Fosphenytoin Alleviates Herpes Simplex Virus Infection-Induced Provoked and Spontaneous Pain-Like Behaviors in Mice

Abstract

In this study, we investigated the effects of fosphenytoin (fPHT), a water-soluble prodrug of phenytoin, on the pain responses of a mouse herpes zoster (HZ) pain model. Transdermal herpes simplex virus type 1 (HSV-1) inoculation induced mechanical allodynia and hyperalgesia of the hind paw and spontaneous pain-like behaviors, such as licking the affected skin. Intravenous injection of fPHT (15 and 30 mg/kg) alleviated HSV-1-induced provoked pain (allodynia and hyperalgesia). The suppressive effects of fPHT on provoked pain were weaker than those of diclofenac and pregabalin which were used as positive controls. fPHT, diclofenac, and pregabalin significantly suppressed HSV-1-induced spontaneous pain-like behaviors. Among them, high-dose fPHT (30 mg/kg) showed the strongest suppression. Intravenous fPHT may become a viable option for an acute HZ pain, especially for spontaneous pain.

INTRODUCTION

Herpes zoster (HZ) is caused by the reactivation of human herpesvirus 3 (varicella zoster virus, VZV) in the sensory ganglion in human subjects and is characterized by clustered vesicles and severe pain.¹⁾ Patients with HZ complain of continuous severe spontaneous pain, such as burning pain, and allodynia, which refers to pain caused by innocuous stimuli. Although HZ usually resolves in two to four weeks, some patients experience pain for a long time even after the resolution of HZ.²⁾ Acute HZ pain is thought to be a risk factor for postherpetic neuralgia (PHN).^2,3) Therefore, it is important to control acute HZ pain effectively in order to prevent PHN.

Fosphenytoin (fPHT), a water-soluble prodrug of phenytoin, is an anti-epileptic drug recommended as the second-line therapy for patients with status epilepticus.^4,5) After intravenous injection, fPHT is converted to phenytoin by serum phosphatases with a conversion half-life of approximately 10 min. Advantages of fPHT include more convenient and rapid intravenous administration, availability for intramuscular injection, and low toxicity at injection sites.^4,5) Several lines of evidence suggest that fPHT is effective in the treatment of trigeminal neuralgia.^6,7) Although phenytoin has been reported to be effective for PHN,⁸⁾ there are no reports for the effectiveness of fPHT on acute HZ pain and PHN.

Previously, we have established mouse models of acute HZ pain^9,10) and postherpetic neuralgia.¹¹⁾ As a first step to clarify the efficacy of fPHT for HZ-associated pain, in this study, we examined the effects of fPHT on acute HZ pain.

MATERIALS AND METHODS

Animals

Female C57BL/6J mice weighing 18–20 g (6 weeks old at the start of experiments; Japan SLC, Shizuoka, Japan) were used. They were housed in standard polycarbonate cages (four mice per cage) under controlled temperature (22 ± 1 °C) and humidity (55 ± 10%) with a 12-h light–dark cycle (lights on at 07:00) and were allowed to access food and water freely. The animal experiments were approved by the Animal Care Committee of the University of Toyama.

Virus Inoculation

The mice were inoculated with herpes simplex virus type 1 (HSV-1) as described previously.^9–11) Briefly, the depilated shin skin of the right hind paw (5 × 5 mm) was scarified and inoculated with HSV-1 (7401H strain; 1 × 10⁶ plaque-forming units). The contralateral hind paw was not inoculated.

Drug Administration

Fosphenytoin (Fostoin^®, Nobelpharma Co., Ltd., Tokyo, Japan) was diluted with physiological saline, and administered intravenously (i.v.). Diclofenac (Sigma-Aldrich, St. Louis, MO, U.S.A.) and pregabalin (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were dissolved in distilled water and administered orally. All mice were injected with i.v. fPHT or saline (control), and were then administered oral (p.o.) diclofenac, pregabalin, or water (control) within 5 min after the i.v. injection.

Behavioral Tests

Mechanical allodynia and hyperalgesia were assessed using von Frey filament with a bending force of 0.17 or 1.20 g (North Coast Medical, Inc., Morgan Hill, CA, U.S.A.), respectively.^9,12) After at least 30 min acclimation, von Frey filament was pressed perpendicularly against the plantar skin and held for one to three seconds with it slightly buckled. Responses to the punctate stimulation were ranked as follows: 0 = no response; 1 = move away from von Frey filament; 2 = immediate flinching or licking of the hind paw. The stimulation of the same intensity was applied six times at intervals of several seconds and the average score served as pain-related score.

In measuring spontaneous pain, the behaviors of the mice were videotaped for 30 min to determine the duration of licking (an index of pain).¹³⁾

Statistical Analysis

Statistical analysis was performed using the SigmaPlot Software version 14 (Systat Software, Ltd., Chicago, IL, U.S.A.). Data are presented as mean ± standard errors of the mean (S.E.M.). Statistical significance between groups was analyzed using one-way ANOVA or two-way repeated measures ANOVA followed by a post hoc multiple comparison test of Dunnett’s Method or Holm–Sidak method; a p value of <0.05 was considered significant.

RESULTS

HSV-1 inoculation on the hind paw produced HZ-like skin lesions in the same dermatome from day 5 after inoculation; the skin lesion score peaked around day 7 following inoculation, which is consistent with previous reports.^9,10) HSV-1 inoculation induced mechanical allodynia (increased responses to 0.17-g stimuli) and hyperalgesia (increased responses to 1.20-g stimuli) of the hind paw (Figs. 1, 2), and also induced spontaneous pain-like behavior, such as licking the affected skin (Fig. 3).

Fig. 1. Effects of Fosphenytoin (fPHT) on Mechanical Allodynia and Hyperalgesia Induced by Transdermal HSV-1 Inoculation in Mice

The unilateral hind paw was inoculated with HSV-1 (A, B) and the contralateral hind paw was without inoculation (C, D). A and C; pain score to 0.17-g stimuli. B and D; pain score to 1.20-g stimuli. fPHT (15 and 30 mg/kg) was injected intravenously at day 6 after inoculation. The data presented are means and S.E.M. (n = 8–16). * p < 0.05 vs. VEH (SLN + WTR) no adjustment for multiple time points comparison.

Fig. 2. Effects of Diclofenac (DIC) and Pregabalin (PGB) on Mechanical Allodynia and Hyperalgesia Induced by Transdermal HSV-1 Inoculation in Mice

DIC (30 mg/kg) and PGB (3 mg/kg) were administered orally at day 6 after inoculation. The data presented are means and S.E.M. (n = 8–16). * p < 0.05 vs. VEH (SLN + WTR) no adjustment for multiple time points comparison.

Fig. 3. Effects of Fosphenytoin (fPHT), Diclofenac (DIC), and Pregabalin (PGB) on HSV-1 Inoculation-Induced Spontaneous Pain-Like Behaviors in Mice

The time spent in licking behavior was measured for 30 min from 1 h after administration. The data presented are means and S.E.M. (n = 8–16). * p < 0.05 vs. VEH (SLN + WTR) when analyzed using one way ANOVA followed by Dunnett’s Method for multiple comparisons. One way ANOVA results for factor of Between Groups; DF = 4, Sum of Squares = 389121.7, Mean Square = 97280.4, F-value = 5.467, p = 0.002.

The effects of fPHT (15 and 30 mg/kg, i.v.), diclofenac (30 mg/kg, p.o.), and pregabalin (3 mg/kg, p.o.) on mechanical allodynia and hyperalgesia observed on day 6 following inoculation were examined. A dose 30 mg/kg of fPHT showed a tendency to suppress allodynia from one hour after administration, but it was not significant (Fig. 1A, Table 1). In the 15 mg/kg fPHT group, significant suppression was observed at three hours after administration compared with the vehicle control group (Fig. 1A, Table 1). For hyperalgesia, both doses of fPHT showed a significant inhibitory effect around two to four hours after administration (Fig. 1B, Table 1). Diclofenac showed a tendency to suppress allodynia and significantly reduced hyperalgesia (Figs. 2A, B, Table 2). Pregabalin showed potent and significant analgesic effects on both allodynia and hyperalgesia (Figs. 2C, D, Table 2). No significant change was observed in the response of contralateral hind paw with any of the drug administrations (Figs. 1C, D).

Table 1. Two Way Repeated Measures ANOVA (One Factor Repetition) for fPHT (15 and 30 mg/kg, i.v.)

Drug administraton	Factor	DF	Sum of squres	Mean square	F-Value	p-Value
0.17 g lpsi	Treatment	2	0.269	0.134	3.180	0.044
	Subject (Treatment)	45	2.509	0.0557
	Time	6	0.871	0.145	3.874	0.001
	Treatment × Time	12	0.644	0.0536	1.431	0.160
	Residual	126	4.722	0.0375
	Total	191	8.281	0.0434
	Comparison for treatment*		Diff of mean	t	p	p < 0.05
	VEH vs. 15 mg/kg		0.180	2.159	0.071	No
	VEH vs. 30 mg/kg		0.119	1.428	0.160	No
1.20 g lpsi	Treatment	2	1.410	0.705	6.147	0.003
	Subject (Treatment)	45	10.217	0.227
	Time	6	1.250	0.208	2.793	0.014
	Treatment × Time	12	0.908	0.0757	1.014	0.440
	Residual	126	9.402	0.0746
	Total	191	23.326	0.122
	Comparison for treatment*		Diff of mean	t	p	p < 0.05
	VEH vs. 15 mg/kg		0.405	2.402	0.041	Yes
	VEH vs. 30 mg/kg		0.299	1.777	0.082	No

*Multiple Comparisons versus Control Group (Holm–Sidak method) : Overall significance level = 0.05.

Table 2. Two Way Repeated Measures ANOVA (One Factor Repetition) for Diclofenac (30 mg/kg, p.o.), and Pregabalin (3 mg/kg, p.o.)

Drug administraton	Factor	DF	Sum of squres	Mean square	F-Value	p-Value
Diclofenac 0.17 g lpsi	Treatment	1	0.0627	0.0627	1.445	0.232
	Subject (Treatment)	30	1.305	0.0435
	Time	6	0.267	0.0445	1.026	0.414
	Treatment × Time	6	0.0653	0.0109	0.251	0.958
	Residual	84	3.642	0.0434
	Total	127	5.873	0.0462
Diclofenac 1.20 g lpsi	Treatment	1	0.818	0.818	8.291	0.005
	Subject (treatment)	30	3.340	0.111
	Time	6	1.369	0.228	2.423	0.033
	Treatment × Time	6	1.252	0.209	2.216	0.049
	Residual	84	7.909	0.0942
	Total	127	17.649	0.139
Pregabalin 0.17 g lpsi	Treatment	1	0.215	0.215	7.366	0.008
	Subject (treatment)	29	0.930	0.0311
	Time	6	0.479	0.0798	2.812	0.015
	Treatment × Time	6	0.257	0.0428	1.508	0.186
	Residual	81	2.300	0.0284
	Total	123	4.761	0.0387
Pregabalin 1.20 g lpsi	Treatment	1	2.103	2.103	24.203	<0.001
	Subject (treatment)	29	4.141	0.143
	Time	6	1.669	0.278	4.234	<0.001
	Treatment × Time	6	1.037	0.173	2.630	0.022
	Residual	81	5.323	0.0657
	Total	123	18.458	0.150

Next, the effects of fPHT, diclofenac, and pregabalin on spontaneous pain-like behaviors were examined. The behavior was measured for 30 min starting 1 h after drug administration. On day 6 following inoculation, HSV-1-inoculated mice showed spontaneous pain-like behaviors such as licking of the affected skin (Fig. 3). All medications significantly decreased the time spent in licking behaviors. Notably, fPHT (15 and 30 mg/kg, i.v.) suppressed spontaneous pain-like behavior in a dose-dependent manner, and although statistically not significant, the 30 mg/kg dose suppressed the behavior most strongly compared with all trial drugs.

DISCUSSION

In the present study, we demonstrated for the first time that intravenous injection of fPHT alleviates the HSV-1-induced mechanical hyperalgesia and spontaneous pain-like behaviors. Although the effects of fPHT on provoked pain (allodynia and hyperalgesia) were slightly weaker than those of diclofenac and pregabalin, 30 mg/kg fPHT suppressed spontaneous pain-related behavior more potently than diclofenac or gabapentin. “Spontaneous pain” in HZ is severe, autonomous and inescapable, which makes the patient very uncomfortable, whereas “provoked pain” can be avoided by not touching or moving the affected area. Patients with HZ often complain of spontaneous pain such as burning, tingling, or numbness of the skin. In the treatment of acute HZ pain, suppression of spontaneous pain is an important issue. Therefore, it is considered to be clinically meaningful that fPHT potently suppressed spontaneous pain-related behaviors and its effect was potent than that of diclofenac or pregabalin.

The mechanism of action of fPHT is related to the blockade of voltage-operated sodium channels.⁴⁾ Nerve blockade with local anesthetics (e.g., bupivacaine) provides pain relief from HZ.^1,14,15) It has been reported that HSV-1-infected rat dorsal root ganglion (DRG) neurons generate spontaneous activity, and the VZV infection results in increased protein expression of sodium channels Nav1.3 and Nav1.8 in the rat DRG.^16,17) Taking into consideration that spontaneous pain is thought to be the result of ectopic firing of sodium channels in the nervous system, the involvement of sodium channels in acute HZ pain, especially spontaneous pain, is strongly suggested. Further studies are required to clarify the subtype of sodium channel (i.e., Nav1.3, 1.7, 1.8 etc.) and the location where it is expressed in HSV-1-inoculated mice.

It has been reported that intravenous fPHT results in almost complete and long-lasting pain relief of patients with trigeminal neuralgia refractory to various medications such as carbamazepine, lamotrigine, or gabapentin.^6,7) In the present study, fPHT potently inhibited spontaneous pain-like behaviors. In the treatment of severe HZ pain, intravenous fPHT may become a viable option for an acute HZ pain, especially for spontaneous pain.

Acknowledgments

The authors would like to thank Ms. Izumi Hamada, a statistician at Nobelpharma Co., Ltd., for her help in statistical analysis. The authors also thank all the staff members of the Division of Animal Resources and Development, Life Science Research Center, University of Toyama.

Conflict of Interest

The authors declare that this study received funding from Nobelpharma Co., Ltd. Drs. Yoshimi Kitada and Saori Arai are full-time employees of Nobelpharma Co., Ltd. The other authors declare no conflict of interest.

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