Phase I Study of a Topical Skin Protectant Against Chemical Warfare Agents

INTRODUCTION

Sulfur mustard is a potent vesicant that penetrates the human skin easily and although blisters appear within hours after exposure, the molecular damage occurs within minutes.1-5 Despite continuing efforts there are still no antidotes available.1-3,5-8 Several nerve agents (e.g., VX) have the ability to penetrate the human skin, thus exerting their toxic effects. VX specifically is considered as one of the most potent chemical warfare agents (CWAs).1,9 Current chemical, biological, and radio-nuclear (CBRN) personal protection equipment includes a mask, protective clothing, gloves, and overboots. Any penetration of the above mentioned CWAs through gaps in the protective equipment entails a potential life-threatening risk.

A topically applied pretreatment may act as a dermal barrier, preventing the absorption of these agents through the skin and thus their respective toxicity.10 Such skin protectant may serve as an additional means of protection in conjunction with other physical protection components. Towards this end, the primary application areas should include the integration areas of the personal protective equipment, i.e., the neck, armpits, groin, waist, wrists, and ankles, which are also considered to be relatively sensitive skin areas.1,2,5 The "topical skin protectant" strategy has been adopted by several countries so far, including Israel.10

IBI, a topical skin protectant resistant to CWAs that was developed at the Israel Institute for Biological Research (IIBR), was successfully tested in various animal models.4 Using the well-established primary skin irritation Draize test,11 the protectant was demonstrated to be nonirritating in guinea pigs and in rabbits. No adverse dermatological or systemic effects were found in a long-term toxicological study in pigs following an overdose application of the protectant (covering >20% of the skin surface, applied three times per day for 2 weeks). Evaluation included a clinical follow-up for 1 month, blood and urine biochemistry, including magnesium (Mg) levels, and pathology. No detectable levels of the protectant ingrethents were found in blood and urine during the entire monitoring period.4 Also, blood and urine Mg levels were not increased. Irritation and sensitivity tests were performed on 50 human volunteers (11 males, 39 females; 10 at the ages of 18-35 years, 14 at the ages of 36-45, 26 at the ages of 46-65) and were not found to be associated with any skin irritation or with any allergic sensitization.4

Here we tested the medical safety of repeated topical application of the IB1 lotion in a randomized placebocontrolled double-blind phase I clinical study in young healdiy volunteers. Since the lotion includes a relatively high content of magnesium sulfate, potential changes in serum Mg levels were monitored, as well as other hematological and biochemical parameters, local dermatological adverse effects, and convenience of use.

MATERIALS AND METHODS

The study was approved by the review board of the Israeli Defense Force's (IDF) Medical Corps and was conducted according to the Declaration of Helsinki. All volunteers gave their written informed consent.

Preliminary Application Test

To determine the amount of IBl lotion needed for each application, an application test was performed before the main study. In this preliminary study 10 volunteers received small glass vials containing 20 ml of the IBl and were asked to cover relevant areas as described in Figure 1. The application was performed in the presence of the researchers. After application, the remaining lotion was weighed. On average, ~10 ml (containing 0.37 g of Mg) were required to cover all relevant areas, and this amount was thus determined as the experimental dose.

Volunteers

Volunteers were recruited from a medic's course in the IDF's military medical school. Inclusion criteria allowed only healthy soldiers with normal physical examination, normal baseline complete blood count, liver function tests, and renal function tests to participate. Soldiers with any chronic or acute disease (especially dermatological diseases) or receiving any medical treatment were excluded. This excluded soldiers with asthma, atopic dermatitis, previously known allergy, allergic rhinitis, allergic conjunctivitis, sun bums, skin lesions at the application areas, and soldiers that for any reason could not replace contact lenses with eyeglasses. Every volunteer had the right to stop participation at any time during the study. In addition, each volunteer filled and signed an informed consent form and a medical questionnaire.

Blinding Procedures

The study was designed as a randomized placebo-controlled double-blind study. The IBl and the placebo lotions were prepared at the HBR and were coded by medical personnel at the IDF's Medical Corps, who were not involved in any other part of the experiment, including design, performance, or analysis. Codes were locked throughout the experiment. The researchers, who enrolled the volunteers, drew the blood, took vital signs, examined the volunteers, and were present in every application, as well as a dermatologist, were blinded to the lotion composition throughout the experiment until after all analyses were completed. Coding was revealed only at the end of the study by the same personnel that coded them before the study.

The placebo was the vehicle of the IB 1 lotion, consisting of 87% glycerin (Merck, Israel) and 13% Aqua without any magnesium. Both placebo and IBl lotions were indistinguishably odorless, colorless, and with the same consistency. Both were supplied in identical vials. The researchers received a single box containing batches of coded vials that were randomly assigned to the volunteers during the preparation day of the study (see below "Experimental Protocol"). Each volunteer was assigned one vial per application.

Experimental Protocol

A preparation day preceded the actual study by 1 week. Study protocol included 4 consecutive days in which a total of nine applications were carried out, once every 8 hours. The preparation day included signing an informed consent form, full medical examination, assigning the coded batches to the volunteers, and obtaining baseline blood tests (baseline Mg levels, complete blood count, renal functions, and liver functions). The dosing schedule was defined on the basis of the pre-clinical experiments.4 The lotion was applied on six body areas: the neck, axillae, waist, groin, wrists, and ankles (Fig. 1). Volunteers were instructed to evenly apply the lotion on these areas and use all 10 ml supplied. All application sessions were performed in the presence of the researchers to verify that all of the volunteers followed the instructions by marking relevant checklists and drawings. Volunteers washed their hands after every application, and only after several minutes during which the lotion was absorbed into the skin surface did they re-dress. Worth noting, the vials were collected by the researchers after each application.

Complete blood counts and liver function tests were obtained in baseline and poststudy assessments. The baseline test was required for inclusion and exclusion criteria. Further, since the IBl lotion contains magnesium sulfate, renal function tests were taken as baseline and at the end of the study, and blood Mg levels were monitored daily to verify that repetitive IBl application does not lead to a dangerous increase. Because of the diurnal changes in Mg levels,12,13 blood samples were drawn at the same time every day between 12:30pm and 1 :30pm, except for the baseline tests, which were obtained between 10:30am and 11:00am. Baseline test results were used for inclusion/exclusion of candidates. Hence, a total of four blood samples were obtained from each volunteer. The first and the last included a complete blood count, plasma Mg level (total serum level), and liver and renal functions. Two additional samples for plasma Mg levels were obtained from each volunteer on days 2 and 3. Blood samples were transferred immediately in a chilled receptacle to a medical laboratory facility adjacent to the base for analysis.

The volunteers were allowed to have one shower a day, before the night application. No restrictions on any activity, including physical trainings, or on any clothing, including uniforms, were placed on the volunteers. Each volunteer was fully examined by a dermatologist every day. The aim of this examination was to identify any topical adverse effects consistent with either allergic contact dermatitis or other local toxic dermal effect. These were documented in a daily chart, including an image similar to Figure 1, on which the exact area involved could be marked.

Each volunteer filled out a daily questionnaire concerning adverse effects. At the end of the study they all filled out a second questionnaire on the influence of the lotion on daily activities and their comments about the product (data not shown).

Statistical Analysis

The minimal number of subjects for the study was decided on the basis of the following considerations: significance level (a) 0.05; statistical power (l-ß) 0.8; anticipated effect size (f^sup 2^) 0.4. The minimal number accordingly was calculated to be 22 (1 1 in each group); however, since several dropouts were expected, die study was started with 51 subjects (of which 17 indeed dropped out).

Statistical analyses were performed using SAS for Windows version 9.1 (SAS Institute, Ine, Cary, NC). Normality of variables distribution was tested with the Smirnov-Klimigorov test. Plasma Mg levels of the two groups (IB 1 lotion and placebo) were compared for each day of lotion application with die Mann-Whitney nonparametric test. In addition, differences between Mg levels at each stage and the baseline levels were calculated and compared between the two groups. Analysis of variation with repeated measures was fitted for each group, using Proc Mixed in SAS. Statistical significance was set at 0.05. Data are shown as mean (±SD).

RESULTS

Of 130 soldiers approached, only 51 had volunteered. After screening, 2 volunteers were excluded because of a history of asthma, 2 other volunteers were diagnosed with mild anemia at me baseline complete blood count (10.2 mg/dL and 10.6 mg/ dL), 1 volunteer had a high bilirubin level (3.2 mg/dL), and 1 volunteer had a relatively high baseline Mg level (2.9 mg/dL). The remaining 45 volunteers were randomized for treatment groups. During the study, an additional 2 volunteers dropped out because of acute tonsillitis with fever that appeared on the second and third days of the study, and an additional 9 volunteers dropped out of the study at various stages because of nonmedical reasons according to their request. The 34 volunteers who completed the full study protocol were ages 18-20 (mean 18.9 ± 0.7). Decoding revealed mat 21 volunteers received the IBl and 13 received placebos: 26 males (17 in the IBl group, 9 in the placebo group) and 8 females (4 in each application group). The effect of gender on the results could not be measured because of small sample size.

No serious adverse effects were reported throughout the study. Forty percent of the male volunteers in the IB 1 group and 60% of the male volunteers in the placebo group reported a short-lasting (up to 30 minutes) burning sensation when applying the lotion after shaving, with no residual or apparent dermal irritation upon examination by the dermatologist and no pathological findings in their daily medical examinations. No significant differences were found in the complete blood counts between baseline and poststudy samples, except for a slight increase in the white blood count, which was observed in both groups (Table I). The mild increase was statistically significant in the placebo group (p-value = 0.04) and showed a strong trend in the IBl group (p- value = 0.06). There were no significant differences among renal and liver function tests between the baseline and the poststudy analyses (Table I).

After completion of the experimental protocol all volunteers reported that there was no interference with any daily activity or performance, except for very mild interference in wearing clothing and performing power drills, which was reported by the volunteers in the IBl group mainly (Table II). However, these interferences did not seem to impair the solthers' overall physical performance and hence may not have any practical implication. AU volunteers stated that the instructions were clear and simple, there was no need for buddy aid, and that it had no effect on their ability to concentrate during lectores (Table II).

Blood Mg levels were within the normal range in all sample points in both groups (1.53 mg/dL^2.6 mg/dL), except for two sporadic samples from two different volunteers in the IBl group (2.9 mg/dL in the third sample in one volunteer and 2.7 mg/dL in the fourth sample in the other). No significant differences in Mg levels were found between the placebo group and the IBl group in any of the tested time points (Table ??). Individual analysis of each volunteer did not reveal any clinically significant change over time in the Mg levels (data not shown). Differences between mean Mg levels at each time point and baseline levels were calculated and compared between the two groups. The differences were found to be insignificant (Table III). Of note, analysis of variation with repeated measurement revealed that Mg levels on day 1 were significantly lower than other measurements in the same group in both placebo and IBl groups (p-value < 0.001 and/p-value < 0.01, respectively). However, these differences were very small and within the normal Mg range.

DISCUSSION

Many highly toxic CWAs exert their effect through the dermal exposure route, either by vapors or droplets.1-3,5,14 Consensus protective measures include mainly physical protection with specialized outfits. Yet, integration areas, such as neck, armpits, groin, waist, wrists, and ankles are a weak link for penetration and exposure to CWAs. Previous preclinical studies indicated that G?1 lotion, a passive protective lotion against CWAs, significantly reduced the toxicity of sulfur mustard and VX.4 The IBl lotion contains ingrethents approved for human use and has successfully passed the safety testing on animals and humans.4 A major concern regarding future use of the topical skin protectant was the possibility, although faint, that dangerous amounts of magnesium might accumulate in plasma when applying the lotion repeatedly. Thus, the main goals of the current study were to test the dermatologica! and systemic safety of this product in repeated applications and especially to rule out the possibility of reaching dangerous levels of plasma Mg.

Magnesium is the fourth most common cation in the body and the third most common intracellular cation (mainly in muscle and liver).1516 The normal plasma concentration ranges between 1.5 and 2.3 mg/dL (1.2-1.9 mEq/L; 0.62-0.94 mmol/L), with some variation between clinical laboratories. Only 1-2% of body magnesium is extracellular (60% ionized; 15% complexed; 25% protein bound). Magnesium is a necessary cofactor for hundreds of enzymes. It is important for membrane stabilization and nerve conduction.15 Renal excretion is the principal regulator of magnesium balance. Clinically significant hypermagnesemia, albeit unusual, is almost always secondary to excessive intake. The kidneys' ability to excrete excessive magnesium is diminished in patients with chronic renal failure. Massive parenteral infusions of magnesium salts, which overwhelm renal excretory mechanisms, have been reported with inadvertent intravenous infusion, urologie procedures involving irrigation with magnesium salts, and ingestion of large quantities of magnesium-containing antacids and cathartics.16 To the best of our knowledge, there is no published scientific evidence on transdermal absorption of magnesium. The symptoms of hypermagnesemia correlate roughly with serum concentrations but depend on the rate of increase and host factors.16

Usually, symptoms do not appear when plasma magnesium levels are <4.5 mg/dL.15 Hypermagnesemia impairs neuromuscular junction transmission by decreasing acetylcholine release from the presynaptic membrane, mus producing hypotonia, hyporeflexia, and weakness.15 Paralysis occurs at high concentrations. Direct central nervous system depression causes letiiargy and sleepiness. It is also associated with hypotension and flushing owing to vascular dilation. Hypotension can be profound at plasma levels of 4-5 mEq/L because of a direct effect on cardiac function. At die same levels hypermagnesemia typically results in loss of deep tendon reflexes. At levels >5 mEq/L central nervous system depression may range from drowsiness to coma. Electrocardiogram changes occur at levels of 5-10 mEq/L, including prolonged P-R, QRS, and Q-T intervals. Serum levels of 9-12 mEq/L may cause apnea and cardiovascular collapse.15 Severe hypermagnesemia includes nausea, vomiting, hypocalcaemia, and can cause parasympathetic blockade, fixed dilated pupils, and neuromuscular blockade mimicking a midbrain syndrome.15

We found no significant rise in blood Mg levels during repetitive applications. Allegedly, a significant rise could be observed between die first test obtained a week before die study and die other tests obtained during the study. However, this difference was very small and within the normal Mg range (no clinical relevance) and may be attributed to the different hour in which die samples were obtained. Indeed, baseline samples were obtained at 1 0:30am- 1 1 :00am and me other tests at 12:30pm-l:30pm. Two samples in which a higher than normal level of Mg was recorded in two volunteers applying the IBl lotion might be explained by me normal diurnal change in blood Mg levels, although blood samples were drawn at die same time every day during the application study. Our main concern was from much higher blood Mg levels tìiat might cause dangerous symptoms, even if only transiently elevated. Importantly, these slightly above normal values were far from being considered dangerous, especially in young healdiy individuals with normal renal functions. Although there is a possibility that a larger sample size will uncover a group of subjects with a significant rise in blood Mg levels, we think mat the existing evidence supported by animal studies4 is sufficient to conclude that the use of IBl lotion does not lead to increased serum Mg levels and is safe to use from this perspective.

Members of the review board of the Israeli Defense Force's Medical Corps raised concerns of a potential effect of IBl application on liver functions, as well as on complete blood count, because of an inflammatory response, although mere was no evidence of any such changes in animal studies. Hence, complete blood counts and liver function tests were taken as part of the screening procedures and to rule out clues for inflammation that might follow dermal application of the lotion. There was no evidence of any such changes in the animal stuthes, and as estimated, there were also no significant changes in the complete blood count and liver function measurements in either group in our study, except for a very mild increase in white blood count observed post study in bom groups. However, as the difference was very small and absolute values were within the normal range, it was probably unrelated to me chemical composition of me IBl and might reflect a mild stress, perhaps because of repeated drawing of blood. No significant values could be observed in the volunteers who complained of a transient burning sensation (data not shown). Renal function tests were obtained to make sure the basic status of me volunteers was normal and was not altered during the study. No significant changes were recorded in renal function tests, in eitiier group. We conclude that repetitive application of IBl does not cause measurable inflammatory changes or renal or liver abnormalities.

Before this study, irritation and sensitivity tests were performed in a well-recognized medical institute in the country, which showed neither irritation nor sensitivity among 50 participants.4 Here, however, several volunteers reported a shortlasting burning sensation when applying the lotion on freshly shaved skin, yet there were no persistent signs of irritation as defined by a dermatologist. The volunteers noted that it was present for only a short period of time and resembled the feeling of applying an aftershave. No difference could be identified between the placebo and the IB 1 groups. As was mentioned earlier, no changes were found in the complete blood count of these volunteers, as might appear in an inflammatory reaction.

The second goal of the study was to find out whether the topical skin protectant interferes with routine daily tasks. The volunteers' impression was that repetitive applications do not interfere with various daily activities, including writing, jogging, and other sports activities, as detailed in Table II. We have no explanation for the difference concerning the wearing of clothing and performing power drills, especially as there was no difference in the consistency of the IB 1 lotion and the placebo. These differences may not reflect any practical difficulties or differences. Alternatively, the study eventually included a limited number of subjects. To attain confidendy significant results a larger group of volunteers is needed.

The limited number of volunteers in our study is one of its major limitations. Though the final number of volunteers participating in the study was statistically sufficient, it was advisable to have a larger sample size. Unfortunately, >80% of potential volunteers on the base did not want to take part in this study, most of them because of the requirement of drawing several blood samples during the study.

CONCLUSIONS

With the combination of previous animal stuthes, irritation and sensitivity tests, and results that show safety and convenience, we believe that the IB1 lotion should be considered as a safe adjunct to other CBRN protective measures of field solthers.

ACKNOWLEDGMENTS

We thank Dr. Tamar Kadar and the teams of the Pharmacology and Organic Chemistry Departments at the Israel Institute for Biological Research, for their invaluable help in performing this study.

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