Efficacy of Lazolex® Gel in the Treatment of Herpes Simplex Mucocutaneous Infections and the Prevention of Recurrences: A Pilot Study

Background Previous in vitro and in vivo studies indicated that walnut extract has a therapeutic effect on herpes simplex infections. This study aimed to evaluate the efficacy and tolerance of Lazolex® Gel (Iveriapharma, Tbilisi, Georgia), an emollient gel to treat mucocutaneous lesions caused by herpes simplex virus. Methods A single-center, single-arm, open-label, phase II clinical trial was conducted with 30 patients divided into two groups: 15 patients with herpes simplex virus type 1 (HSV-1) infections and 15 with herpes simplex virus type 2 (HSV-2). All received topical treatment with Lazolex® Gel four times a day for 10 days. The efficacy and tolerance of the treatment were evaluated on day 10 and day 20 of the study. Recurrence rates were also evaluated both prior to treatment with Lazolex® and over a 4-year follow-up period subsequent to treatment. Results The median effective time to resolution of symptoms (itching, burning, and pain) was 1.97 days in the HSV-1 group and 3.11 days in the HSV-2 group. The median effective time for vesicles and erosion to disappear was 3.64 days in the HSV-1 group and 3.88 days for the HSV-2 group. Finally, the median effective time for inflammatory signs to disappear was 5.70 and 4.32 days, respectively. Following treatment with Lazolex® Gel, the frequency of outbreaks decreased from a median of 2.00 and 1.00 times per year in the HSV-1 and HSV-2 cohorts to 0.25 and 0.00 (p=0.001 and p=0.003), respectively. Conclusions Topical treatment with Lazolex® Gel applied to lesions four times a day for 10 days was shown to be effective and safe in the treatment of herpes simplex mucocutaneous infections and dramatically reduced the rate of recurrence. Clinical trial was approved by Drug Agency of Ministry of Labour, Health and Social Affairs of Georgia, registration # DA Nº CT-000032, date of approval 01.10.2007.


Introduction
Herpes simplex virus (HSV) belongs to the Herpesviridae family and the Alphaherpesvirinae subfamily [1]. HSV mainly afects the skin and mucosal membranes [2]. Tere are two types of HSV: type 1 (HSV-1), which is transmitted primarily by oral-to-oral contact and commonly causes herpes labialis (also known as orofacial herpes, cold sores, or fever blisters), and type 2 (HSV-2), which is predominantly transmitted sexually (it is considered a Sexually Transmitted Infection) and constitutes the leading cause of genital lesions [2][3][4][5]. In exceptional cases, HSV-1 could be responsible for genital herpes and HSV-2 could cause herpes labialis [6]. HSV is highly prevalent in humans. It is estimated that 70% and 10% of the global population is infected with HSV-1 and HSV-2, respectively. While the proportion of world population expected to have symptomatic HSV-1 and HSV-2 infections is approximately 16% and 5%, respectively [1]. Most commonly, the virus replication is limited to epithelial cells and establishes latency in enervating sensory neurons, where it can remain quiescent for an extensive period of time [2,[7][8][9]. Infection is lifelong, with reactivation of the latent virus causing subsequent outbreaks at the infection site. Tese are usually milder than the primary infection and lesions heal at 10 days if no treatment is undertaken [1,3]. On average, reactivations occur six times a year [1], but frequency and severity decrease over time [2,9]. Te recurrence rate is higher for HSV-2 infection compared to HSV-1 [1,5]. Several stimuli can trigger HSV reactivation, such as ultraviolet radiation, abnormal hormone levels, fatigue, stress and traumatic events, as well as immunosuppression [1,10]. Te development of herpes labialis and genital herpes lesions is sequential and begins with a prodrome showing erythema (usually accompanied by itching) [1,2,11], then papules, vesicles, ulcers or soft crust, hard crust, residual abnormalities (dry faking skin and residual swelling and erythema), and fnally reepithelization and healing (normal skin) [11]. Although HSV infections are usually benign [7], this virus can also cause severe diseases such as encephalitis, lymphocytic meningitis, blindness, and systemic disease in neonates and immunocompromised patients [7,12]. While the diagnosis of HSV is often based on clinical presentation [5], laboratory confrmation is strongly recommended [2,13].
Once infected with HSV, there is no cure. Treatment can, however, attenuate the clinical course, suppress recurrences, as well as reducing viral shedding and complications [2,9]. In general, the prevention and treatment of HSV are managed with systemic or topical drugs [2]. At present, there are many antiviral drugs to treat HSV infections. Te mainstay of antiviral therapy over the past 40 years has been nucleoside analogs such as acyclovir [1,10,14]. And while these drugs have provided substantial improvement in the treatment of HSV infections, the intensive use of nucleoside analogs has led to the emergence of drug-resistant strains, with their long-term toxicity posing a threat [7,8,15,16]. Consequently, the development of new antiviral agents has gained much interest in recent years [10,15,17,18]. Several natural products have shown antiviral efects against HSV, such as extracts, fractionated compounds, and isolated molecules obtained from plants, among others [1,11,15,18]. Furthermore, many plant-derived extracts have been reported to inhibit HSV replication [1,17]. Natural products with potential anti-HSV activity have the advantage of producing less resistance, side efects, and toxicity [10,15].
Intensive research has been carried out in Georgia to develop new natural therapeutical products produced from endemic plants. Based on traditional medicine recipes, the latest biotechnological approaches have been applied to develop Lazolex ® Gel (Iveriapharma, Tbilisi, Georgia) [19,20]. Lazolex ® Gel is an emollient gel for dermatological use composed of three diferent silicones and walnut extract. Tis study aimed to evaluate the efcacy and tolerance of Lazolex ® Gel to treat mucocutaneous lesions caused by HSV. Participants were enrolled in the study based on the following criteria: (1) men or women aged 18-65, (2) diagnosis of acute or chronic herpes simplex mucocutaneous infection, (3) mild course of disease (defned as body temperature <37.2°C and without signs of general infrmity), (4) application of last treatment for herpes simplex infection >3 months, (5) available to cooperate during the study, and (6) provision of written informed consent. Exclusion criteria included the following: (1) abnormal laboratory results, (2) hypersensitivity to the product or its components, (3) pregnancy or breastfeeding, (4) acute/ chronic renal or liver failure, (5) history of migraine, (6) organic brain lesion, (7) generalized anxiety disorder, (8) blood supply disturbance in the vertebrobasilar pool, (9) stage 3 essential hypertension, (10) concomitant acute or decompensated disease that could afect the study results, (11) intake of acyclovir, antibiotics, immunosuppressants, antimetabolites, or glucocorticosteroids during 3-month period prior to the study, and (12) concomitant participation in another clinical trial.

Materials and Methods
Criteria for withdrawal of patients from the clinical trial were the following: (1) individual intolerance, (2) severe adverse efects, (3) general worsening condition, (4) need for the prescription of other treatments during the treatment period of the clinical trial, and (5) patient refusal to continue participation.
Te patients were divided into two groups according to the infectious agent: HSV-1 and HSV-2.

Intervention.
Regardless of their diagnosis, all participants were assigned to receive topical treatment with Lazolex ® Gel.
Patients were given the gel at the end of the screening visit (day 0) by the attending physicians. Te gel was applied to the lesion four times a day over a 10-day period, with the frst application at 9 am and the fourth at 9 pm. Patients administered Lazolex ® Gel to the afected areas themselves.
During the study, participants continued taking their usual medicines, and this information was duly recorded. Administration of the following medications was not allowed during the treatment period of the study: topical treatments, virucides, antimycotic agents, antibiotics, antimetabolites, glucocorticosteroids, and immunosuppressants.

Visit Schedule and Data Collection.
Te clinical trial included six visits: the screening visit (day 0), three visits during the treatment period (days 1, 5, and 10), and two visits during the follow-up period (day 20 and approximately four years after fnishing the treatment).
Te data evaluated by physicians during the trial are shown in Table 1. Te symptoms monitored included itching, pain, and burning at the infection site, which were classifed as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe). Physical examination included body temperature, heart rate, blood pressure, cardiac and pulmonary auscultation, and examination of regional lymph nodes. Te lesion is also reported in terms of size (afected area in cm 2 ) and evidence of the following: erythema, edema, vesicles, erosion, and crusts. Laboratory tests included blood tests (blood count, biochemistry test including AST, ALT, bilirubin, and ammonia, as well as enzyme immunoassays for the detection of immunoglobulins M and G against HSV-1 and HSV-2) and urine tests. All data were recorded on individual patient registration forms.
Te last visit consisted of a phone call to determine the frequency of outbreaks during the 4-year follow-up period and to fnd out the degree of satisfaction regarding the longterm efects of Lazolex ® Gel. Patients received no additional Lazolex ® Gel treatment during these 4 years.
Records and documentation related to the clinical trial were kept in the archives of the JSC Skin and Venereal Diseases Research Institute.

Evaluation of Efcacy and Tolerance Outcomes.
Efcacy was assessed according to the following criteria: (1) course of the disease and (2) reduction in outbreak frequency. Lazolex ® Gel was classifed as efective if herpes lesions improved or healed after 10 days of treatment and as inefective if the lesions did not improve after this period. Te median efective time (ET50), defned as the time (in days) needed to reach complete recovery in 50% of the patients, was used as the main variable for Lazolex ® Gel efcacy. Secondary efcacy outcomes included the degree of patient satisfaction with treatment efcacy (categorized as high, medium, or low).
Tolerance outcomes were evaluated using objective and subjective criteria. Te objective criteria included the comparison of laboratory tests and physical examination before and after Lazolex ® Gel treatment. Te subjective criteria included complaints and symptoms reported by patients. In the event of adverse reactions, they were assessed by the attending physicians. Individual tolerance was categorized into very satisfactory (no clinically signifcant changes in physical examination or laboratory tests; no adverse reactions), satisfactory (insignifcant changes in physical examination or laboratory tests, or mild adverse reactions that do not require a change in treatment), and unsatisfactory (signifcant changes in physical examination or laboratory tests and/or occurrence of adverse reactions that require the withdrawal of the product as well as prescribing treatment to address the adverse reaction).

Statistical Analysis.
A sample size of 15 participants was established for each group according to Julious et al. recommendations for pilot studies [21].
Statistical analyses were performed using BioStat 2008 (AnalystSoft Inc., Alexandria, VA, USA). Te analysis was per-protocol. ET50 was calculated both for subjective criteria (symptoms) and objective criteria (herpes lesions). In the case of the objective criteria, two ET50 were calculated separately regarding the stage of the lesions. Tus, an ET50 was estimated for erythema and edema clearance (considered as infammatory signs), while another ET50 was calculated for vesicles and erosion disappearance. ET50 was obtained using linear regression analysis. Recurrence rates before and after treatment with Lazolex ® were compared using Wilcoxon signed-rank test. Statistical signifcance was set at p < 0.05. For the assessment of laboratory results before and after treatment, means and standard deviations were calculated and descriptive statistics used.

Patient Characteristics.
A total of 30 subjects that were screened for eligibility met the inclusion criteria. According to the serological test results, 15 were infected with HSV-1 and 15 with HSV-2. All participants in the HSV-1 group were diagnosed with herpes labialis and all of the HSV-2 group had genital herpes. No patient withdrew from the study and all participants completed the follow-up. Te baseline characteristics of the 30 participants are shown in Table 2. One participant in the HSV-2 cohort was taking Tirozol ® (Tiamazole) as their usual treatment for hyperthyroidism.
Among the participants with genital herpes, eight had lesions on the mucosa (seven on the glans and a woman on the labia minora) and seven had lesions on the skin (fve on the prepuce, one on the labia majora, and one in the anal region). Table 3, 14 patients (93%) with HSV-1 herpes labialis had itching and burning at the infection site before treatment. Among these, 10 (64%) reported that both itching and burning disappeared after 1 day of treatment. Similarly, 11 patients (73%) had pain before starting Lazolex ® Gel treatment and, of these, 10 patients (91%) reported complete resolution of pain after 1 day of treatment. Nearly all patients experienced symptom relief a few minutes after applying the gel to the lesion. On day 5, 13 patients (87%) reported no symptoms at all. Te ET50 to symptoms resolution was 1.97 days (95% confdence interval [CI]: 0.11-3.83). While all patients with HSV-2 genital herpes had itching, 14 (93%) also had burning, and 12 (80%) had pain at the infection site before treatment. On day 5, 14 patients (93%) reported no symptoms at all. Te ET50 (95% CI) to symptoms resolution was 3.11 days (1.22-4.99). Figure 1 shows the evolution of lesion stages during the study treatment period for the HSV-1 group (herpes labialis). On day 5, two patients out of 15 (13%) were at the beginning of the crust stage, in eight patients (53%), the crust was already formed, and in fve (33%) patients, the crust had fallen of. Also on day 5, among the patients who had already formed the crust, one participant showed a new herpes simplex lesion on the chin. Complete healing was observed in 5 patients (33%) on day 5, and in 13 patients (87%) on day 10. Te ET50 (95% CI) for vesicles and erosion to disappear was 3.64 days (2.45-4.83) and the ET50 (95% CI) for infammation signs to disappear was 5.70 days (3.96-7.44). Similarly, Figure 2 shows the evolution of the stages of the lesion during the treatment period for the HSV-2 group (genital herpes). Complete healing was observed in 12 patients (80%) on day 5, and in all patients on day 10. Te ET50 (95% CI) for vesicles and erosion to disappear was 3.88 (1.22-4.99) and the ET50 (95% CI) for infammation signs to disappear was 4.32 (2.13-6.45). To illustrate these results, images showing the progression of the lesions during the treatment period are presented in Figures 3 and 4.   outbreaks as high. Among the participants asked about the symptoms of outbreaks that occurred during the 4-year follow-up, fve patients (63%) from the HSV-1 group (herpes labialis) and four (80%) from the HSV-2 group (genital herpes) reported that the intensity of symptoms was milder when compared to outbreaks prior to Lazolex ® Gel treatment.

Assessment of Tolerance.
Laboratory test results remained within the corresponding reference intervals both before (day 0) and after treatment (day 10). Prior to treatment, two patients had the enlargement of regional lymph nodes (one in the HSV-1 group and the other from the HSV-2 group), whereas after treatment their size was reported as normal.
In the HSV-1 group (herpes labialis) and the HSV-2 group (genital herpes), no side efects or adverse reactions were reported. Product tolerance was classifed as highly satisfactory for the 15 patients in each group.

Discussion
Te present study evaluates the efcacy and tolerance of Lazolex ® Gel to treat herpes simplex mucocutaneous infections. Our fndings show that Lazolex ® Gel applied to lesions four times a day for 10 days is efective and safe in patients with HSV infections, as well as considerably reducing the rate of recurrence during a post-treatment period of 4 years.
As reported by patients, Lazolex ® Gel treatment soothes symptoms associated with herpes labialis and genital herpes, such as itching, burning, and pain. Tus, according to our results, symptoms are expected to resolve in 50% of patients 2 and 3 days after treatment initiation for HSV-1 (herpes labialis) and HSV-2 (genital herpes), respectively. Furthermore, our results show that most patients will be free of symptoms after 5 days of treatment (in our study, 87% and 93% of patients with HSV-1 and HSV-2 mucocutaneous infections, respectively). It is worth mentioning that nearly all participants felt symptom relief a few minutes after applying the gel. Te immediate efect of Lazolex ® Gel could be greatly appreciated by patients since itching, burning, or pain are bothersome symptoms associated with HSV infections and their swift relief is highly welcomed [22]. In our study, the characteristics of lesions also started to improve on the very frst day of treatment. Both vesicles and erosion disappeared in half of the patients on day 4 for both the HSV-1 (herpes labialis) and HSV-2 (genital herpes) groups. Similarly, the resolution of infammatory signs (erythema and edema) took place on day 6 and day 4-5 in half of the patients infected with HSV-1 and HSV-2, respectively. By day 5, most patients treated with Lazolex ® Gel are expected to achieve complete healing (in our study, 33% and 80% of patients with HVS-1 and HSV-2 infections, respectively). Lazolex ® Gel is an emollient gel for dermatological use that contains three diferent silicones and walnut extract.
Silicones have been widely used in topical applications for decades [23]. Tey are considered safe and have countless benefts, such as being easy to spread, long-lasting, resistant to washing-of, biocompatible, producing an antiinfammatory efect, and ofering a flm-forming ability that protects against chemical and microbial invasion [23][24][25]. Furthermore, silicones provide a pleasant, silky, nongreasy, nonstaining texture [25,26]. All these benefts are due to the unique physicochemical properties of silicones: emollience, chemical and thermal stability, low glass transition temperature, low viscosity and near zero surface shear viscosity, low surface tension and moderate interfacial tensions, and high gas permeability [23,25]. While many mechanisms of action have been proposed for silicones, it is universally accepted that they produce occlusion by forming a fexible hydrophobic barrier that decreases transepidermal water loss, and thus facilitates and maintains the hydration of the stratum corneum [23,24,27,28]. Walnut extract, in contrast, has traditionally been used to treat ulcers, burns, and warts [29]. It has a polyphenol content that provides antioxidant, antimicrobial properties [30][31][32]. Lazolez ® Gel acts by forming a flm that protects the mucocutaneous lesions caused by HSV from dryness, irritation, and external agents. Tis barrier maintains a favorable environment for wound healing. By reducing small contaminants inside the lesion (dead cells, cellular debris, virus, and bacterial particles, among others), hydrating, and cleaning the wound, Lazolex ® Gel reduces pain and irritation in addition to expediting healing.
Satisfactory results have been achieved with other topical products, both synthetic and natural, in the treatment of herpes simplex mucocutaneous infections. Boes et al. [33] compared three diferent products to treat herpes labialis in a randomized clinical trial: a flm-forming patch, a set of semiocclusive hydrocolloid patches, and acyclovir 5% cream. Te mean healing times (95% CI) in days were 9.30 (8.75-9.85), 9.67 (9.11-10.22), and 9.80 (9.30-10.30), respectively. In addition to walnut extract, used to manufacture Lazolex ® Gel, several plant extracts have been shown to have antiviral efects. For example, extracts of Aglaia odorata, Moringa oleifera, and Ventilago denticulate have been shown to have antiviral activity against HSV in cell cultures and animal models [1]. Essential oils extracted from plants such as Glechon spathulate, Glechon marifolia, and those from the Labiatae and Verbenaceae families have been described to have antiviral activity against HSV in cell cultures as well [1]. However, clinical trials testing botanical extracts have yet to be carried out [34]. Lemon balm cream was evaluated in 66 individuals with recurrent herpes labialis in a randomized, double-blind, placebo-controlled study. Lesions healed faster with lemon balm cream compared to placebo, and patients had less pain and vesicles [34,35]. Te combination of sage and rhubarb extract was tested in a randomized placebo-controlled study that included 149 patients with herpes labialis. Te patients were treated topically with acyclovir, sage extract, or a combination of sage and rhubarb extract. Tis combination was more  efective than sage alone, and as efective as acyclovir. Te mean healing times were 7.6 for sage cream, 6.7 days for rhubarb-sage cream, and 6.5 days for acyclovir cream [36]. A parallel controlled open-label randomized clinical study that included 756 adults compared kanuka honey (obtained from the kanuka tree) with acyclovir 5% for the treatment of herpes labialis. Te efcacy of topical medical-grade kanuka honey was no diferent to topical acyclovir. Te median (95% CI) healing times in days were 8 (8-9) for acyclovir and 9 (8-9) for kanuka honey [37]. However, to our knowledge, there are no published data regarding the long-term efcacy of natural products in treating herpes simplex mucocutaneous infections, so our study is groundbreaking in this regard. While other topical antiviral agents such as acyclovir 5% cream (with or without hydrocortisone) and penciclovir 5% cream are not efective in preventing recurrent herpes labialis [38], our data showed that Lazolex ® Gel dramatically reduced the number of infection outbreaks in the years following treatment for both herpes labialis and genital herpes. Te frequency of outbreaks signifcantly decreased from a median of 2.00 and 1.00 times a year to 0.25 and 0.00 in the HSV-1 (herpes labialis) and HSV-2 (genital herpes) cohorts, respectively. Te diagnosis of herpes has important lifelong psychological implications [2,9] and the prevalence of reactivations is estimated at an average of six times per year [1], so these fndings may have a very positive impact on patient quality of life. Furthermore, reducing the rate of recurrence could have favorable consequences for health systems. In this regard, Xia et al. [39] calculated the costs associated with HSV infection in emergency rooms in US hospitals. From 2006 to 2013, the costs were estimated at 543 million dollars, going from 45 million dollars in 2006 to 91 million in 2013, mainly due to a 24% increase in the number of patients.
In our study, most of the patients considered the treatment highly efective at the end of the 4-year follow-up period. Several patients who had outbreaks during these years stated that the intensity of symptoms was milder compared to those prior to receiving Lazolex ® Gel treatment. However, it should be noted that herpes recurrences tend to be less frequent over time [9]. When assessing patient satisfaction, there are other factors to consider in addition to efcacy, such as aesthetic aspects. Gels are widely accepted as convenient therapeutic products. In this regard, as reported by Skulason et al., the majority of patients prefer transparent gels to white creams due to the aesthetic benefts they ofer [40].
Although standard therapies with acyclovir and other synthetic drugs are available, the safety of these drugs is limited due to the development of adverse efects, such as renal failure, hepatitis, and anaphylaxis [8,41]. Our fndings confrmed that tolerance to treatment with Lazolex ® Gel applied to lesions four times a day for 10 days was good. Tere were no adverse reactions or side efects in the HSV-1 group (herpes labialis) or HSV-2 group (genital herpes). Given these results and those of the toxicological studies previously performed by the manufacturer, Lazolex ® Gel is considered safe. Novel treatment approaches for herpes simplex infections are needed not only because of the safety issues mentioned above, but also due to the emergence of virus strains resistant to commonly used antiviral drugs [8]. Tus, Lazolex ® Gel could be an efective alternative treatment for herpes simplex mucocutaneous infections.
While this is the frst study to investigate Lazolex ® Gel as a treatment for herpes labialis and genital herpes, previous studies have evaluated its potential to treat recurrent stomatitis. In the comparative study by Gogotishvili et al. [19], which included 50 patients with recurrent aphthous stomatitis, Lazolex ® Gel was more efective than treatment with Solcoseryl ® dental adhesive paste, A and E vitamins, and cedar oil. Lazolex ® Gel has been shown to accelerate tissue regeneration and increase the duration of remission in patients with recurrent aphthous stomatitis and recurrent herpetic stomatitis [20,42]. Among these studies, the only one which focused on the safety profle for the treatment reported no adverse events, a fnding consistent with the positive tolerance observed in our study [42].
Te present study has several limitations. Firstly, the study was conducted with no control group. Tis pilot study was designed as an initial step to assess the efcacy and tolerance of Lazolex ® . Tanks to the preliminary information this trial obtained, a randomized, double-blind, placebo-controlled study is currently underway to evaluate the efectiveness and safety of Lazolex ® in patients with recurrent genital herpes (LAZOGENHER study). In the HSV-1 cohort (herpes labialis), the only sex represented was women (100%). Tis fact can be considered a limitation of the study, but can be explained by the higher prevalence of clinically manifested orofacial herpes simplex in females compared to males [33]. Tis clinical trial also ofers important advantages, such as the evaluation of long-term efcacy. Furthermore, unlike other studies [33], the progression of the lesions was evaluated by an attending physician.

Conclusions
Topical treatment with Lazolex ® Gel applied to lesions four times a day for 10 days has proven to be efective and safe to treat mucocutaneous lesions caused by HSV-1 and HSV-2 and to reduce the frequency of recurrence. It is efective in controlling pain and infammation as well as shortening the duration of typical symptoms such as itching and burning. Lazolex ® Gel is a promising candidate for the topical treatment of herpes infections, particularly given the emerging problem of drug resistance. It would be advisable to design more extensive clinical trials to confrm the ET50 reported in the present study. In addition, randomized placebo-controlled studies are needed, not only to confrm our fndings, but also to reinforce the efcacy of plant extracts in treating HSV infections.

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