**5.2.3 Hair growth cycle modifiers**

#### **5.2.3.1 Cyclosporine A**

60 Topics in Cancer Survivorship

applied to the skin to improve heat conductance. Heat treatment at 48-48.5°C for 20 minutes increases Hsp70 and subsequently protects against CIA in response to various treatments as summarized in Table 3. The protective effect of heat treatment was confirmed in an adult mouse model receiving cyclophosphamide. Additionally, localized heat treatment was shown not to interfere with the anti-tumor activity of drugs. These findings suggest that localized activation of stress proteins in the hair follicles might be an effective strategy

**Chemotherapeutic agents Dosing Protective** 

I.P. 2.5 µg/g twice I.P. 35.5 µg/g twice

Table 3. Localized, heat-induced protection against CIA in neonatal rats.

animals but their clinical use will require further investigations.

S.C. 5 µg/animal, twice

Currently, there are no FDA-approved drug treatments for CIA but several pharmacological strategies have been proposed. Many of these strategies have shown promising results in

Differences in the molecular machinery of normal cells and tumor cells as a result of cell transformation dominate the tumor targeting delivery arena. Tumor-specific ligands and antibodies have been used to provide targeting ability to drug carriers such as liposomes. Accordingly, these liposomes can protect patients from the side effects of chemotherapy, including hair loss. Examples of the targeting moieties are folate receptor (FR) for ovarian, colorectal, and breast cancer; transferrin for pancreatic cancer; anti-HER2 antibody for breast cancer; anti-CD19 for malignant B cells; anti-GD2 for neuroblastoma and melonoma; and prostate-specific membrane antigen (PMSA) aptamer for prostate cancer and tumor vascular

MAD11 monoclonal antibody (MAb) is an anti-anthracycline antibody that reacts with doxorubicin and other anthracycline chemotherapeutics. Topical administration of liposomes containing MAD11 MAb was shown to prevent CIA in doxorubicin-treated neonatal rats at the frequency of 31 in 45 rats (Balsari et al., 1994). MAD11 MAb was encapsulated into liposomes to facilitate absorption through the stratum corneum and to delay systemic distribution of the antibody. Topical MAD11 MAb was found to be nontoxic and does not induce systemic activation of cytokines. Thus, MAD11-loaded liposomes might be an effective strategy in preventing anthracycline-induced alopecia in cancer patients. However, the advantage of this strategy is limited in combination therapy since the

I.P. 20-30 µg/g once/2.5-4.5 µg/g twice

**Frequency** 

0.94 (45/48) 0.97 (29/30) 1.0 (56/56) 1.0 (7/7)

against CIA without affecting the anti-tumor efficacy.

Etoposide

Taxol

Cyclophosphamide

Cyclophosphamide/doxorubicin

**5.2 Pharmacological prevention** 

**5.2.1 Tumor targeting delivery** 

**5.2.2 Drug-specific antibodies** 

endothelium (Huges et al., 2001; Yu et al., 2009).

antibody could not react with the other drugs in combination.

Cyclosporine A is an immunosuppressive immunophilin ligand used in the treatment of autoimmune diseases and in post-organ transplantation to reduce patients' graft rejection. In Tlymphocytes, cyclosporine A forms complex with cyclophilin and inhibits calcineurin, leading to the inhibition of Go to G1 cell cycle transition and proliferation. The use of cyclosporine A in alopecia originates from its common side effect of excessive hair growth called hypertrichosis. Cyclosporin A induces anagen and inhibits catagen of the hair cycle, leading to the promotion of hair growth under normal and pathologic conditions such as alopecia areata and androgenetic alopecia (Paus et al., 1989; Taylor et al., 1993; Lutz et al., 1994).

The effect of cyclosporine A on CIA has been investigated in neonatal rat and adult mouse models. In neonatal rats, topical administration of cyclosporine A prevents CIA induced by cyclophosphamide, cytosine arabinoside and etoposide (Hussein et al., 1995). In adult mice given cyclophosphamide, topical or systemic administration of cyclosporine A retards CIA, prevents the progression of damaged hair into telogen, and thus induces faster hair regrowth.

#### **5.2.3.2 AS101**

AS101, ammonium trichloro (dioxoethylene-o,o') tellurate, is a synthetic immunomodulator that has been shown to protect mice from hemopoietic damage caused by chemotherapeutic agents such as cyclophosphamide, 5-fluorouracil, doxorubicin and etoposide. In phase II clinical trials, AS101 was shown to protect against CIA in patients with non-small cell lung cancer (NSCLC) receiving a combination therapy of carboplatin and etoposide (Sredni et al., 1996). The results of this study are summarized in Table 4.


Table 4. Prevention of alopecia in AS101-treated NSCLC patients.

The mechanism of action of AS101 was investigated in neonatal rats receiving cytosine arabinoside (Sredni et al., 1996). The study demonstrated that the protective effect of AS101 was through macrophage-derived factors such as interleukin-1 (IL-1). IL-1 induces the secretion of other cytokines such as keratinocyte growth factor (KGF) which stimulate the proliferation and differentiation of keratinocytes within the hair follicles.

#### **5.2.3.3 Minoxidil**

Minoxidil is one of the FDA approved drug for the treatment of androgenetic alopecia. Topical minoxidil shortens the telogen phase by inducing the entry of resting hair follicles into the anagen phase, thereby stimulating hair growth (Messenger and Rundegren, 2004). Minoxidil also prolongs the duration of anagen phase and enlarges hair follicles, probably by its proliferative and anti-apoptotic effects on dermal papilla cells (Han et al., 2004). Several studies have also investigated the effect of minoxidil on CIA. In neonatal rats, local injection of minoxidil protects against CIA induced by cytosine arabinoside but not by cyclophosphamide. However, topical minoxidil (2%) does not protect against CIA. In one

Chemotherapy-Induced Alopecia 63

Rapid proliferation of keratinocytes during the anagen phase of hair follicle is one the main predisposing factors of CIA. Thus, one approach to protect against CIA is to arrest the cell

Multiple effects of calcitriol (1,25-dihydroxyvitamin D3) on keratinocytes, i.e., inhibition of DNA synthesis, Go/G1 cell cycle arrest, and induction of cell differentiation, have been reported (Kobayashi et al., 1998; Wang et al., 2006). Thus, it is likely that calcitriol induces changes in keratinocyte proliferation and/or terminal differentiation, subsequently altering cellular susceptibility to apoptosis. In neonatal rats, topical administration of calcitriol reduces CIA induced by cyclophosphamide, etoposide, and a combination treatment of cyclophosphamide and doxorubicin (Jimenez and Yunis., 1992). In adult mice receiving cyclophosphamide, topical calcitriol however fails to prevent or retard CIA, but somehow reduces massive apoptosis of hair matrix keratinocytes, a key feature of CIA, and enhances the regrowth of normal hair shaft. (Paus et al., 1996; Schilli et al., 1998). In humans, calcitriol has a protective effect against CIA induced by paclitaxel (Jimenez and Yunis, 1996), but not by a combination of 5-fluorouracil, doxorubicin and

Cyclin-dependent kinase 2 (CDK2) is a member of the serine/threonine protein kinase family that plays a key role from late G1 to late G2 of the cell cycle. Potent small inhibitors of CDK2 have been synthesized and tested for their effect on CIA. One of these synthetic inhibitors was shown to inhibit the progression from late G1 into S phase in human diploid fibroblasts and also inhibit apoptosis induced by etoposide, 5-fluorouracil, taxol, cisplatin and doxorubicin. In neonatal rats, topical application of the inhibitor reduces hair loss at the site of application in 50% of the rats having etoposide-induced CIA and in 33% of the rats with CIA induced by cyclophosphamide and doxorubicin (Davis et al., 2001). Histological examinations of the skin from etoposide-treated rats show that the inhibitor increases the number of viable hair follicles and dermal papilla, reduces the level of inflammation and amount of damage to epithelium, reduces the thickening of epidermis and decreases the number of apoptotic cells in the hair follicle matrix. However, in subsequent studies the authors reported that they were unable to reproduce the results in the neonatal rat model (Davis et al., 2002), thus the use of this inhibitor in CIA becomes questionable, although the

Various chemotherapeutic agents induce apoptosis of hair follicle cells and cause CIA, although the underlying mechanisms are unclear. Caspase-3 is a key executor of apoptosis and its activation is normally used as an indicator of caspase-dependent apoptosis (Porter and Janicke, 1999). M50054, 2,2'-methylenebis, is an inhibitor of caspase-3 activation that was shown to inhibit etoposide-induced apoptosis in human monocytes. In neonatal rats, topical administration of M50054 reduces CIA induced by etoposide (Tsuda et al., 2001).

**5.2.6 Cell cycle or proliferation modifiers** 

cyclophosphamide (Hidalgo et al., 1999)

idea of using CDK2 inhibitors is still ongoing.

**5.2.7 Inhibitor of apoptosis 5.2.7.1 Caspase3 inhibitor** 

**5.2.6.2 CDK2 inhibitor** 

cycle and inhibit cell proliferation.

**5.2.6.1 Calcitriol** 


randomized clinical trial, topical minoxidil (2%) was shown to shorten the duration of CIA in breast cancer patients receiving 5-fluorouracil, doxorubicin, and cyclophosphamide (Duvic et al., 1996). The results of this study is summarized in Table 5.

Table 5. Minoxidil shortens duration of CIA.

#### **5.2.4 Cytokines and growth factors**

Hair follicle cells express receptors for multiple cytokines and growth factors that regulate hair growth cycle (Trueb, 2002). These regulators include fibroblast growth factors (FGF), transforming growth factors (TGF), insulin-like growth factors (IGF), epidermal growth factors (EGF), interferon and interleukins (Stenn and Paus, 2001). Moreover, hair cycle is regulated by androgens and parathyroid hormone (PTH) (Sawaya, 2001).

IL-1 and ImuVert, a biological response modifier derived from *S. Marcescens*, were reported to protect against CIA induced by cytosine arabinoside and doxorubicin in neonatal rats (Hussesin, 1993). Both agents can induce the release of multiple cytokines and growth factors. It was suggested that the protection of CIA by ImuVert is mediated through IL-1. Similarly, EGF and FGF-1 have been shown to protect against CIA induced by cytosine arabinoside but not by cyclophosphamide in neonatal rats (Jimenez and Yunis, 1992). In contrast, FGF-7 and KGF partially protect against CIA by cytosine arabinoside by retarding hair loss (Danilenko et al., 2000). In organ-culture human scalp hair follicles and HaCaT keratinocytes, KGF protects against the cytotoxicity of mafosfamide, the cell culture active derivative of cyclophosphamide (Braun et al., 2006). The mechanism of action of KGF has been proposed to involve specific signaling pathways including PI3K and ERK1/2.

PTH antagonists reduce cell apoptosis in the hair bulb matrix and delay the onset of CIA in adult mice, whereas PTH agonists enhance the apoptosis and accelerate hair regrowth after CIA. However, neither PTH agonists nor antagonists prevent CIA (Peters et al., 2001).

#### **5.2.5 Antioxidants**

Broad spectrum antioxidant *N*-acetyl cysteine (NAC), an analog and precursor of glutathione, when administered topically or parenterally, protects against CIA induced by cyclophosphamide in neonatal rats. In contrast, NAC could not protect adult mice from CIA induced by doxorubicin (Wang et al., 2006).


#### **5.2.6 Cell cycle or proliferation modifiers**

Rapid proliferation of keratinocytes during the anagen phase of hair follicle is one the main predisposing factors of CIA. Thus, one approach to protect against CIA is to arrest the cell cycle and inhibit cell proliferation.

#### **5.2.6.1 Calcitriol**

62 Topics in Cancer Survivorship

randomized clinical trial, topical minoxidil (2%) was shown to shorten the duration of CIA in breast cancer patients receiving 5-fluorouracil, doxorubicin, and cyclophosphamide

Hair follicle cells express receptors for multiple cytokines and growth factors that regulate hair growth cycle (Trueb, 2002). These regulators include fibroblast growth factors (FGF), transforming growth factors (TGF), insulin-like growth factors (IGF), epidermal growth factors (EGF), interferon and interleukins (Stenn and Paus, 2001). Moreover, hair cycle is

IL-1 and ImuVert, a biological response modifier derived from *S. Marcescens*, were reported to protect against CIA induced by cytosine arabinoside and doxorubicin in neonatal rats (Hussesin, 1993). Both agents can induce the release of multiple cytokines and growth factors. It was suggested that the protection of CIA by ImuVert is mediated through IL-1. Similarly, EGF and FGF-1 have been shown to protect against CIA induced by cytosine arabinoside but not by cyclophosphamide in neonatal rats (Jimenez and Yunis, 1992). In contrast, FGF-7 and KGF partially protect against CIA by cytosine arabinoside by retarding hair loss (Danilenko et al., 2000). In organ-culture human scalp hair follicles and HaCaT keratinocytes, KGF protects against the cytotoxicity of mafosfamide, the cell culture active derivative of cyclophosphamide (Braun et al., 2006). The mechanism of action of KGF has

been proposed to involve specific signaling pathways including PI3K and ERK1/2.

PTH antagonists reduce cell apoptosis in the hair bulb matrix and delay the onset of CIA in adult mice, whereas PTH agonists enhance the apoptosis and accelerate hair regrowth after CIA. However, neither PTH agonists nor antagonists prevent CIA (Peters et al., 2001).

Broad spectrum antioxidant *N*-acetyl cysteine (NAC), an analog and precursor of glutathione, when administered topically or parenterally, protects against CIA induced by cyclophosphamide in neonatal rats. In contrast, NAC could not protect adult mice from CIA


**Mean (days)** 

Minoxidil Placebo

50.3 187.2 136.9 155.3

61.8 148.5 86.7 131.2 *p***-value** 

0.15 0.07 0.03 0.40

(Duvic et al., 1996). The results of this study is summarized in Table 5.

regulated by androgens and parathyroid hormone (PTH) (Sawaya, 2001).

Maximal hair loss to first regrowth (period of baldness)

Baseline to first moderate or dense hair growth

Table 5. Minoxidil shortens duration of CIA.

**5.2.4 Cytokines and growth factors** 

**5.2.5 Antioxidants** 

induced by doxorubicin (Wang et al., 2006).

others reported no protective effect of vitamin E (Batchelor, 2001).

**Interval** 

Baseline to maximal hair loss Baseline to maximal regrowth Multiple effects of calcitriol (1,25-dihydroxyvitamin D3) on keratinocytes, i.e., inhibition of DNA synthesis, Go/G1 cell cycle arrest, and induction of cell differentiation, have been reported (Kobayashi et al., 1998; Wang et al., 2006). Thus, it is likely that calcitriol induces changes in keratinocyte proliferation and/or terminal differentiation, subsequently altering cellular susceptibility to apoptosis. In neonatal rats, topical administration of calcitriol reduces CIA induced by cyclophosphamide, etoposide, and a combination treatment of cyclophosphamide and doxorubicin (Jimenez and Yunis., 1992). In adult mice receiving cyclophosphamide, topical calcitriol however fails to prevent or retard CIA, but somehow reduces massive apoptosis of hair matrix keratinocytes, a key feature of CIA, and enhances the regrowth of normal hair shaft. (Paus et al., 1996; Schilli et al., 1998). In humans, calcitriol has a protective effect against CIA induced by paclitaxel (Jimenez and Yunis, 1996), but not by a combination of 5-fluorouracil, doxorubicin and cyclophosphamide (Hidalgo et al., 1999)

#### **5.2.6.2 CDK2 inhibitor**

Cyclin-dependent kinase 2 (CDK2) is a member of the serine/threonine protein kinase family that plays a key role from late G1 to late G2 of the cell cycle. Potent small inhibitors of CDK2 have been synthesized and tested for their effect on CIA. One of these synthetic inhibitors was shown to inhibit the progression from late G1 into S phase in human diploid fibroblasts and also inhibit apoptosis induced by etoposide, 5-fluorouracil, taxol, cisplatin and doxorubicin. In neonatal rats, topical application of the inhibitor reduces hair loss at the site of application in 50% of the rats having etoposide-induced CIA and in 33% of the rats with CIA induced by cyclophosphamide and doxorubicin (Davis et al., 2001). Histological examinations of the skin from etoposide-treated rats show that the inhibitor increases the number of viable hair follicles and dermal papilla, reduces the level of inflammation and amount of damage to epithelium, reduces the thickening of epidermis and decreases the number of apoptotic cells in the hair follicle matrix. However, in subsequent studies the authors reported that they were unable to reproduce the results in the neonatal rat model (Davis et al., 2002), thus the use of this inhibitor in CIA becomes questionable, although the idea of using CDK2 inhibitors is still ongoing.
