Efficacy and Safety of Clinacanthus nutans Lindau Cream vs. Podophyllin for the Treatment of Adults with Condyloma Acuminata

Human papillomavirus (HPV) infection causes condyloma acuminata (CA). Podophyllin is the standard treatment. Clinacanthus nutans Lindau (C. nutans), a medicinal plant, has potent anti-inflammatory and antiviral effects. C. nutans cream is widely used in Thailand to treat the herpes simplex virus. We proposed that C. nutans might also induce CA clearance. There are no studies of C. nutans treatment of CA. This randomized controlled trial at Siriraj Hospital, Thailand, was conducted between January 2018 and December 2019. CA samples were obtained from 10 men with at least two CAs 1 centimeter apart. Each wart was randomized to a 4-week treatment with either C. nutans or podophyllin. The participants were 24 to 72 years old. Most HPV types were low-risk HPVs (HPV 11, HPV 6). Median CA clearance with podophyllin was a 97% CA clearance with podophyllin and 82% with C. nutans. C. nutans may be an alternative treatment for CA.


Introduction
Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide [1]. More than 200 types of HPV have been identified [2]. ey can be subdivided according to their oncogenic potential into high-risk (HR) or low-risk (LR) types. e immune system clears most HPV infections within 2 years of onset, and persistent HPV infection is essential for tumorigenesis, transformation, and progression [3]. HR types are strongly associated with malignant diseases such as cervical, anogenital, and oropharyngeal cancers [4], while LR types, such as HPV 6 and 11, are detected more frequently in condyloma acuminata (CA) [5]. HPV induces hyperplasia and hyperkeratosis, which are present clinically as warts [6].
CA has a significant impact on the quality of life [7]. Its treatment is usually lengthy and painful. Most current treatment options work by destroying affected tissues. e options consist of cytotoxic therapies (trichloroacetic acid, podophyllin, and 5-fluorouracil) or physically ablative therapies (cryotherapy, carbon dioxide laser, electrodesiccation, and surgical excision) [8,9]. e location and number of lesions and the cost of treatment are considered when determining the extent of the therapy required for a patient.
Podophyllin is a plant-derived compound that causes tissue necrosis by arresting cell division and mitosis. Podophyllin may be applied directly to CA once a week, and it should be washed off 4 hours after treatment. e purified form is preferred as it has a good safety profile [10].
Clinacanthus nutans Lindau (C. nutans) is another herb belonging to the Acanthaceae family and is used as a wellknown herbal medicine. In Malaysia, Indonesia, ailand, and China, the plant is used as an alternative medicine for insect bites, skin rashes, herpes simplex virus (HSV) and varicella-zoster virus infection, diabetes, and gout [11,12]. Because several experiments have reported antiviral activities, C. nutans is also recommended as an antiviral agent against HSV and VZV [13][14][15]. C. nutans has biological properties, such as antioxidant activity, antiviral activity, anti-inflammatory activity, and immunomodulatory effects [16]. C. nutans is safe as in vivo study on the toxicity of C. nutans did not show any toxicity [12].
In ailand, C. nutans cream has been approved by the ai Ministry of Public Health and is available in the market. To our knowledge, there is no study on the effectiveness of C. nutans cream for CA treatment. Our objective was to compare the efficacy of C. nutans and podophyllin for the treatment of HPV-infected CA by determining the CA size reduction in ai males.

Study Population.
is randomized controlled trial enrolled ai men between January 2018 and December 2020. e inclusion criteria were as follows: (1) ai men attending the Sexually Transmitted Disease Clinic at Siriraj Hospital; (2) 18 years or older; (3) willing to participate and give their written consent; (4) a diagnosis of anogenital warts by clinical manifestation; and (5) the presence of at least two CAs that were 1 centimeter apart. e exclusion criteria were as follows: patients (1) with HIV, (2) taking immunosuppressive drugs, or (3) with an autoimmune disease.

Product
(1) Podophyllin was produced by Vidhyasom Co Ltd, Bangkok, ailand. e concentration was 25% podophyllin which constitutes of podophyllum resin 25 g in compound tincture of benzoin 100 ml.
(2) C.nutans cream was produced by the Government Pharmaceutical Organization (GPO), Bangkok, ailand, and the ai Ministry of public health has approved C. nutans cream on the National list of essential medicine. e constitution was C. nutans extract powder 4.343 g in cream 100 g.

Procedure.
Before starting its trial, its protocol was approved by the Siriraj Institutional Review Board of the Faculty of Medicine Siriraj Hospital (approval number 550/ 2559 [ECI]). Written informed consent was taken from the eligible patients, and questions were asked and clinically evaluated. eir medical history was taken, and a physical examination was performed. e lesion sites were the shaft of the penis, glans of the penis, neck of the penis, and glans of the penis. e participants were randomly assigned by a block of four. Specimens swabbed from lesions in the genital area were collected and kept in cryopreserved tubes at −80°C.
HPV genotyping was performed by the linear array HPV genotyping test (Roche Diagnostics, Switzerland), based on a reverse hybridization of amplicons to immobilize membrane-bound probe. HPV DNA in the swabbing sample was detected by multiplex PCR targeted to amplify the conserved L1 region of the viral genome. e system involves coamplification of the β-globin gene as an internal control. e PCR, performed in a 100 μL reaction volume, was composed of a 50 μL linear array master mix and a 50 μL of DNA, as per standard protocol. After amplification, the biotinylated-PCR products were denatured immediately by adding 100 μL NaOH to each PCR tube. Hybridization of the denatured amplicons and genotyping were performed on the GT-Blot 48, as per the manufacturer's instruction. Only samples that give a positive signal for β-globin were considered for analysis. Using the linear array HPV reference guide, the strips were manually interpreted using the linear array HPV reference guide, by reading the individual types down the length of the strip. e 37 HPV types that were analysed for comprised 13 HR-HPV types (16, 18,   e mean, standard deviation, mode, median, maximum, and minimum were analyzed and reported as percentages. Pretreatment and posttreatment values for podophyllin and C. nutans were compared using nonparametric Wilcoxon signed-rank tests and paired t-tests. Statistical significance was accepted for P values less than 0.05.

Results.
e ages of the 10 male participants ranged from 24 to 72 years, with a mean (SD) of 38.7 (16.2) years. Table 1 details the demographic data of the participants. Half were single, 40% were married, and 10% were divorced. Approximately 40% of the participants reported having a vocational education. Sixty percent of the participants were business owners. Only 20% of the participants had an underlying disease (allergic rhinitis in all cases). All participants had had multiple partners in their lifetime, had their last sex during the previous 6 months, and sometimes used condoms. Regarding sexual orientation, 90% reported being heterosexual, and 10% reported being bisexual. Twenty percent of the participants had a history of other sexually transmitted diseases.
All participants (100%) were HPV positive. ere were both low-and high-risk HPV types. Mostly, the HPV types were HPV 11, followed by HPV 6. Approximately 50% of the patients were coinfected with multiple types of HPV, and 40% had multiple genital warts (Table 2). ere was no specific clinical difference between the HR-HPV group and the LR-HPV group.
At the end of the treatment course, 9 patients showed more than a 75% reduction in CA lesion size, compared to baseline, with podophyllin treatment. ree out of 10 patients (ID 01, 05, and 10) achieved a 100% reduction with podophyllin treatment. Regarding C.nutans treatment, 6 of the 10 patients showed a more than 75% reduction, but only 1 patient completed the treatment with a 100% reduction ( Table 3 and Figure 1). e median total clearance for the podophyllin treatment was 97% CA reduction due to the CA clearance. However, the median total clearance for the C. nutans treatment was an 82.5% CA reduction (Table 3). ere was a statistically significant difference for podophyllin treatment over C. nutans treatment (P < 0.001).

Discussion
CA is a benign disease caused by LR HPV. Our study showed that the most common types of HPV in CA were HPV 6 and 11.
is result is consistent with the previous studies in ailand, the United States, Brazil, Mexico, and China [17][18][19][20]. We also found that coinfections with multiple types of HPV were frequent, which is in agreement with prior studies [21].
To our knowledge, this was the first controlled clinical trial to compare the efficacy of podophyllin and C. nutans in clearing HPV-infected CA. We evaluated the efficacy of the 2 treatments using 10 patients.
Treatments for CA include topical therapies applied by the patient or the clinician and cytotoxic or physically ablative therapy approaches. Recurrence rates have been reported to range from 25% to 67% [22]. One-third of patients have been reported to clear spontaneously [23]. In ailand, patient-applied treatment is imiquimod, and clinician-applied methods are podophyllin, trichloroacetic, cryotherapy, carbon dioxide laser, electrosurgery, and surgical excision. Currently, evidence for more effective alternative treatments is limited. Another consideration is that the costs of the various treatments available differ markedly. According to an evidence-based review, the first-line destructive treatment is cryotherapy, but the more expensive surgery and electrodesiccation options are more effective than cryotherapy. Similarly, first-line topical treatments are currently Evidence-Based Complementary and Alternative Medicine 3 podophyllin and imiquimod [22]. However, since imiquimod is expensive in ailand, we prefer to use podophyllin. Podophyllin is derived from the roots of the mayapple plant (Podophyllum peltatum). Podophyllin is a cytotoxic agent that binds to the tubulin subunit of spindle microtubules and arrests mitosis in metaphase, eventually disrupting viral activity [24]. e use of podophyllin is an effective, safe, and noninvasive method for CA treatment. In randomized controlled trials, podophyllin yielded moderate clearance rates of 41% to 77% and surprisingly high recurrence rates between 25% and 70% [25]. e side effects of podophyllin include local skin reactions and the potential for  systemic absorption, including neurological toxicity, bone marrow suppression, teratogenicity, mutagenicity, and death [26]. Pregnancy is an absolute contraindication to treatment. Due to its adverse effect profiles, podophyllin should be treated with provider-administered treatment. However, podophyllin is still used for the treatment of CA due to its easy availability [27]. nutans is a medicinal plant known as "Praya-yo" in ailand. It has been reported to have anti-inflammatory, antiviral, antimicrobial, and antivenom activities [28]. In ailand, it is a well-known treatment for the herpes simplex virus, and the cost of treatment is reasonable. Limited evidence from a systematic review and meta-analysis of randomized clinical trials showed some beneficial effects of C. nutans preparations for treating herpes genitalis [29]. In ailand, the GPO of the Ministry of Public Health produces 4% C. nutans cream for patient-applied treatment. e medication is applied 4 times daily.
is study evaluated the effects of treating HPV-infected CA with podophyllin (the gold standard; applied weekly by doctors) and 4% C. nutans cream (applied 4 times daily by patients). e clearance rate of the podophyllin treatment was noticeably higher than that reported by an earlier study [30]. As to the C. nutans treatment group, the clearance rate was lower than that of the podophyllin group for all patients.
However, the efficacy of the C. nutans treatment was lower than that of the podophyllin treatment. We suggest combining both to treat CA, for example, having patients apply C. nutans cream at home every day and visit their doctor weekly for podophyllin treatment.
Some limitations were in our study. First, as there was a small sample size, a larger cohort is needed to validate the optimal dosage of C. nutans for the treatment of CA. Furthermore, the recommended application of C. nutans cream 4 times a day for an HPV infection may not be convenient or practical for some patients.

Conclusions
Our study found that C. nutans may be beneficial for the treatment of CA. C. nutans may be used as an adjuvant treatment for CA.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.