In vivo evaluation the efficiency of nitazoxanide with cationic Gemini surfactant on Cryptosporidiosis

: Infection with cryptosporidiosis endangers the lives of many people with immunodeficiency, especially HIV patients. Nitazoxanide is one of the main therapeutic drugs used to treat cryptosporidiosis. However, it is poorly soluble in water, which restricts its usefulness and efficacy in immunocompromised patients. Surfactants have an amphiphilic character which indicates their ability to improve the water solubility of the hydrophobic drugs. Our research concerns the synthesis of new cationic Gemini surfactants that have the ability to improve the solubility of the drug Nanazoxide. So, we synthesized cationic Gemini surfactants. N 1 ,N 1 ,N 3 ,N 3 -tetramethyl-N 1 ,N 3 -bis(2-octadecanamidoethyl)propane-1,3-diaminium bromide (CGSPS18) and 2,2 ‘ -(ethane-1,2-diylbis(oxy))bis(N-(2-octadecanamidoethyl)-N,N-dimethyl-2-oxoethane-1-aminium) dichloride (CGSES18) and the detection of their chemical composition by spectroscopic methods, as well as studying the properties of their surfaces and their toxicity. Furthermore, the efficacy of nitazoxanide in infected mice was studied in conjunction with three different doses of surfactants. To assess the effect of nitazoxanide and surfactants, the infection was parasitologically counted before and after treatment, and the intestinal, liver, and lung tissues were also examined histopathologically. In this study, it was found that the combination of the drug nitazoxanide with surfactants, especially the compound (CGSPS18) at a concentration of 25% increased the efficacy and resulted in a percentage reduction of 90.8%. Histopathological examination revealed that the group treated with the drug nitazoxanide in combination with CGSPS18 showed the best results exhibiting an almost normal villous pattern. This study demonstrated an increase in the effectiveness of nitazoxanide when combined with surfactants, and this suggests a promising future for the use of surfactants as an adjunct to enhance the effectiveness of nitazoxanide for the treatment of cryptosporidiosis in immunocompromised patients, particularly HIV patients.


Introduction:
Cryptosporidium spp. is one of the most common intestinal parasites. It was first detected in mice in 1907 but was not identified as a major cause of diarrheal disease in humans until 1976, whether in healthy children or immunocompromised adults. The infection can be life-threatening and has been detected in more than 70 countries. The pathogen can be transmitted by many routes, including water and food, but is the most common cause of waterborne disease. It is estimated to have a high prevalence in children under five years of age, low-income people, people with gastrointestinal symptoms, the elderly, and people with immunodeficiency, especially HIV patients [1][2][3] . Cryptosporidium parvum (C. parvum) is a coccidian protozoan that causes cryptosporidiosis. It is immune to all chlorination levels, and it is a pathogen that lives only inside cells. The parasite's development is mainly limited to the intestine; in immunocompromised hosts, the biliary system, pancreas, and lungs may also be affected 4,5 . There is also evidence that cryptosporidiosis is associated with the occurrence of cancer 6 .
To date, there is no fully effective therapy for Cryptosporidium spp. treatment options are minimal, despite the overall burden of cryptosporidiosis. Nitazoxanide is the only drug approved by the U.S. Food and Drug Administration, and it is not effective in patients with immunodeficiency and not approved in infants younger than 1 year 7 . It acts by inhibiting pyruvate ferredoxin oxidoreductase (PFOR), thereby impeding anaerobic energy transfer reactions 8 . As nitazoxanide is used to treat cryptosporidiosis in children, its efficacy is limited in malnourished people and compromised immune systems, this highlights the significance of research towards medication development for the treatment of cryptosporidiosis. Nitazoxanide, a high-dose waterinsoluble antiprotozoal drug, was developed with the aim of modulating gastro-retentive dosage forms using super porous hydrogel composites, which resulted in improved stability, drug content, and drug release. Furthermore, loading of chitosan nanoparticles with nitazoxanide increased their efficacy in both infected immunocompetent and immunosuppressed mice, resulting in a decrease in the shedding of Cryptosporidium oocyst and an improvement in the histopathological changes caused by the infection in the mice's liver, intestine, and lungs 9,10 .
Surfactants were used as one of the components to prepare oil-in-water nanoemulsion, which improves the solubility of nitazoxanide and medical bioavailability, increases the therapeutic effect of the drug, reduces the dosage of the drug and increases the activity of resistance to parasites, worms, bacteria, etc., and is used to treat diseases caused by protozoa in humans and livestock, besides, it is safe to use and easy to prepare 11 . In another study, the dissolution enhancement of nitazoxanide was studied and compared with its two derivatives nitazoxanide-glutaric acid and nitazoxanide-succinic acid in the presence of two types of cellulosic polymers 12 .
Surfactants have an amphiphilic character, containing both hydrophobic and hydrophilic portions. Often, aggregate at interfaces and are associated in solutions (water or oil phases) to attempt to isolate their different portions from the phase interaction. There are clearly many areas where applied chemistry can help in the future development of drug encapsulation by surfactants 13 . in an early study, two types of surfactants were used as antiprotozoal agents and showed antiprotozoal properties in sheep rumen 14 . Various drug delivery mechanisms and drug targeting have been studied with the aim of reducing drug degradation and losses, preventing adverse effects, and improving drug bioavailability 15 . One of the most classical techniques to improve the water solubility of hydrophobic drugs is solubilization in surfactant micelles, which is still employed in the pharmaceutical industry. [15][16][17] .
Cryptosporidium spp. poses a threat to the lives of many people around the world. Therefore, we focused our attention on the most common drug for its treatment, namely nitazoxanide, but it is poorly soluble in water, which affects its efficacy. Our study aimed to prepare a cationic Gemini surfactant with high efficiency and minimal toxicity. A comparison of the efficacy of nitazoxanide on cryptosporidiosis alone, and in combination with surfactants at different concentrations was performed in C.parvum infected mice after induction of immunosuppression.
Parasitological and histopathological examinations were performed to evaluate the effect of the drugs and surfactants.

Experimental:
Synthesis of cationic Gemini surfactant Synthesis of (CGSES18) It was prepared according to the approach described in Bin-Hudayb et al. 18 . (CGSPS18) was synthesized in two steps as follows preparation of N- (2-(dimethylamino)

ethyl) octadecanamide (DAEO)
At 35-40 o C, add dropwise octadecanoyl chloride 2.51 g, 0.0083 mol to a mixture of N,Ndimethyl ethylenediamine 2.42 g, 0.0275mol in 15ml dry ether. The reaction was agitated for 2 hours, and at the end of this period, the solvent had been evaporated under reduced pressure then filtered off as a precipitate and recrystallized with pure ethanol after washing in water. The final product was vacuum-dried 19 .
preparation of (CGSPS18) N-(2-(dimethylamino) ethyl) octadecanamide 7.09 g, 0.02 mol and 1,3-dibromopropane (PS) 2.02 g, 0.01 mol were placed in ethyl acetate (as solvent) in a 100 ml quick-fit flask equipped with a condenser. The flask was magnetic stirred under reflux for 24 hours. Once the solvent has been removed by evaporation under reduced pressure, the final product was dried under vacuum after recrystallization with petroleum ether see schema in

Preparation of oocyst
The molecular laboratory of Theodor Bilharz Research Institute provided the Cryptosporidium parvum oocysts. By utilizing the appropriate primers in a nested PCR reaction, this strain's molecular identity was established from the start of the cycle, and the PCR result was subsequently sequenced 21 . Samples were first washed by 10 ml of N-saline centrifuged for 10 minutes at 3000 rpm, the supernatant was removed, and this process was done twice. The sediment was then re-suspended with Shether's solution and allowed to stand for 10 minutes. Next, collection of the supernatant, and it was saline-washed twice. The remaining oocysts in the deposit were counted using a hemocytometer to calculate the dose needed for infection. The dosage of infection was 10 3 C. parvum oocysts 22 .

Animals Animal source and handling
Fifty-four white albino female mice of the CDI strain were healthy, lab-bred, and between 6 and 8 weeks old, weighing between 25 and 30 grams (all obtained from Theodor Bilharz Research Institute animal house). The experiment was carried out in a plastic cage with clean Sawdust for bedding and a decent ventilation system at the biological section of Theodor Bilharz Research Institute. Mice were housed under standard environmental factors (12/12 h light/dark cycle), with a temperature of 24°C and a relative humidity of 50%. They were fed with a commercial diet and water and protected from direct sunlight to maintain optimum sanitation. All mice received Praziquantel 600mg at a single dose of 40 mg/kg and were allowed to rest for 7 days before the start of the experiment to ensure that the mice were free from the parasitic infection 23 .

Groups of Animals
This study was carried out on fifty-four healthy, (lab-bred) white albino female mice, which were divided into nine groups, with six immunocompromised mice in each group. Mice in groups II to XV were infected with C. parvum oocysts at a dose of 10 3 oocysts/0.2ml/mouse orally via a nasogastric feeding tube. Group. I: non-infected and non-treated (negative control). Group. II: infected and non-treated (positive control). Group. III: infected and treated with nitazoxanide in a dose of 200 mg/kg daily for 7 successive days after infection 10 . Group. IV: infected and treated with CGSES18 in a concentration of 25% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it for 3 times\week for 7 successive days post infection. Group. V: infected and treated with CGSES18 in a concentration of 50% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it for 3 times\week for 7 successive days post infection. Group. VI: infected and treated with CGSES18, in a concentration of 100% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it for 3 times\week for 7 successive days post infection. Group. VII: infected and treated with CGSPS18 in a concentration of 25% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it 3 times\week for 7 successive days post infection. Group. VIII: infected and treated with CGSPS18 in a concentration of 50% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it for 3 times\week for 7 successive days post infection. Group. IX: infected and treated with CGSPS18 in a concentration of 100% at a half dose of 100 mg\ml 24 with nitazoxanide half the dose from it for 3 times\week for 7 successive days post infection. Doses were computed based on the table of (Barnes & Paget) 25 .

Immunosuppression
Dexamethasone sodium phosphate (Dexazone), 0.25 ug/g/day, was supplied through nasogastric feeding tube to suppress the immune system. This dosage was given every day for two weeks before receiving an oral injection with Cryptosporidium spp. oocysts. All mice groups received a weekly maintenance dosage of dexazone throughout experiment 26,27 .

Infection
Using a nasogastric feeding tube, mice were orally infected with C. parvum oocysts at a dosage of 10 3 oocysts/0.2 ml/mouse 22 . After conducting a pilot investigation, it was found that 10 2 oocysts/0.2 ml/mouse was insufficient to produce infection whereas 10 4 oocysts/0.2 ml/mouse resulted in the death of the mice.

Drugs and therapeutic doses
Medications were given orally, via a nasogastric feeding tube.
 Nitazoxanide: was given in a dose of 200 mg/kg daily for 7 successive days after infection 10 .  Surfactant CGSES18 & CGSPS18: was given in a dose of 100 mg\ml 3 times\week for 7 successive days post infection 24 .

Experimental evaluation of treatment
Parasitological examination Stool samples were collected from each group 7 days after infection. The therapeutic impact of the medications was then assessed using stool samples obtained 7 days after therapy. After being stained with the modified Ziehl-Neelsen (MZN) stain, samples were examined for parasites 28 . We used a microscope to count the C.parvum oocysts in each sample. The percentage (%) reductions in oocysts were calculated using the following Eq.1: 1 %R: % reductions, C: control group and E: experimental groups of mice 29 .

Histopathological examination
All mice were scarificed 10 days after treatment. The ileocecal region, lungs, and liver were located and fixed in 10% buffered formalin solution and processed into paraffin blocks. Sections 4-5 um thick were cut using a rotatory microtome (Leica, Germany) in the pathology department of TBRI. Routine staining was done using Hematoxylin and Eosin(H&E) for demonstrating the different pathological changes using a light microscope (Zeiss, Axio) 30 , with an attached digital camera Micr5 for taking microphotographs for interesting findings.

Statistical analysis
Data were analyzed using paired sample t-test and independent sample t-test by using predictive analytic software IBM SPSS Statistics (Version 25), P value is considered significant if < 0.05 31 .

Results and discussion: Chemistry
Two types of cationic Gemini surfactants were prepared. The first contains an ester spacer (CGSES18) and the second a propane spacer (CGSPS18). They were prepared by acylation of N,N-dimethyl ethylenediamine with octadecanoyl chloride in dry ether, leading to the production of (DAEO) as intermediates. These were agitated separately with ethane-1,2-diylbis(2chloroethanoate) or 1,3-dibromopropane, respectively, to synthesize the required surfactants after 24 h.
Construction explanation: Fourier-transform infrared (FTIR), nuclear magnetic resonance (NMR), and mass spectroscopy (MS) were used to determine the structures of all the cationic Gemini surfactants (CGSES18) and (CGSPS18) prepared. The (FTIR) spectra of the (DAEO) substance indicated a strong absorption band at 1624-1644 cm -1 , typical of carbonyl amide, and a strong signal in the region of 3002-3056 cm -1 , which is characteristic of NH stretching. There was a significant peak at 1761 cm -1 corresponding to the absorption of carbonyl of the ester group for CGSES18, in addition to the distinctive intermediate peaks (DAEO) as shown in Table 1  According to the 1 H-NMR data for DAEO, CGSES18, and CGSPS18 in Table 2, Figs. 5,6,7. The chemical shifts for protons (CH3CH2) of the lipophilic tail of the cationic Gemini surfactants CGSES18 and CGSPS18 were 0.88, 0.89, and 1.24, 1.26 ppm, respectively. At δ (2.21 -2.88) ppm, there was a methylene proton peak before the carbonyl (C=O) group, that belongs to the fatty chain. At δ 3.55, 2.68 ppm a singlet of (CH3)2 protons directly linked to the positively charged quaternary nitrogen [N + (CH3)2] was detected. The methylene protons between the two nitrogen atoms, i.e., N-CH2 -CH2-N + , are responsible for the peaks at 3.94, 2.93, and 3.68, 2.90 ppm. At δ 4.51 and 2.32 ppm for CGSES18 and CGSPS18, respectively, typical resonance protons of the methylene group (-CH2-) were found to be directly linked to the quaternary nitrogen that has a positive charge, which is a part of the spacer. CGSES18 has a methylene proton peak next to the ester group, which is part of the spacer, at δ 5.14 ppm, and CGSPS18 has a methylene proton signal next to N + -CH2 -CH2 at 3.35 ppm. In contrast to CGSPS18, which had a singlet at 7.72 ppm, the resonance of a proton bonded to nitrogen in CGSES18 is displaced to the up field and is impacted by the ester group of the spacer, that was a singlet at 8.48 ppm.    Fig. 9. In mass spectroscopy, the chemical structure of all evaluated contemporary cationic Gemini surfactants was validated using mass spectral data. The calculated m/z values for the molecular weight of cationic Gemini surfactant 924.27 and 911.14 were in perfect agreement with the practical values for (924, 911) for CGSES18 and CGSPS18, as shown in Table 3 Figure 11. Mass spectrum for CGSPS18

Surface tension measurements Critical Micelle Concentration ( Ccmc ) and Effectiveness ( πcmc )
The Ccmc value was calculated by plotting the surface tension (γ) against (LnC) as shown in Fig. 12. It was determined using the breakpoints of the graphs presented in Table 4. It was observed that with increasing surfactant concentration up to Ccmc, a significant linear decrease in surface tension was observed for CGSES18 and CGSPS18, with a greater decrease in surface tension for CGSES18 than CGSPS18. Thus, the effectiveness (πcmc) at Ccmc is higher for CGSES18 than for CGSPS18 and is estimated using the following Eq.2 32-34 . π cmc = γ o − γ cmc 2 Where γo is the surface tension of pure water and γcmc are the surface tension at Ccmc.

Ln(concentration) in double-distilled water at 25 o C Surface Excess (Γmax) and Minimum Surface Area (Amin)
The efficiency of adsorption at the interface is called the maximum surface excess concentration Γmax. And the minimum surface area occupied by any molecule adsorbed at the air-solution interface is called Amin. Γmax and Amin were calculated using the Gibbs adsorption Eqs. 3 and 4, which is as follows [35][36][37] : Plotting γ versus lnC, the slope refers to dγ/dlnC. In comparison to the bulk solution, the surfactant concentration near the surface was always greater because the surfactants in the solution prefer the air/water interface over the bulk solution 38 .  39 .
We found that the various spacers have little effect on the calculated Amin values. There is a significant difference in the calculated Amin values between CGSPS 18 and CGSES18, due to CGSPS18 with propane spacer attached to the quaternary ammonium group has a greater Amin value than that of similar ester functionalized Gemini surfactants. The polar character of the spacer in CGSES18, which has a stronger affinity for the aqueous phase, is to blame for this behavior, that makes CGSPS18 stretch at the air-water interface. As shown in Fig. 13 40,41 . Surfactant molecules dissolve in water at a concentration greater than the critical micelle concentration (CMC), forming micelles, while the hydrophilic heads remain on the micelle's outer surface to increase water interaction, the hydrophobic tails congregate at its center to reduce water contact 42 .The aqueous phase penetrates the micelle beyond the hydrophilic head groups, with the hydration sphere consisting of the first few methylene groups close to the head as the hydration sphere 15,43 . As a result, micelles can improve the solubility of sparingly soluble compounds in water, which is particularly useful in the pharmaceutical industry. Solubilization is the reversible interaction between a substance's micelles and a surfactant to cause a material to dissolve spontaneously in water 42 . Since the drug nitazoxanide has an intermediate solubility, it should be placed in the middle of the micelle, e.g., between the CGSES18 and CGSPS18 micelles' hydrophilic head groups 44 . The solubilization of this drug could be mostly owing to adsorption at the micelle-water interface rather than incorporation into the micelle interior 45 .

Evaluation of cytotoxicity against WI-38 cell line
Inhibitory activity against normal human lung fibroblast cells was observed for CGSES18 under these experimental conditions with CC50 = 21.33 ± 1.08 µg/ml as shown in

Parasitological examination
Cryptosporidiosis is a parasite that causes diarrheal diseases that affect the majority of people worldwide 48 . In both developed and developing countries, it is considered a serious problem, especially for immunocompromised patients 49 , particularly children under five years of age, the elderly, and people with immunosuppression, especially HIV/AIDS patients 2,50 . In addition to the numerous side effects of available drugs, and the weakness of their efficacy. Our study addressed the drug nitazoxanide as one of the most important drugs for the treatment of Cryptosporidium spp. 7 , and its weak efficacy is due to its poor solubility in water, resulting in low bioavailability and delivery problems 9,51 . Therefore, cationic Gemini surfactants with a solubilizing effect have been developed for poorly soluble drugs 52 . The effects of CGSES18 and CGSPS18 at different concentrations on Cryptosporidium spp. in combination with nitazoxanide were investigated. The results were compared with the untreated positive control group and the group taking nitazoxanide alone. The parasitological studies revealed that there were highly significant statistical differences between the untreated positive control and all groups, regardless of whether they had taken nitazoxanide alone or in combination with surfactant, they also show highly significant statistical differences between the group that took nitazoxanide alone and the groups that took nitazoxanide with surfactant at all different concentrations (P-value < 0.05), as shown in Tables  5,6.  It was observed that the lowest number of oocyst excretion 143.50± 3.450 with a percentage reduction of 90.8% was observed in group VII treated with CGSPS18 at 25% concentration in combination with nitazoxanide. Followed by group IX with a percentage reduction of 89.8% treated with (CGSPS18) at a concentration of 100% in combination with nitazoxanide. Followed by group VIII with a percentage reduction of 89.1% treated with (CGSPS18) at a concentration of 50% in combination with nitazoxanide, and then group III with a percentage reduction of 88.1% treated with nitazoxanide as shown in Fig. 16. The parasitological study showed that the combination of surfactants with nitazoxanide reduced the excretion of oocysts, especially in CGSPS18. This is due to the ability of cationic Gemini surfactants to solubility enhancement 53 . Our results went in agreement with Gaggero et al. 54 who use surfactants to enhance in vitro dissolution of poorly soluble drugs like praziquantel which use for the treatment of schistosomiasis. Also shown with Kolenyak-Santos et al. 55 .

Histopathology assessment
Our work was the first to evaluate the combined therapeutic effect of cationic Gemini surfactant with nitazoxanide on cryptosporidiosis.

Examination of intestine
Histopathological examination of the ileocecal region of the mice in the different groups showed various degrees of pathological and neoplastic lesions among them. These findings were in accordance with Abdelhamed et al. 56 , and Fahmy et al. 57 53 , has the ability to enhance solubility. Group III treated with nitazoxanide alone showed revealed conventional histopathological features with normal villous architecture, absent or mild inflammation and normal mucin secreting pattern as shown in Fig. 17 c.

Examination of liver sections
Histopathological examination of liver sections from different groups was concerned mainly with changes in hepatic architecture, hepatocytic changes, liver fibrosis, and inflammatory changes. Additionally, dysplastic liver changes following C. parvum infection has been observed in SCID mice 60 . The histopathological findings were mildly variable between different groups. Hepatocytic cloudy swellings and hydropic changes were minimal in negative and positive control mice as shown in Fig.  17-e,f respectively. It was most apparent in group IX treated with the nitazoxanide drug in combination with CGSPS18 at a concentration of 100% as shown in Fig. 17-g and mild in all other groups as in group VI treated with the nitazoxanide drug in combination with CGSES18 at a concentration of 100% as shown in Fig. 17

Examination of lung sections
Histopathological examination of lung sections from the different groups was concerned mainly with changes in the pulmonary vasculature and alveolar patterns as well as inflammatory changes. The histopathological findings were mildly variable between different groups like in negative group (I), positive group (II), a group treated with CGSES18 at a concentration of 50% in combination with nitazoxanide (V) and group treated with CGSPS18 at a concentration of 50% in combination with nitazoxanide (VIII) as shown in Fig. 17-i, j, k, l respectively. These findings were in agreement with Moawad et al. 10 .

Conclusion:
Two types of cationic Gemini surfactants were utilized in this study, one with an ester spacer (CGSES18) and the other with a propane spacer (CGSPS18). The efficacy of the drug nitazoxanide in this investigation was enhanced when it was combined with surfactants, particularly with the surfactant CGSPS18 at a concentration of 25%, which gave a percentage reduction of 90.8%.
Histopathological examination showed that the group treated with the drug nitazoxanide in combination with CGSPS18 had the greatest efficacy and showed an almost normal villous pattern at all drug concentrations. This paves the way for the use of surfactants in the treatment of cryptosporidiosis with nitazoxanide to increase efficacy, as surfactants increase water solubility.