Incorporating ifosfamide into salvia oil-based nanoemulsion diminishes its nephrotoxicity in mice inoculated with tumor

Introduction

Ifosfamide (IFO), an alkylating antineoplastic agent and a prodrug used in adults and children,^1,2 has a broad-spectrum anticancer activity against solid tumors of soft tissue, bone, and lung.³ One of the major side effects of IFO is the formation of the cytotoxic metabolite, chloroacetaldehyde (CAA), during the IFO metabolism which may induce nephrotoxicity almost in the form of proximal tubular damage and glomerular filtration rate (GFR) reduction.⁴ IFO may cause Fanconi syndromeas a result of the renal damage, characterized by the glycosuria, phosphaturia, aminoaciduria, bicarbonaturia, and kaluria.

Nanoemulsion (NE) is a dispersion system that consists of oil, water, surfactant and most frequently cosurfactant.⁵ The diameter of the resulted dispersed nanodroplet is usually in the range of 20-200 nm. The major advantages of NEs in drug delivery system are their ability to improve the drug bioavailability and ameliorate the drug efficacy which leads to the reduction of the total drug dose and hence may result in eliminating the unfavorable side effects of the drug.^5-7 Several studies have formulated IFO in nanocarriers including solid-lipid nanoparticle, nanostructured lipid nanoparticles, self-assembled polymeric nanoparticles, span 80 nano-vesicles, and self-microemulsifying drug delivery systems.^8-12

NEs are considered potential carriers for the essential oils and have several disadvantages due to their hydrophobic nature, volatility, and ability to degradation. NEs may improve the bioefficacy, cellular uptake, and tissue distribution of the essential oils.¹³ It has been demonstrated recently that the incorporation of different essential oils into the NEs may induce apoptosis and cytotoxicity in breast cancer cell line (MCF-7) and cervical cancer cell line (HeLa).^14,15 Several studies have demonstrated the antitumor potential of the essential oils against a broad spectrum of cancers.¹⁶ Recently, a large number of research studies have reported the potent effect of salvia (SAL) plant as anticancer, antimicrobial, antimutagenic, antioxidant, and anti-inflammatory.¹⁷

In this study, IFO loaded in NE containing SAL essential oil, which has anticancer and antioxidant beneficial properties, was evaluated in vivo with the aim to assess the nephrotoxicity effect induced by IFO.

Materials and Methods

Results

Assessment of the therapeutic efficacy of drug formulas

The therapeutic efficacy of the drug formulas was monitored by measuring the MST and the amount of tumor collected following the sacrifice of the mice as illustrated in Table 1. The least amount of tumor was collected from the SAL-IFO group while it had the maximum MST at which its survival percentage (%S) was greater than the IFO group by two-fold. In terms of the effect of drug formulas on the food consumption and % change in body weight (Fig. 1), the entire tested groups were comparable within 60 days except for SAL and EAC groups that had got changed by the time periods. Relative to the other tested groups, SAL group started to consume more food after 28 days (P< 0.05) and markedly gained weight after 11 days (P< 0.001), whereas EAC group had considerably gained weight after 10 days and its food intake had varied after 17 days (P< 0.05).

Fig. 1

The effect of drug formulations on the mean of (a) the body weight change (%) and (b) food intake (g/mice/day in EAC bearing mice) of the experimental groups (n = 10 animals/group). Error bars represent the standard error of the mean (SE).

Table 1. The effect of drug formulations on the collected ascetic fluid of the sacrificed animals, the mean survival time (MST), and survival percentage (%S) for the experimental groups (n =10 animal/group)
Animal Groups	Ascetic fluid (mL)	MST (days)	%S
Normal		60 ± 0.00***b,c	100
EAC	1.05±0.03**c	18.5 ± 0.3***a,c	0
IFO	1.14±0.05***c	32.5 ± 5.07 ***a,b	30
SAL	1.17±0.03*b,***c	23.50 ± 2.10***a,c	10
SAL-IFO	0.89±0.057**b	36.00 ± 4.90***a,b	60
Note. The ascetic fluid and MST data were expressed as mean ± SE. Significant differences (*0.01≤ P < 0.05, **0.001≤ P<0.01, and ***P<0.001) were indicated between the individual group and the normal (a), EAC (b), and SAL-IFO (c) groups.

Detection of kidney function

Serum and urine analysis

As illustrated in Table 2, the IFO group exhibited the highest elevations in the serum levels of CRT and BUN when compared to the other tested groups. On the other hand, the administration of SAL-IFO into the mice had reduced the levels of serum CRT and BUN to be remarkably less than IFO groups. The serum level of GLU was markedly decreased in EAC and IFO groups when compared to the normal group whereas the SAL-IFO group reversed this effect to be almost comparable to the normal group. In terms of the electrolytes levels, there were no significant differences in serum Ca⁺², Mg⁺² and Na⁺ levels among the experimental groups. However, the Cl- level of SAL-IFO group was lower than that of EAC and IFO groups but it was comparable to the normal group. The K⁺ level was elevated in SAL and SAL-IFO groups when compared to the other tested groups. On the other hand, the SAL-IFO group had lowered the levels of U_p relative to the other tested EAC groups but it was comparable to the normal group. Although the levels of U_CO2 were raised in the treated EAC groups relative to the untreated EAC and normal groups, they were significantly reduced in the mice treated with SAL and SAL-IFO when compared to the IFO treated mice.

Table 2. Drug formulations effect on the kidney function of the tested mice
Groups	Normal	EAC	IFO	SAL	SAL-IFO
CRT (mg/dL)	118±2***b	149.3±8.6***a,c	190±1.6***a,b,c	106.6±1.7*a,***b,**c	120.6±4.3***b
BUN (mmol/L)	9.1±0.07***b,c	10.6±0.06***a,c	12±0.11***a,b,c	12.5±0.09***a,b,c	11.4±0.08***a,b
GLU (mg/dL)	117.16±2.6***b	83.49±3.3***a,c	100.16±2.9***a,b,c	110.23±4.9**a,**b,*c	118.15±0.9***b
Ca+2 (mmol/L)	2.22±0.01	2.27±0.009	2.150.02	2.19±0.008	2.05±0.016
Mg+2 (mmol/L)	1.1±0.004	1.2±0.007	1.1±0.005	1.18±0.009	1.06±0.007
Na+ (mmol/L)	148±3.3	151±1.5	149±3.6	148±2.9	143±4.1
Cl- (mmol/L)	113±1.4**b	118±2.2**a***c	116±1.9*b,**c	115±1.32**c	110±2.3***b
K+ (mmol/L)	4.4±0.29*c	4.4±0.32*c	4.6±0.1	5.1±0.22**a,b	5±0.15*a,b
UCO2 (mmol/L)	1±0.0001**b,***c	1.5±0.28**a,c	2.5±0.28***a,b,**c	2±0.0001***a,**b	2±0.0001***a,**b
UP (mmol/L)	62.3±1.06***b	72.6±2.08***a,c	68.19±0.19**a,**b,c	64.4±3.6***b,*c	59.3±0.3***b
Relative kidney weight	0.011±0.001	0.012±0.001	0.013±0.002	0.012±0.002	0.012±0.001
Note. The data were expressed as mean ± SE, (n= 10 animals/group). Significant differences (*0.01≤P <0.05, **0.001≤P<0.01, and ***P<0.001) were indicated between the individual group and the normal (a), EAC (b), and SAL-IFO (c) groups. CRT: creatinine; BUN: blood urea nitrogen; GLU: glucose; Ca+2: calcium; Mg+2: magnesium; Na+: sodium; Cl-: chloride; K+: potassium; UCO2 : urine bicarbonate; Up: phosphate

Antioxidant activity in kidney tissues

In terms of the percentage changes of the levels of GSH and GR in the kidney tissues relative to the normal group, shown in Fig. 2A-2B, EAC group exhibited a remarkable increase in GSH level (34%) with a slight decrease in GR activity (4%) while the administration of IFO into the mice resulted in significantly depleted GSH (50%) and GR (43%) activity levels. Interestingly, treating the mice with SAL-IFO had reduced this depletion as the level of GSH decreased to about 35% with the restoration of the GR enzyme level. The level of GSH in the SAL group was almost similar to the normal group whereas the GR enzyme level tended to increase.

Fig. 2

The oxidative stress analysis of the kidney tissues of (a) reduced glutathione (GSH), (b) glutathione reductase (GR), (c) lipid peroxidation (MDA) and (d) catalase enzyme for the experimental groups (n= 10 animals/group). The data were expressed as mean ± SE (error bars). Significant differences (***, P< 0.001) were indicated between the individual group and the normal (^a), EAC (^b), and SAL-IFO (^c) groups.

Regarding the levels of MDA in the kidney tissues, presented in Fig. 2C, they were significantly higher (~ 23%) in IFO treated group than those of the normal group while the combination formula, SAL-IFO, reversed this elevation in the mice to be comparable to the normal and EAC groups. Additionally, SAL-IFO treatment had restored the catalase activity which was comparable to the normal group, but it was considerably decreased in the EAC, IFO and SAL-treated groups by 25%, 33%, and 19%, respectively (Fig. 2D).

Histopathological changes in kidney tissues

The histological kidney structure of the normal mice, shown in Fig. 3A, exhibited normal Bowman's space surrounding the Bowman's capsule and the renal tubules including proximal tubule and distal tubule. Tissues of EAC tumor group revealed kidney damage including Bowman’s capsule rupture and distortion of the proximal tubule and distal tubule (Fig. 3B). In addition to the changes caused by the tumor, free IFO treatment had caused renal injury due to the presence of lymphocytic infiltration (Fig. 3C). In contrast to the tissues of EAC and IFO groups, the tissues of the mice treated with SAL exhibited moderate damage in Bowman's capsule and renal tubules (Fig. 3D). Interestingly, there were no signs of injury and the structure of the kidney tissue of the SAL-IFO group was ameliorated to be similar to the normal group (Fig. 3E).

Fig. 3

Photomicrograph of the histological structure of kidney for the experimental groups of (a) normal group, displaying normal Bowman's space (BS) surrounding the Bowman's capsule (BC), proximal tubule (PT), and distal tubule (DT). (b) EAC group, showing ruptured Bowman’s capsule (black arrow) with distorted feature of proximal tubule and distal tubule (red arrow), (c) IFO group, showing ruptured Bowman’s capsule (black arrow) with lymphocytic infiltration (orange arrow) and injury in proximal tubule and distal tubule (red arrow), (d) SAL group, showing moderate damage in proximal tubule and distal tubule (black arrow) with slight rupture of Bowman's capsule, and (e) SAL-IFO group, showing nearly the same histological structure as the normal group. H&E stain, 400 x magnifications, 20 µm.

Discussion

Alkhatib et al¹⁸ had previously formulated and evaluated the physical characterization and the antitumor activity of SAL and SAL-IFO in MCF-7 and HeLa cells. SAL and SAL-IFO exhibited negative zeta potentials of -7.9±0.11 mV and -1.14 ± 0.08 mV, respectively, while their z-average diameters were in the range of 52.12-56.18 nm and 56.64-64.62 nm, respectively. The polydispersity indexes of the nanodroplet sizes indicated that both of SAL and SAL-IFO were homogeneously distributed. According to the in vitro antineoplastic activity of SAL and SAL-IFO, the incorporation of IFO into SAL-NE had markedly ameliorated the efficacy of IFO. In the current study, the MST of the EAC cell-bearing mice treated with IFO at the dose 60 mg/kg body weight for three consecutive days was greater than SAL-IFO treated groups by two folds. Interestingly, SAL-IFO group exhibited a double higher survival percentage (60%) when compared to the free IFO group (30%). The antitumor activity of IFO is attributed to its ability to alkylate and produce DNA cross-links which cause damage to the DNA.²⁴ The potent effect of the combination of IFO with the SAL oil-based NE when compared to the free IFO and other tested groups can be due to the antiproliferative action, antimigratory and antiangiogenic effects of SAL.^25,26 Several studies on SAL plant had demonstrated the possible antitumor activity against numerous cancerous cell lines and cancer animal models.^27-33

Besides, drinking Sage tea, a natural substance made from the leaves of SAL officinalis, had been reported to prevent the onset of colon carcinogenesis phases.³⁴ Growing evidence had shown the ability of SAL to act as a mutagenesis inhibitor through the methanolic extract. It had been demonstrated that SAL had a protective effect against cyclophosphamide- IFO analog- induced genotoxicity in rats.³⁵ The cytotoxicity and anticancer activity of SAL stem from its constituents. In vitro and in vivo studies reported that the SAL components such as caryophyllene, α-humulene, manool, ursolic acid, and rosmarinic acid had the potential to inhibit tumor cells growth in different cell lines, prevent skin tumors formation, impede angiogenesis phases such as proliferation, migration, adhesion and tube formation.^{28,306,32,36,36-38}

One of the major side effects of IFO is nephrotoxicity that can be attributed to the formation of IFO CAA metabolite. As a consequence, glomerular toxicity may develop due to the increase in blood CRT concentration and the reduction of GFR. Additionally, Fanconi syndrome may arise as a result of proximal tubules toxicity which is characterized byaminoaciduria, glycosuria, phosphaturia, bicarbonaturia, and kaluria.⁴ Several in vivo studies reported the nephrotoxicity coupled with IFO.^39-42 In the present study, serum CRT, BUN, U_CO2, and U_P were elevated while serum GLU level had decreased in the IFO group. On the other hand, the combination therapy, SAL-IFO, had eliminated the adverse side effects caused by IFO according to the results of biochemical analysis and histological study of the kidney. Perhaps loading IFO in a NE containing SAL oil had improved the permeation of both IFO and SAL into the cancer cells.⁴³

It had been reported previously that the nephrotoxicity of IFO metabolites including CAA was attributed to the depletion of stored glutathione associated with significant increases of lipid peroxidation leading to the defect of the antioxidant defense system.^39-42,44 In the current study, glutathione depletion in the IFO treated group was detected by the reduction in GR and catalase activities and elevation in the MDA when compared to the normal group. Interestingly, loading IFO in NE-based SAL oil reversed the side effect of the free IFO on the renal antioxidant defense system. Recently, it had been confirmed by in vitro studies that the incorporation of the essential oils into NE had potentiated their inhibitory actions against the cancer cells growth.^14,15

Cells are protected against the overproduction of reactive oxygen species by natural antioxidants. Several studies reported that SAL possessed a powerful antioxidant activity which was explained by the SAL protective effect on the DNA through its antioxidant activity.^35,45 Furthermore, it had been found that rat hepatocytes resistance against oxidative stress was increased through drinking SAL water.⁴⁶ Many reports demonstrated that SAL constituents had powerful antioxidant compounds such as carnosol, rosmarinic acid and caffeic acid.^47-51

Conclusion

The incorporation of IFO into NEcontaining SAL oil had improved the efficacy of IFO and eliminated its nephrotoxicity. It is recommended to make further studies on the adverse effect of IFO-SAL on other organs.

Acknowledgment

The authors wish to express a sincere thanks and appreciation to King Abdulaziz City for Science and Technology for its financial support.

Funding source

This research project designated by a number (1-17-01-009-0068) was supported by King Abdulaziz City for Science and Technology.

Ethical statement

The animals were acclimatized as recommended by King Abdulaziz University's policy to a well-ventilated laboratory environment in polypropylene cages, at a temperature of 25 ± 1° C and facilitated with 12 hours light/dark cycles. They were nourished by standard pellet diet and fresh water before and during the experiment. The animals were kept and handled according to King Abdulaziz University's policy and the international ethical guidelines on the care and use of laboratory animals. The ethical approval was obtained from the Research Ethics Committee in the Faculty of Medicine at King Abdulaziz University (1-17-01-009-0068).

Competing interests

The authors report no conflicts of interest.

Authors’ contribution

MHA and SMA wrote the concept of the study and designed the experiments. SMA performed the experiments. MHA and SMA analyzed and interpreted the data. All authors drafted and revised the manuscript. The whole study was performed under the supervision of MHA and HMA.

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