louisiana, USA).
CYP was obtained from the Ochsner Clinic Foundation Cancer Infusion Center in solution (20 mg/ml) and used within 24 h to ensure full bioactivity. PTH-CBD and PTH(7–33)-CBD were diluted for injection in a collagen-binding buffer vehicle (50 mmol/l Tris HCl, 5 mmol/l CaCl2, pH 7.5).
Pilot study – depilation only
Twenty-one mice were depilated by shaving, followed by waxing using Sally Hanson wax strips (Wal-Mart, Luling, Louisiana, USA). Mice were then divided equally into three groups: (a) vehicle control, which received an injection of vehicle only; (b) PTH-CBD, which received a single subcutaneous injection of 320 μg/kg of PTH-CBD; and (c) PTH antagonist, which received a single subcutaneous injection of 320 μg/kg of PTH(7–33)-CBD. All injections were administered at the time of depilation. Mice were observed for 18 days and then killed.
Study 1: prophylactic effects of PTH-CBD on chemotherapy-induced alopecia
Thirty mice were divided equally into three groups: (a) no chemo, which received injections of vehicle only; (b) chemo, which received CYP injections at a dose of 50 mg/kg at 0, 2, and 4 weeks and buffer at week 0; and (c) chemo+PTH-CBD prophylaxis, where chemo+PTH-CBD was received as injections of 50 mg/kg CYP at 0, 2, and 4 weeks and a single injection of 320 μg/kg of PTH-CBD at week 0. All injections were administered intraperitoneally. The mice were observed for 15 months and then killed.
Study 2: treatment effects of PTH-CBD on chemotherapy-induced alopecia
Twenty mice were injected intraperitoneally with three separate doses of CYP (120, 120, and 150 mg/kg) at 0, 2, and 6 weeks. The mice were observed periodically for the general health status, signs of alopecia as well as change in hair color. After 4 months, the mice developed variable hair loss and loss of pigmentation. In addition, we removed hair from a small region on the lower dorsal surface of mice using Nair cream. Mice were divided randomly into two groups of 10 each, a chemotherapy-only group (chemo) receiving a subcutaneous injection of vehicle and a treatment group (chemo+PTH-CBD therapy) receiving a subcutaneous injection of PTH-CBD (320 mcg/kg) at the site of hair removal. The mice were observed for 21 days and then killed.
Study 3: prophylactic versus treatment effects of PTH-CBD for chemotherapy-induced alopecia
Twenty-four healthy female mice received three doses of CYP by an intraperitoneal injection (150 mg/kg at 0, 1, and 2 weeks). Of these, eight mice (chemo+PTH-CBD prophylaxis) received a single dose of subcutaneous injection of PTH-CBD (320 μg/kg) together with the first course of chemotherapy (week 0). After 12 months, mice receiving chemotherapy alone were divided into two groups, one receiving a single subcutaneous injection of PTH-CBD (320 mcg/kg) (chemo+PTH-CBD therapy) and one receiving vehicle (chemo). The mice were observed for 4 months and then killed.
All the three study protocols have been summarized in Table 1.
Photo-documentation was obtained to monitor the changes in hair growth. For gray scale analysis, images were captured using the Kodak Gel Logic 100 Imaging System (Eastman Kodak, Rochester, New York, USA) on a Spectroline Bi-O-Vision UV/white light transilluminator (Spectronics Corporation, Westbury, New York, USA).
Photographs were taken with exposure 0.2 s, F-stop 2 mm, and magnification 15 mm to maintain the hair texture in the linear range for analysis. For gray scale analysis, an elliptical region of interest was selected on the dorsal skin of the mice, covering as much of the back of the mouse as possible. This densitometry value was normalized to the average of those obtained from two background region of interests placed on either side of the mouse.
Mice were killed and skin regions from the nape of the neck to the middle of the back were obtained for histological analysis. The skin was fixed in 10% buffered formalin and processed for routine histology using hematoxylin and eosin staining. Skin samples were sectioned along the longitudinal and cross-sectional axes of the hair follicles. The number of anagen VI hair follicles and the number of these hair follicles in each skin layer per high-power field were determined by three independent observers who were blinded to the treatment status of the animals. The high-power fields with maximum follicular density were selected for counting: one with follicles predominantly on long section and one with follicles predominantly on the cross-section selected per slide.
Measurements were analyzed by analysis of variance, followed by post-hoc tests, either Bonferroni’s or Dunnett’s, as indicated for each comparison. The software used for this statistical analysis is GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, California, USA).
Pilot study – depilation only
To determine whether the positive effects of PTH-CBD on hair regrowth in a depilated mouse model of chemotherapy 18 might be partially attributable to positive effects on depilation injury, we examined the effects of our treatments in mice depilated by waxing with no chemotherapy. Vehicle-treated mice showed anagen response on day 9–10 as expected (figure not shown), and by day 18 had a thin coat of hair covering the depilated region (Fig. 1a). PTH-CBD-treated animals had an earlier anagen response at the injection site (day 7), early hair eruption by day 10 as expected, and by day 18 had a thicker coat of hair (Fig. 1a). Antagonist-treated animals had an anagen eruption at the site of injection that was earlier still, on day 5, followed by modest hair eruption over that location (figure not shown). However, further hair growth appeared to have been inhibited such that by day 18, the injection sites appeared relatively bare (Fig. 1a). On histological examination, both vehicle control-treated and agonist-treated animals showed abundant normal-appearing anagen follicles on day 18 (Fig. 1b), as expected following the depilation injury. Antagonist-treated animals had fewer hair follicles at the site of injection, which appeared to be dystrophic, with evident melanocyte clumping (Fig. 1b). These histological changes bear a remarkable resemblance to those observed in the depilated CIA animals 18, making it difficult to distinguish drug effects in response to depilation injury versus those in response to chemotherapy injury.
Thus, although depilation has the advantage of synchronizing the hair follicles to provide maximal and more uniform chemotherapy-induced damage, the depilation itself causes an injury response in the hair follicles that can be modulated by treatment with CBD-linked PTH analogs. We therefore proceeded with the following experiments to determine whether our previous findings in the depilated chemotherapy mouse model 18 could be confirmed in a model of chemotherapy alopecia that does not include a depilation step. As the antagonist compounds showed obvious deleterious effects on hair growth, we did not conduct further testing of these compounds in treating chemotherapy alopecia.
Study 1: prophylactic effects of PTH-CBD on chemotherapy-induced alopecia
In a study carried out primarily to evaluate the effects of PTH-CBD on chemotherapy-induced osteoporosis 23, we observed differences in hair loss patterns between treated and control animals. At the termination of the study (15 months after the initial treatment), the group receiving CYP chemotherapy alone (chemo) showed variable hair loss and depigmentation, whereas the group receiving CYP and PTH-CBD (chemo+PTH-CBD prophylaxis) showed normal hair that was indistinguishable from that of mice receiving no chemotherapy (no chemo) (Fig. 2).
Histological examination showed morphological changes in the hair follicles after CYP therapy (chemo), which were more superficially located and showed clumped melanocytes around the bulb, characteristics of the dystrophic anagen and catagen phase 24 (Fig. 3). PTH-CBD pretreatment (chemo+PTH-CBD prophylaxis) led to deeper rooting and reduced melanocyte clumping, thus reversing the dystrophic changes. Hair follicle counts from skin samples obtained at the end of the study period showed a trend toward greater number of hair follicles in the subcutaneous fat layer (9.3±6.8 vs. 0.6±0.5, NS), which, on the basis of the anatomical location, can be presumed to be in the anagen VI phase. The greater number of anagen VI hair follicles and deeper-rooted hair follicles are consistent with the observed increases in hair growth. These results provided indications that PTH-CBD pretreatment could prevent the hair loss from CYP chemotherapy.
Study 2: treatment effects of PTH-CBD on chemotherapy-induced alopecia
After observing these prophylactic effects of PTH-CBD in chemotherapy-induced hair loss, we next tested whether PTH-CBD administration can treat CIA if administered after the hair loss develops in this non-depilated mouse model. Mice administered a higher dose of CYP than in the first study developed variable hair loss, thinning, and decreased pigmentation 4 months after chemotherapy (Fig. 4). Twenty-one days after the administration of PTH-CBD (chemo+PTH-CBD therapy), mice showed evidence of partial regrowth and repigmentation of hair, whereas those receiving vehicle alone (chemo) showed continued hair loss (Fig. 4). Skin samples obtained from the site of injection at the termination of the study showed relatively fewer dystrophic hair follicles in the chemo group, similar to those in the first study (not shown). The PTH-CBD-treated mice (chemo+PTH-CBD therapy) showed more numerous hair follicles with reversal of the dystrophic changes, again similar to those in the first study (not shown). Histological sections of regions of the tail, where the hair coat is normally thin, were indistinguishable between groups (not shown). Thus, it appears that administration of PTH-CBD after hair loss occurs can reverse some of the chemotherapeutic-induced changes, although there is a suggestion of only partial recovery.
Study 3: prophylactic versus treatment effects of PTH-CBD for chemotherapy-induced alopecia
Having observed the beneficial effects of PTH-CBD when administered before or after development of chemotherapy-induced hair loss, we next conducted an experiment to directly compare prophylactic versus therapeutic administration of PTH-CBD on hair growth after CYP administration. Animals receiving chemotherapy alone (chemo) showed evidence of hair loss beginning 6–12 months after treatment, whereas animals treated with PTH-CBD at the time of receiving chemotherapy (chemo+PTH-CBD prophylaxis) showed no evidence of hair loss at any time point (Fig. 5). Animals treated with PTH-CBD after the hair loss had developed (chemo+PTH-CBD therapy) showed partial recovery of hair growth over the next 4 months.
When quantified by gray scale analysis, animals treated with PTH-CBD either prophylactically (chemo+PTH-CBD prophylaxis) or therapeutically (chemo+PTH-CBD therapy) showed significantly greater average light absorption on the dorsum compared with those receiving chemotherapy alone (Fig. 6, P<0.05). As the hair on C57BL/6J is naturally black, this finding indicates a combination of increased hair and/or increased hair pigmentation in the PTH-CBD-treated animals. There was also a nonsignificant trend toward greater light absorption in animals treated with PTH-CBD prophylactically versus those treated after hair loss had developed.
Histological examination of the skin samples from the chemotherapy-only group (chemo) again showed small, dystrophic hair follicles with melanin clumping (not shown). Prophylactic administration of PTH-CBD (chemo+PTH-CBD prophylaxis) resulted in reversal of the dystrophic changes, and mice in the treatment group (chemo+PTH-CBD therapy) showed intermediate histological features (not shown). Hair follicle counts were highly variable, presumably because the hair follicles were not synchronized by depilation. Although analysis by two-way analysis of variance was not significant, there was an apparent trend toward greater number of anagen VI hair follicles in the subcutaneous region with prophylactic PTH-CBD administration (chemo+PTH-CBD prophylaxis) versus chemotherapy alone (chemo) (15.6±12.3 vs. 0.1±0.1, NS), but not with animals treated with PTH-CBD after developing alopecia (chemo+PTH-CBD therapy) (0.1±0.1 vs. 0.1±0.1, NS) (Fig. 7). There appeared to be a trend toward a greater number of hair follicles in the dermal layers in the therapy group versus the no chemo group (5.0±2.8 vs. 10.2±0.4, NS).
PTH-CBD is a fusion protein of the active portion of parathyroid hormone and a bacterial collagen-binding domain. This compound was designed to promote the distribution and retention of PTH(1–33) to high collagen-containing tissues with relatively high blood flow, such as bone and skin 19,23. We have shown previously that PTH-CBD promotes more rapid regrowth of hair in a depilated mouse model of CIA 18. However, we observed in those studies that depilation itself induces anagen VI phase transition of the hair follicles in control animals not receiving chemotherapy, 20,21 and here, we show that the response to this hair follicle injury is modulated by PTH-linked CBD compounds, raising concerns that the positive effects of PTH-CBD observed previously in the depilated mouse model of CIA may have resulted from improved repair of depilation injury rather than of chemotherapy injury. Interestingly, this study also showed what appeared to be previously unknown deleterious effects of PTH antagonists on hair growth, inducing dystrophic changes in hair follicles and inhibiting normal regrowth of hair after depilation. These findings provide evidence that the agonist PTH-CBD and the antagonist PTH(7–33)-CBD do indeed have opposing effects on hair follicles.
We observed in studies of chemotherapy-induced osteoporosis that a chemotherapeutic regimen that more closely mimics standard regimens for cancer therapy in humans also results in long-term hair loss in mice. Presumably, cyclical administration of chemotherapy captures a different set of hair follicles in the anagen phase with each cycle. We therefore proceeded to test the effects of PTH-CBD in this model of CIA in mice in the absence of depilation. When administered prophylactically, a single subcutaneous injection of PTH-CBD prevented CYP-induced hair changes, such as thinning and color change. These changes were grossly apparent in the photo-documentation record; in fact, the chemotherapy-treated mice that received PTH-CBD prophylaxis were indistinguishable from those that did not receive chemotherapy (Fig. 2). Histologically, the mice treated with CYP alone showed hair follicles predominantly in the dystrophic anagen and catagen phases, whereas those pretreated with PTH-CBD showed deeper rooting and reduced melanocyte clumping, thus reversing the dystrophic changes, similar to animals that did not receive chemotherapy (Fig. 3).
When administered as a therapy after hair loss develops from CYP administration, PTH-CBD treatment promoted regrowth of hair, although 21 days after PTH-CBD treatment the hair loss had not completely reversed in most animals. Histologically, the PTH-CBD administration again resulted in more numerous hair follicles and in resolution of the dystrophy induced by CYP treatment. On directly comparing prophylactic versus therapeutic administration of PTH-CBD, we again observed no hair loss after prophylactic PTH-CBD administration; therapeutic administration of PTH-CBD resulted in correction of hair loss that appeared to be less complete. Although PTH-CBD administration resulted in resolution of hair follicle dystrophy when administered either before or after the hair loss event, we only observed apparent increases in anagen VI hair follicles with prophylactic PTH-CBD administration (Fig. 7). There did appear to be a greater concentration of hair follicles in the dermal layers in the therapy group, which appeared to be more mature telogen follicles. Importantly, there was no apparent change in hair growth, nor were there any evident histological changes, in regions of skin (tail, ears) that do not normally have a full coat after PTH-CBD administration.
CYP is an alkylating agent that induces DNA damage, thus preferentially affecting cells that are dividing more frequently. PTH would not be expected to have any direct effects in inhibiting this process; the prevention of hair loss presumably occurs as a result of physiologic antagonism of the chemotherapy-induced hair loss effect. The increase in anagen VI follicles after PTH-CBD treatment is consistent with known effects of PTH in activating WNT signaling and increasing the production of β-catenin 25, which induces hair follicle transition to the anagen phase 26,27. However, given that CYP primarily damages anagen VI hair follicles 8, it is surprising that prophylactic administration of PTH-CBD provides protection, rather than enhancing chemotherapy damage. We postulate that continued anagen phase induction after this initial damage would result in more rapid replacement of the damaged hair follicles, with an overall net positive effect on hair growth. This would predict that PTH-CBD should be effective in preventing hair loss from a variety of chemotherapeutic agents.
Although various approaches have been available for the prevention and treatment of chemotherapy-induced alopecia, these generally have limited efficacy. Prevention includes the use of scalp tourniquets or scalp hypothermia 28. Scalp tourniquets decrease the blood flow to the scalp, which in turn reduces the delivery of chemotherapeutics to the hair follicles 29; however, it also causes significant patient discomfort and would prevent chemotherapeutics from treating tumor cells in the scalp, and is therefore not in widespread use. Scalp cooling is intended to reduce hair loss by reducing blood flow and by decreasing metabolic activity of the hair follicles, decreasing their susceptibility to chemotherapeutic damage 30. Most studies show decreased hair loss with scalp cooling (WHO grade 0, 1, 2 or ‘no wig required’), although most of these are not randomized trials 30. Specific results depend on the type of chemotherapeutic used, the amount of cooling in different protocols, and the chemotherapeutic protocol utilized 31,32. Cooling decreases the effectiveness of chemotherapeutics in the scalp, and is contraindicated in patients with extensive hematological malignancies 33. Other side effects include headaches, uncomfortable sensation, and claustrophobia from the devices, leading to more than 10% of patients discontinuing the treatments in some studies 34,35. The overall effect of scalp cooling as an available therapy for CIA is a moderately effective method to prevent hair loss during most chemotherapy regimens 36.
Although there are currently no approved drug treatments for CIA in humans, attempts have been made to utilize existing treatments for other forms of hair loss in chemotherapy patients. Topical minoxidil has been approved for the treatment of androgenic alopecia and alopecia areata, but it has very limited efficacy in CIA 37,38. Among all the agents that have been evaluated in humans for preventing CIA, only minoxidil was able to reduce the severity or shorten the duration of CIA, but it could not prevent hair loss 33,37. Although calcitriol was considered a promising agent, it did not prevent or treat CYP-induced alopecia in mice 7,39. Furthermore, topical administration may result in contact dermatitis 40.
Overall, it appears that PTH-CBD is effective in preventing or reversing hair loss in a non-depilated mouse model of CIA. Prophylactic therapy appeared to be more effective than treatment after hair loss develops. As there are no known or suspected interactions with PTH-CBD on the chemotherapeutic effects of CYP, the reversal of hair loss appears to be the result of the direct effects of PTH-CBD on the hair follicles. Treatment with PTH-CBD results in an increase in the number of anagen VI hair follicles, likely through a mechanism of activation of WNT signaling. With continued study, PTH-CBD may develop as a promising new therapy for CIA.
The authors are grateful to Dr Alan Burshell, section head, department of endocrinology at Ochsner Clinic Foundation, New Orleans, Louisiana, for his kind support and scientific advice throughout the studies. The authors also acknowledge support, in whole or in part, by the National Institutes of Health Center for Protein Structure and Function Grants NCRR COBRE 1 P20RR15569 and INBRE P20RR16460. This work was also supported by the AR Biosciences Institute (ABI) and a grant-in-aid for scientific research (C) from the Japan Society for the Promotion of Science and Kagawa University Project Research Fund 2005–2006.
Conflicts of interest
PTH-CBD is patented and exclusively licensed to Biologics MD, LLC. Robert Gensure is Chief Medical Officer of Biologics MD. Robert Gensure, Tulasi Ponnapakkam, Osamu Matsushita, and Joshua Sakon have a stock ownership in Biologics MD. Ranjitha Katikaneni has no conflicts of interest.
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