Efficacy and safety of a modified combination regimen of phenothrin and ivermectin lotion in patients with head lice in Tsukuba, Japan

The combination therapy of phenothrin (PHT) and ivermectin (PI regimen) was effective for pyrethroid‐resistant head lice in Okinawa; however, further investigations have been required to improve the regimen. This study aimed to investigate the efficacy of the modified PI (mPI) regimen, which increased the maximum number of PHT treatments in the PI regimen by one, in Tsukuba, which has a low mutation rate compared to Okinawa.


| INTRODUC TI ON
Head lice are transmitted by head-to-head contact and are more common among young children 1 ; 90% of patients with head lice are children aged 0-11 years 2 . The head lice become larvae after the eggs hatch in about 7-12 days, and after 3 molts, they become adults in about 2 weeks 3 . Females live up to 30 days and are expected to lay about 100-200 eggs during their lifetime 4 . Recently, the spread of pyrethroid-resistant head lice has increased in Western countries [4][5][6][7][8][9][10] and is also becoming a problem in Okinawa, Japan. Head lice are treated with many types of topical drugs, including the pyrethroid series compound permethrin, ivermectin, spinosad, and dimethicone 11 . Currently, the only treatment for head lice in Japan is pyrethroid series 0.4% phenothrin, which is an over-the-counter (OTC) drug.
Pyrethroid resistance has been associated with amino acid substitutions M815I, T917I, and L920F in the knockdown (kdr) gene encoding the α-subunit of voltage-sensitive sodium channel (VSSC) 12 .
In a previous study, the efficacy and safety of a combination regimen comprising 5% phenothrin lotion (Sumithrin Lotion 5%, Kracie Inc; PHT) 15 and 0.5% ivermectin lotion (Sklice lotion 0.5%, Arbor Pharmaceuticals Inc; IVM) 16 , designated as a PI regimen, was investigated in head lice patients in Okinawa, Japan. The PHT formulation, which has a 12.5-fold higher concentration of phenothrin than the OTC drug, was approved in 2014 as a scabies treatment for medical use in Japan, and the IVM formulation was approved in 2011 for pyrethroid-resistant head lice in the United states. In the Okinawa study, all head lice had kdr mutation and PHT were effective against some pyrethroid-resistant head lice, and IVM was significantly effective against PHT-ineffective head lice 17 . Tsukuba City, where we conducted this study, is located on the mainland of Japan and is located 1611 km north of Okinawa, the southernmost island in Japan. Thus, all of the head lice isolated in the Okinawa study had the kdr mutation 17 , and the efficacy of the regimen in the presence of a mixture of lice with and without kdr mutation has not yet been confirmed.
The aim of the present study was to investigate the efficacy of the modified PI (mPI) regimen, which increased the maximum number of PHT treatments in the PI regimen by one, in Tsukuba with a lower mutation rate compared to Okinawa 9 and the correlation of kdr mutations at VSSC loci and GluCl-encoding loci with drug resistance.

| Medicines
PHT was purchased from Toho Holdings Co., Ltd. IVM was purchased from The Coghlan Group Inc. The import of IVM was approved by Kanto-Shinetsu Regional Bureau of Health and Welfare.

| Declarations
The protocol for this research project has been approved by a suitably constituted Ethics Committee of the institutions, and it conforms to the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines. The name of the committee is the Ethics Review Committee for Medical Research, Tokyo University of Science, and the approval number is 16037. Individual written informed consent was obtained from all patients and/or guardians.

| Study design
The study was an open-label, uncontrolled, exploratory study and conducted at the Department of Dermatology, University of Tsukuba Hospital (Ibaraki, Japan), in cooperation with the local medical associations and pharmacist associations. The target cases were 12 cases, and the eligibility criteria were patients diagnosed with head lice infestation aged 6 months or older in which 2 or more lice bodies and 5 eggs were observed. Exclusion criteria were as follows: (a) any history of head lice treatment in the prior 4 weeks; (b) a history of allergy to any of the components of ivermectin or phenothrin; or (c) if estimated as ineligible by the investigators. Eligible patients/parents agreed not to use any other treatment for head lice, comb out nits, or cut or chemically treat hair during the study. Patients older than 20 years received a clear explanation of the aims of the study. If the patients were younger than 20 years, the investigator explained the regimen to patients in an easy-to-understand manner.
Patient IDs were assigned in the order of meeting the eligibility criteria. Patients received sufficient explanation about PHT, focusing on the following: (a) It is approved as a scabies medicine; (b) the ingredient is the same as found in 0.4% Sumithrin shampoo, 18 with the concentration being about 12 times higher; and (c) the application is limited to the head unlike whole-body application for scabies.
Similarly, patients received adequate explanation about IVM, focusing on the following: (a) The formulation is not yet approved in Japan but is an approved and prescribed head lice therapeutic in the United States and (b) safety and efficacy are both unknown due to its first use in Japanese subjects. To assess any influence on efficacy, investigators confirmed the use of concomitant medications and the patient's hair type. Hair length was recorded as "short" when above the shoulder, "long" when below the shoulder, and "very long" when below the waist. Hair texture was considered "dry" when the moisture content was low, "oily" when greasy, and "normal" if neither.
Hair shape was confirmed as "straight" or "curly".
The scheme of the mPI regimen is illustrated in Figure 1. The determination of the second medicine received (PHT or IVM) was assessed in the same way as in the Okinawa study 17 . In brief, the medicine to be applied on the second visit (day 8) was determined after collecting head lice 20 times with a comb. If the body of head lice was not found, PHT was used two more times (PHT × 3 group), and if more than one body was found, PHT was switched to IVM 6 | SATO eT Al. and used once (PHT-IVM group). The investigator followed up each treatment and collected head lice bodies and eggs at the fifth visit on day 29. If lice bodies were found on day 29, IVM was applied once, even in the PHT × 3 group, to confirm the disappearance of lice bodies after one week.
On day 1 (before the first treatment), patients' blood was collected in both groups, on day 8 (before the IVM treatment) and on day 15 in the PHT-IVM group, and on day 22 in the PHT × 3 group, for biochemical blood tests (optional). The investigators submitted data to the clinical study electronic data capture software (OpenClinica, LLC) customized for the study. on a whiteboard (297 × 420 mm), pasted with an adhesive tape in the same way described for the Okinawa study 17,19 . The presence or absence of head lice bodies on the board determined the efficacy of the treatment. If a head lice body was not observed, the investigator reported the treatment as effective. If the body was found, the investigator reported the treatment as ineffective. After the final visit, the whiteboards were sent to the study's data managers at the Tokyo University of Science, who confirmed the number of bodies and counted eggs of head lice. All eggs were examined microscopically (×200) to see whether they were before hatching. Then, the nonempty eggs were counted as "eggs". The visual analogue scale (0: do not feel it, 5: feel it the strongest) was used to assess the itch grade for evaluating itching.

| Evaluation of efficacy and safety
Adverse events involving the skin and scalp were evaluated on days 1 (baseline), 8,15,22, and 29, and biochemical blood tests were performed to assess aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels on days 1 (baseline for both groups), 8 (PHT-IVM group), 15 (PHT-IVM group), and 22 (PHT × 3 group). In the Okinawa study, there was no problem in the blood test of all. Therefore, the blood test was not mandatory in this study.
Investigators assessed the adverse events (skin, scalp, and ocular irritation), reported by the patients or observed by the investigators, and evaluated the correlation between the adverse events and PHT or IVM. The assessed adverse events were scored using Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The study was discontinued if an adverse event was scored 3 or higher.

| End points
The primary end point was the rate of patients free from lice bodies on day 29. The secondary end point was the difference in the number of lice bodies and eggs collected and the itch grade on days 15, 22, and 29 compared with those at baseline and the reduction rate of bodies and eggs.

| Genotyping of head lice
To determine the presence or absence of kdr mutation in VSSC in each head louse, DNA sequencing analysis was performed using the genomic DNA extracted from the head lice bodies or eggs collected on days 1, 8, 15, 22, and 29 as described previously 17 . We analyzed a total of 9 loci for amino acid substitutions, including 3 loci (M815I, P917I, and L920F) investigated in the Okinawa study 17 and an ad-  The mutations at T236A, A251V, or H272R in GluCl were analyzed by PCR of the genomic DNA using the primers 7203F and 7800R (Table 1) with the same condition as described above for the kdr mutation analysis and then mixed with the primer 7750R (Table 1) for the sequencing analysis. The primers for the GluCl mutation analysis were designed based on a previous report 14 .

| Statistical analysis
Steel's multiple comparison Wilcoxon test was performed to investigate the significance of the changes in the number of bodies and eggs and the itching grade on days 15, 22, and 29 from baseline (before day 1 of administration).  Table 2. On day 8, 1 week after the first PHT treatment, PHT was effective in 7 of the 8 patients ( ID 3,4,5,7,8,9,and 10) and was therefore continued for these patients (PHT × 3 group). For the remaining patient in which PHT was ineffective (ID 6), the treatment was switched to IVM (PHT-IVM group). Table 3

| Safety
In ID 3, eczema was observed on both of the lower limbs 3 days before the fifth visit (day 29). Since the site was not close to the head,

| Genotyping of head lice
The genotype on kdr mutations in VSSC was analyzed for 123 samples;  who were sensitive to the first PHT application, but was higher at 57.9% (11/19) in samples from the patient (ID 6) for whom the first PHT was ineffective. Therefore, the mutation rate more than doubled in head lice collected from the patient for whom PHT was ineffective.
In addition to the previous Okinawa study 17 (Table S1).

| D ISCUSS I ON
In this study, the efficacy and safety of the mPI regimen were evaluated, which comprises PHT and IVM. Since the PHT formulation provided in this study contains a 12.5-fold higher concentration than the OTC PHT formulation for head lice, it could have potential efficacy against pyrethroid-resistant head lice. No lice bodies were found in any of the patients at day 29 after treatment with the mPI regimen ( could also be due to the use of goggles in this study 19 . The efficacy of these treatments was evaluated by the presence or absence of bodies of lice as in the IVM clinical trial 19 , but it is also necessary to consider the presence of eggs. The head lice bodies were disappeared in all patients during the observation period, although eggs were still present in 75% of patients on day 29 (Table 3). In this study, we confirmed that all eggs were prehatched, but it is impossible to determine whether the eggs were actually alive. In ID 9 and 10 who were family members and were being treated at the same time, increases in egg count of over 50 were observed at day 22. The increase may have been due to reinfection in public spaces such as school, but this was not confirmed. More observational data are needed to conclude whether eggs should be observed until disappearance as a measure of continuity of infection. Head lice treatment requires predicting the effective treatment for each patient, using pyrethroid and the alternative IVM properly.
Head lice genotyping is therefore an ingenious method to predict the individual response to PHT. However, the phenotype (response to PHT) does not always match the genotype (kdr mutation), and gene sequencing is time-consuming. In the PI and mPI regimens, the first PHT treatment is used to investigate the response to PHT. This in vivo phenotyping could be one of the best methods to choose an effective treatment for head lice.
In addition, drug cost is an important factor in drug selection.
The cost of PHT is 4436 yen/average single dose (64.1 g), and the TA B L E 4 Number of kdr-type mutated head lice and eggs, and mutation rate

57.9%
Note: Each number shows the ratio of mutated sample to its analyzed sample.  10,21,26,27 . The kdr mutation has a geographic origin that explains the regional variance in the incidence of pyrethroid resistance 28 . In 2009, the frequency of resistance was 96% in Okinawa and was 5% in the mainland areas of Japan 5,9 . The ratio of lice with M815I-L920F mutations was 100% in the Okinawa study 17 , whereas it was 19.6% in the present Tsukuba study. Furthermore, since the resistance rate has increased in both Okinawa and the mainland, the spread of pyrethroid-resistant mutants throughout Japan is concerning.
The genotyping in the Okinawa study 17  In the present study, we additionally analyzed the GluCl mutation, which may be associated with IVM head lice resistance, and found no T236A, A251V, or H272R mutant at three out of five sites reported in the Senegal study 14 . IVM-resistant head lice are not currently reported in Japan; however, the potential of transmission and expansion may require attention.
Overall, our results demonstrate that the mPI regimen is a potential effective treatment for economically combating both PHT-sensitive and resistant head lice. This study revealed four new haplotypes in kdr loci that may contribute to future studies for the development of effective therapeutics against head lice.

ACK N OWLED G M ENTS
We would like to express our sincere gratitude to all the staff of the