**5.1 Physical prevention**

#### **5.1.1 Scalp torniques**

58 Topics in Cancer Survivorship

Although animal models have provided valuable information regarding the pathogenesis of CIA, the animal physiological and pathological conditions do not necessarily mimic human conditions. The first human organ-culture hair follicle model for CIA was developed in 2007 (Bodo et al., 2007). In this model, mature anagen (anagen VI) human hair follicles are microdissected intact from the occipital scalp of healthy adults. The isolated hair follicles are maintained in William's E medium containing L-glutamine, hydrocortisone, and insulin. 4- Hydroperoxycyclophosphamide (4-HC), a key cyclophosphamide metabolite, was used to verify key *in vivo* characteristics of CIA since clinical and animal data of cyclophosphamideinduced CIA are well established. 4-HC was shown to induce melanin clumping and incontinence, reduce keratinocyte proliferation, induce keratinocyte and dermal papilla cell apoptosis, and induce premature catagen, resembling *in vivo* hair follicle dystrophy. Comparison of the test results in adult mouse model and organ culture model in response to cyclophosphamide is shown in Table 2. The human organ-culture hair follicle system could be used to study the pathogenesis and potential treatment of CIA, i.e., to identify key

**Key clinical parameters Adult mouse model Organ culture model** 

Keratinocytes, dermal papilla cells, and melanocytes constitute the major cell types in the hair follicle. In CIA, massive apoptosis of keratinocytes occurs; thereby cultured keratinocytes are commonly used as a predictive model for chemotherapy-induced hair follicle damage. Primary and immortalized normal human keratinocytes (HaCaT) have been used to test the effects of chemotherapeutic agents and protectants (Matsumoto et al., 1995; Braun et al., 2006; Janssen et al., 2008). However, since different hair follicle cells interact and affect each other cell growth and cycling (Rogers and Hynd, 2001), the effects of chemotherapeutic agents on other cell types, e.g., dermal papilla cells and melanocytes, should also be evaluated, i.e., through the use of a co-culture system. Furthermore, current cell culture models lack biological measures of hair growth and cycling; however, they offer

the advantages of gene manipulation, ease of use, high throughput, and low cost.

broadly classified as physical and pharmacological, as described below.

Several approaches have been investigated to overcome CIA. These approaches can be

Not directly Yes

Yes Yes

Yes

Yes Yes

Not assessed

Yes Yes

**4.2 Culture models 4.2.1 Organ culture model** 

molecular targets and inhibitors of CIA.

Inhibition of hair shaft elongation Increased apoptosis of matrix

Decreased proliferation of matrix

Disrupted melanogenesis and

**5. Approaches to prevent CIA** 

Table 2. Comparison of adult mouse model and organ culture model.

keratinocytes

keratinocytes Catagen induction

melanin transfer

**4.2.2 Cell culture model** 

Scalp torniques are the application of bands around the head to occlude the superficial blood flow to scalp, thus reducing the amount of drugs delivered to the hair follicles. The torniques range from 10 mmHg above systolic pressure to 300 mmHg around the scalp. These torniques are applied 5-10 minutes prior to or at the time of chemotherapy until up to 30 minutes after the drug administration (Cline, 1984). Although reports described mild to moderate prevention of CIA induced by vincristine, cyclophosphamide, and doxorubicin, this technique is no longer recommended due to patient discomfort (Wang et al., 2006).

#### **5.1.2 Scalp cooling**

Scalp cooling or hypothermia is the application of cold to the scalp using a device (cap) that is pre-cooled in a freezer or exchanges coolant with reservoir. A period of cooling lasts from 5 minutes prior to chemotherapy until an hour or more after the drug administration. Many studies have shown that the efficacy of scalp cooling can range from 0-90% (for review, *see* Grevelman and Breed, 2005). A recent study reported that scalp cooling helps reduce major CIA in patients receiving doxorubicin (60 mg/m2), docetaxel (80 mg/m2), or combination of 5-fluorouracil (600 mg/m2), epirubicin (60 mg/m2), and cyclophosphamide (600 mg/m2) for 6 to 9 cycles (Auvinen et al., 2010). The current hypotheses of the protective effect are: (i) cooling reduces blood flow to hair follicles by vasoconstriction, resulting in a decrease in the amount of drugs available for uptake; and (ii) cooling decreases cellular metabolism and drug uptake. Scalp cooling to 20°C was shown to reduce blood flow to 20% of normal flow; however, further decrease in the temperature (<18°C) did not result in further decrease in scalp blood flow (Janssen et al., 2007). Recent *in vitro* studies indicate the significant role of temperature on keratinocyte cell viability upon doxorubicin chemotherapy; however, there is no difference in cell survival between 10°C and 22°C (Janssen et al., 2008). Based on these findings, it appears that there is an optimal temperature for scalp cooling (~20°C), and that increasing the cooling will only result in patient discomfort. Other factors affecting the effectiveness of this method include drug regimen, application and duration of cooling, and the cool conductivity (Betcheler, 2001).

Scalp cooling is practically ineffective if the chemotherapeutic agent is administered as a continuous infusion over a prolonged period. Additionally, scalp cooling increases the risk of scalp metastasis, and is therefore contraindicated in patients with hematological malignancies and cutaneous T-cell lymphoma (Dean et al., 1979; Apisanthanarax and Duvic, 2003).

#### **5.1.3 Heat treatment**

Stress protein response is one of the cellular protective mechanisms against various adverse conditions. Enhanced expression of stress proteins such as Hsp90, Hsp70, and Hsp25 has been observed in response to certain physical and chemical stresses, which has been linked to increased stress tolerance. Previous *in vitro* studies have shown that heat treatment and overexpression of stress response proteins, e.g., Hsp70 and Hsp27, could protect against the cytotoxic effects of anticancer drugs such as doxorubicin, cyclophosphamide, etoposide, and taxol (Kampinga, 1995; Jaattela et al., 1998; Kwak et al., 1998; Ito et al., 1999; Xia et al., 1999), leading to the investigation of the protective effect of stress protein activation on CIA in neonatal rats (Jimenez et al., 2008). In this study, heat was locally applied to the skin with a copper cylinder through which heated water was circulated. Conducting gel (Vaseline) was

Chemotherapy-Induced Alopecia 61

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

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

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.,

> 20.4 37.2

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

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

5.1 16.2

**% of patients in alopecia grade** 

28.2 27.9

No Mild Moderate Severe Total

38.5 13.9 7.7 4.6

androgenetic alopecia (Paus et al., 1989; Taylor et al., 1993; Lutz et al., 1994).

1996). The results of this study are summarized in Table 4.

**of patients** 

proliferation and differentiation of keratinocytes within the hair follicles.

30 28

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

**Treatments No.** 

Carboplatin and etoposide

**5.2.3 Hair growth cycle modifiers** 

**5.2.3.1 Cyclosporine A** 

regrowth. **5.2.3.2 AS101** 

+ AS101

**5.2.3.3 Minoxidil** 

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 against CIA without affecting the anti-tumor efficacy.


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

#### **5.2 Pharmacological prevention**

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 animals but their clinical use will require further investigations.

#### **5.2.1 Tumor targeting delivery**

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

#### **5.2.2 Drug-specific antibodies**

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 antibody could not react with the other drugs in combination.
