Nefopam induced dose-related changes in renal and hepatic functions

Nefopam is a non-opioid, centrally acting, non-steroidal analgesic drug. It is used to treat mild to moderate painful conditions. Although developed about ive decades ago, its use has gained resurgence in recent years. This studywas designed to investigate the effects of different analgesic doses of nefopam on the liver and kidneys of mice. Forty albinomice were divided into four groups of 10 mice for each group. Group 1, 2 &3 received a daily intraperitoneal injection of Nefopam at 10, 20, and 30 mg/kg doses, respectively. The fourth group (control group) received injections of normal saline. After twoweeks of treatment, the animals were weighed and sacri iced, and then blood was collected for liver enzymes analysis and renal function test as well as histological assessment. The results revealed that the bodyweight increase ratio was signi icantly lower in group 3 (P-value <0.01). All tested liver enzymes i.e., ALT, AST, and ALP levels showed a highly signi icant (P-value <0.01) change among the tested groups. AlthoughALT remainedwithin normal limits in the irst two groups andnormal group, in group3 (30mg/kg), its level exceeded thenormal value. Liver enzyme changes re lected and supported the histopathological indings in the liver tissue. Group 2 & 3 showed varying degrees of hepatotoxicity, ranging from granulomatous lymphocytic in iltration to micro-vesicular steatosis and apoptotic pictures. Both kidney function test and histopathological examination, on the other hand, illustrated insigni icant effect (P-value >0.05) ofNefopamon the kidneys. Nefopam iswell tolerable by the liver at low analgesic doses butmayhavedetrimental effects at higher analgesic doses and prolonged duration of intake.


INTRODUCTION
The liver is considered to be one of the most vital organs that functions as a center of metabolism for nutrients and excretion of waste metabolites, but mainly providing protection against foreign substances by detoxi ication and elimination (Roy et al., 2012). The liver can metabolize xenobiotics, which can lead to drug-induced liver injury (DILI) and is a potential complication of many drugs. DILI broadly classi ied into two types: intrinsic and idiosyncratic types; intrinsic DILI generally is dose-dependent and predictable (e.g., acetaminophen toxicity), whereas idiosyncratic DILI is unpredictable and does not depend directly on dose (Chalasani and Björnsson, 2010).
Druginduced liver injury is one of the main reasons for the withdrawal of many drugs from the market. The mechanism of hepatotoxicity can be investigated through mitochondrial dysfunction and DNA damage -this imperial function caused by the drug itself or its cytochrome P450 mediated metabolite. Reports on hepatotoxicity suggest that oxidative stress, microvascular steatosis, imbalance energy storage are a major outcome of mitochondrial dysfunction (Au et al., 2011).
The kidney is an essential organ required by the body to perform several important functions, including the maintenance of homeostasis, regulation of the extracellular environment, such as detoxi ication and excretion of toxic metabolites and drugs. Therefore, the kidney can be considered as a major target organ for exogenous toxicants. Therefore, approximately 20% of renal injury or damage is caused by drugs (Kim and Moon, 2012). Renal injury or damage can be diagnosed through a simple blood test. Evaluation of nephrotoxicity through blood tests includes the measurements of blood urea nitrogen (BUN), the concentration of serum creatinine, glomerular iltration rate, and creatinine clearance (Rached et al., 2008). Drug-induced renal injury or damage can be caused by mechanisms, including changes in glomerular hemodynamics, tubular cell toxicity, in lammation, crystal nephropathy, rhabdomyolysis, and thrombotic micro-angiopathy (Ferguson et al., 2008).
Nefopam is a benzoxazine that was developed in the 1960s as a muscle relaxant, but clinical use revealed that it has non-opioid, centrally acting, non-steroidal analgesic abilities, and it was used for relief of moderate pain (Seetohul et al., 2015). Its unique mode of action involves inhibition of the reuptake of serotonin, noradrenaline, and dopamine and may interact with the glutamatergic system (Saghaei et al., 2012). The drug undergoes signi icant irst-pass metabolism to inactive metabolites, and only 4-6% is excreted unchanged in urine (Djerada et al., 2014). Due to the resurgence of its use over the past years for the treatment of chronic painful conditions e.g., neuropathic pain, it gained more attraction from researchers to examine its potential effects on different body organs (Kim and Abdi, 2014). The aim of the study was to investigate the effect of different doses of nefopam on renal and hepatic function in mice.

Animal Grouping
The study was conducted at the pharmacology/toxicology lab in Al-Rasheed University College/ Pharmacy Department after obtaining an animal ethical approval. Forty adult male albino mice were obtained for this study. They had a median age of 8 ± (1.55) weeks. The animals were divided into four groups, 10 mice in each group. All animals in each group were weighed pre, and post-intervention, and the body weight increasing ratio was calculated as well. The intervention included receiving a single daily intraperitoneal (i.p.) injection. The i.p. doses were as following: Group 1 received 10 mg/kg Nefopam i.p.; group 2 received 20 mg/kg Nefopam i.p.; group 3 received 30 mg/kg Nefopam i.p. for 2 weeks; while group 4 (control group) received normal saline i.p. for 2 weeks. Nefopam (Acupan ® ) was used as a 20mg/2 mL injectable ampoule, and the dose was calculated for each animal according to its body weight, and smaller doses were diluted with distilled water.

Serum Biochemical Analysis
At the end of the experiment, the animals were put under anesthesia with diethyl ether, and blood was collected by heart puncture for biochemical analysis. Blood was centrifuged at 4000 rpm for 4 min, and plasma was isolated for the assessment of liver enzymes and renal function by means of spectrophotometry. The assessed liver enzymes included; alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), whilst the kidney function tests were tested through blood urea and serum creatinine.

Histological Examination
The liver and right kidney were excised, weighed, washed, and ixed in 10% formaldehyde for tissue processing and histological examination. Hepatic and renal tissues were prepared for routine H&E staining, according to Bancroft and Stevens (Bancroft and Stevens, 1982). Five sections (5µm thick) from each liver were examined. Digital images were acquired using Micros microscope (Austria) with built-in Micros Camera using the provided NMS Software (v 1.5.3.291) running on Microsoft Windows CE (v 5.00, build 1400).

Statistical Analysis
Data were analyzed using SPSS 23 software (SPSS Inc., Chicago, IL, USA), and the results were presented as mean + SD. Mann-Whitney test was used to compare the differences in body weight, increasing ration, organs weight, and organ/ body ratios of each group with the control group as they show nonnormal distribution. While One-way ANOVA was used to compare the differences in the levels of the biomarkers among the groups. P-values less than 0.05 were regarded as statistically signi icant, while P-values less than 0.01 were regarded as high statistical signi icance.

RESULTS AND DISCUSSION
The results revealed that the average initial body weight of all animals in all groups was 17.98 ± (2.36) gm, while the inal body weight was 21.44 ± (2.38) gm. The bodyweight increase ratio was signi icantly lower in group 3 (P-value <0.01), which was (14%) compared to the control group (21%). The bodyweight increase ratio between group1 and group2 did not show the signi icant change (P-value >0.05) in comparison to the control group, as shown in Table 1. Liver weight in the control group was signi icantly (P-value <0.01) lower than the other groups. However, the liver/body weight ration did not show a statistical signi icance (P-value >0.05) difference between each group and the control group Table 2.
Liver enzyme levels revealed a highly signi icant difference in their levels among all groups (P-value <0.01) Figure 1. However, ALT remained within normal value in all groups except in group 3 (30 mg N), which was signi icantly higher when compared to the other groups, and showed abnormally elevated value (>120 IU/l) (Otto et al., 2016). AST remained within the normal limit (191 IU/l) in all groups. ALP remained within normal limits (43-64 IU/l) in all groups, although it showed a high statistically significant difference (P-value <0.01) among the groups.
Hepatic tissue examination revealed normal hepatic lobular structure and morphology in control and (10 mg N) groups. Branching and anastomosing cords of hepatocytes were seen radiating from central veins towards portal triads. The cords were separated by narrow sinusoidal spaces. Hepatocytes had vesicular nuclei, and some of them were binucleated (indicated by red arrows). In the (20 mg N) group, hepatic architecture was preserved, and hepatocyte appeared normal, but there were scattered granulomatous lymphocyte in iltration with particular location af inity around the central veins  (Data presented as means ± SD, n = 10, ** high statistical signi icance at P-value <0.01) (yellow arrows and blue surrounded area). Hepatic tissue section of (30 mg N) group revealed that, the tissue showed evidence of hepatic cell injury as shown by several apoptotic pictures (blue arrowheads), peri-venular lymphocyte in iltration, and mid-lobular hepatocytic micro-vesicular steatosis (numerous small lipid droplets are present in hepatocyte cytoplasm) (red arrowheads) as shown in Figure 2.
The analysis of kidney weights revealed that weights (absolute & relative) showed insigni icant (P-value >0.05) change among all the groups, as shown in Table 3.
Renal function tests remained within normal reference values, and there was no statistically signi icant difference in their levels among the treated nor control groups (P-value >0.05) Figure 3. This was complemented by normal histological features of renal tissue Figure 4. The cortical area showed normal glomerular arrangements with normal urinary spaces and The normal analgesic dose of Nefopam in rodents ranges from low analgesia at 10mg/kg to high analgesia at 30mg/kg (Girard et al., 2008).
However, the incidence of adverse effects increases with the dose (Sanga et al., 2016). Among the common side effects are nausea, dizziness, blurred vision, and confusion (Gregori-Puigjane et al., 2012). In this study, only the (30 mg N) group failed to gain weight as the other groups. This may be most likely related to the aforementioned side effects encountered with high analgesic doses. Since Nefopam is non-sedative (Bilotta et al., 2005), its central actions are unlikely to be related to the reward and satisfaction centers in the brain and thus does not affect appetite and food intake directly (Buritova and Besson, 2002). Although liver weights were different in the control group, the liver weight/body weight ratios were comparable in all groups. This is best explained by the difference in growth rates among the animals causing the control group animals that had the median initial weight to catch up with the other groups, i.e., the gain in body weight exceeded the gain organ weight.
Enzymatic changes in different groups are re lective of the dose régime given. Low analgesic doses (10 mg N) and control (placebo) groups had nor-mal comparable results re lecting the ability of the liver to handle the 10mg/kg dose without enzymatic activation. However, at 20mg/kg dose, the enzyme levels increased signi icantly but still within normal levels, and at 30mg/kg, the ALT exceeded the normal levels while AST was at its high normal. ALT is a more speci ic marker than AST in detecting hepatocytic damage since AST is also present in cardiac and skeletal muscles and red blood cells and may be elevated in diseases related to these sites (Ramaiah, 2007). Elevated levels of ALT with low AST/ALT ratio are indicative of liver cell necrosis (Nyblom et al., 2004). Elevated ALP levels are associated with cholestatic liver damage. It appears that hepatocytic necrosis precedes cholestasis or maybe the major form of liver injury caused by the metabolic pathway of Nefopam. Nefopam is mainly metabolized by Ndemethylation but may involve other routes, since the amount excreted unchanged is minimal (Sanga et al., 2016).
The enzymatic changes are complemented by the histopathological indings. While the control and (10 m N) groups showed normal liver histology, the other two groups showed advancing levels of cell damage ranging from granulomatous legions in the (20 m N) group to clear cell apoptosis and micro-vesicular steatosis in the (30 mg N) group. Drug-induced liver damage depends on the type of the drug, dose, and duration of intake and encompasses a range of lesions including granulomas, fatty liver change (steatosis), zonal necrosis, cholestasis, and cholestatic-hepatitis (Lee, 2003). While any drug may cause any lesion, certain drugs are associated with more lesions than others are. Analgesics are more commonly associated with granulomatous hepatitis (Andrade et al., 2005), while valproate and cytotoxic drugs are more associated with steatosis (Amacher and Chalasani, 2014). Both lesions are associated with elevated ALT and AST. The absence of cholestasis is supportive of the normal ALT levels and may indicate that the metabolic pathway of Nefopam does not interfere with cholestatic pathology, at least for the duration and dose of the study.
Regarding the effect of different doses of nefopam on kidney function and histological features, there are insuf icient studies about its effect on kidney function. The study revealed that the kidney function for all animal groups was not affected, and the levels of BUN and creatinine remained within the reference normal values; even the histological examination did not show any feature deviation from the normal. One study can prove this, where Oliver and co-workers in 2010 studied the effect of using nefopam in end-stage kidney disease patients as it was less likely to decline kidney function further, but it should be used with caution due to possibility of elevated serum level (Mimoz et al., 2010).

CONCLUSION
Nefopam is well tolerable by the liver at low analgesic doses but may have detrimental effects at higher analgesic doses with prolonged duration of intake and with no signi icant changes in kidney function.