The Pharmacokinetics and Skin Changes in Rabbit on Selamectin Solution

Background: It found that many antiparasitic drugs have a good treatment effect on the pets skin diseases. whether selamectin solutions have a bad effect on skin tissues needs more research. Objective: In order to investigate the effect of selamectin solution on skin changes in rabbit. Method: the skin of rabbit was given different doses of selamectin solution, then the quantity of per unit area of hair follicles and sebaceous glands and the morphology structure of them and the changes of skin thickness, epidermis thickness and dermis thickness were researched through routine paraffin sections and H. E, Evg, Masson’s, Pas, Toluidine blue staining and Image Pro Plus6.0. The concentration of blood and urine were measured by High Performance Liquid Chromatography after administration. Results: The thickness of skin, per unit of hair follicles and sebaceous glands all had an obviously increasing trend with the increasing of doses. 1 day later, the blood and urine concentration of the selamectin and the metabolite avermectin all were in highest. Many selamectin solution were excluded in the form of prototypes and many were in avermectin. There were a higher blood concentration and metabolite avermectin until 72h. Conclusion: The skin appearance changes were insignificant while the skin thickness and accessory organs increased obviously in microscope after given more selamectin. In 24h, the blood and urine concentration all were in highest. This study, which discussed the influences by selamectin solution in rabbit skin and the pharmacokinetics, was to provide the basis for the deeper study of Antiparasitic drugs effects on pet skin diseases.


Introduction
Selamectin which belongs to macrolides antibiotics is a good insecticide in pet parasites inside and outside. M. Honda el have made a research that selamectin solution was a suitable and economical treatment for guinea pigs infected with the mites in doses of 5.0~7.5mg/head administration [1]. James W el found selamectin solution can be absorbed transdermally through skin and be immediately eliminated in rabbits while the long-term safety and repeated applications must be certified [2]. Jennifer J. D'Agostino el foud selamectin can be absorbed obviously in bullfrogs skin, single doses administration had no significant histologic effects even if in high concentrations [3]. Sang-Hun Kim founed at a dose of 12 mg/kg-1 is effective and convenient for controlling cheyletiellosis in rabbits and may be a practical and well-tolerated treatmentoption in this species [4]. And Arif Kurtdede's study was similar in rabbits in P. cuniculi and S. scabiei [5]. Selamectin also show a good effect on mite infection in masked palm civet and in additional endo-and ectoparasites especially in heartworm disease in cats [6,7]. In all, Selamectin can exert an ideal deworming effects through the skin administration.
The skin in rabbit consists of epidermis, dermis and subcutaneous tissue, hair follicles, sebaceous glands, sweat glands, etc. The skin are often sufferred from infectious diseases, such as bacterium which contains Staphylococcus Necrobacillosis, fungus like dermatophytosis and microsporum canis [8][9][10][11]. Temporary parasites contains Sarcoptidae, Psoroptidae, flea etc which can affect the rest and feeding then causing lowly development and no economic value [12]. Up to now, there is no report on the effect of selamectin solution on rabbit skin tissue structure. This study was to measure the skin thickness of the site in administration and the opposite, to research the changes in hair follicles and sebaceous glands and to measure the blood and urine concentration and metabolite. It could provide a reference for transdermal administration in pets, which summarize the effect of selamectin solution in rabbit skin.

Sample Collection
There were four doses administration groups in this study respectively 6mg/kg, 18mg/kg, 60mg/kg and 120mg/kg. Rabbits and Drugs were bought in Yi Bin Xiehe Yijia animal hospital. Plasma was obtained by centrifuging blood and blood was gathered through heart after administration in 120mg/kg group in 1h, 2h, 6h, 12h, 24h, 48h, 72h. The urines were collected by diapers in 2h, 6h, 12h, 24h, 48h, 72h. In 72h, the rabbits were put to death, then taking skin samples immediately in longissimus site and the opposite.

Samples Preparation
Skin samples were fixed in 4% paraformaldehyde solution, then washed by running water 30min and embedded in paraffin, later making the 5µm thickness continue slices and staining. Plasma samples were collected by heart blood, then stored in -80°C refrigerator after centrifugating. Urine was gathered by diapers, then dissolved in acetonitrile and also stored in -80°C refrigerator.

Data Measurement and Statistics
Per unit of hair follicles and sebaceous glands were calculated and the skin thickness, epidermis thickness and dermis thickness were measured and the average value were counted by Image Pro Plus 6.0 after observing the tissue samples through Microscopic imaging system. Skin average thickness increasing by doses=skin average thickness in high doses-skin average thickness in low doses. Amount of hair follicles and sebaceous glands increasing=amount of hair follicles and sebaceous glands in high doses-amount of hair follicles and sebaceous glands in low doses. The greater changes were more significant in differential values. Everytime samples concentrations were calculated by concentration of reference substance, Peak area of reference substance and Samples peak area all were ensured by the peak time through High Performance Liquid Chromatography.

Skin Thickness Changes in Administration in Rabbits
With the increasing administration, the skin thickness, epidermis thickness, dermis thickness shows a significantly addition trend. The skin thickness ranged from 518 µm~1279 µm, 964 µm average. The epidermis thickness ranged from 9.249µm~29.352 µm, 16.892 µm average, and the dermis were 501.6µm ~1202.7 µm, 926.5 µm. The addition trend was more obvious because of increasing doses. The thickness increasing degree were mainly due to the dermis changes. (Figure 1).

Skin Thickness Changes in Opposite Side in Rabbits
The skin thickness, epidermis thickness, dermis thickness all showed an addition trend in the opposite side. The skin thickness ranged from 495.4 µm~903.83 µm, 707.04 µm average. The epidermis thickness ranged from 5.385 µm~12.305 µm, 8.380 µm average, and the dermis were 467.235 µm~878.407 µm, 664.111 µm. The addition trend was relatively insignificant and the thickness increasing degree was also mainly due to the dermis changes. (Figure 1)

Hair folLicles Changes
The number of hair follicles showed a significantly addition trend in determined area with the increasing doses. Per unit of hair follicles ranged from 54.360 /mm2~140.441 /mm2, 105.358 average, in 60mg/kg, the trend was espeacilly obvious. While the opposite sides were not apparently it ranged from 41.515 /mm 2~6 4.525 /mm 2 , 53.034 average. The number of hair follicles in administration site increased significantly while the opposite were in normal. (Table 1)

Sebaceous Glands Changes
The number of sebaceous glands showed an apparently addition trend in determined area and the opposite side with the increasing doses. Per unit of sebaceous glands ranged from 0.406 /mm2~2.349 /mm2, 1.428 average, the opposite ranged from 0.365 /mm2~2.349/mm2, 1.232 average. The number of sebaceous glands both in administration site and the opposite increased significantly. (Table 1)

The Changes in Skin Thickness and Tissue Structure
In all, the administration site skin shows a greatly increasing trend in skin thickness, dermis thickness, per unit of hair follicles and sebaceous glands through in high doses. Compared with the administration site, the opposite corresponding site trend was relatively weakened in these fields. The structure of skin tissues, hair follicles and sebaceous glands show no obvious changes through administration.

Drug Concentration in Blood and Urine
Selamectin was absorbed in blood through skin by skin administration and entered the whole body in blood circulation system. The selamectin and avermectin in blood were tested after administration, in 24h, both of them were in highest concentration, respectively 12.22µg/mL and 9.64µg/mL. The selamectin and metabolite avermectin in urine which were collected and tested were in growing trend after administration in 2h. Selamectin solution can be absorbed in blood and distributed quickly in whole body which retains a high blood concentration within a certain time frame. Drugs were excreted many in prototype selamectin and many in metabolite avermectin through urine. The concentration in blood and urine were in figure 3.

Discussion
This study suggests that regular concentration selamectin solution can cause clearly thickening in animal skin and the number of hair follicles and sebaceous glands obviously increasing, so the barrier function of the skin can be significantly enhanced. These results are the same with these drugs on skin function such as H. Hernandez's study on the function of pentyl-rhamnoside in skin's keratinocyte, J. Idkowiak 's roport on the starfruit (Averrhoa carambola) leaf extract and M. Hou's study on topical hesperidin which was from the Orange peel [13][14][15]. Some researches on many durgs were harmful to the skin barrier, like the Glucocorticoids, EGFR regulation and Histamine suppresses [16][17][18]. These activated substances had a special mechanism of action which were to repair the barrier function of the skin. The treatment on skin parasitic diseases is to cause rapid death of the worm to achieve the effects of broad spectrum. While in higher concentration and repeated dosing, especially in 80% and 100%, which could cause the morphological changes, acute inflammatory and a large scale occurrence of the autophagic cell death process [19][20][21][22].
Selamectin solution was widely used in pet, such as New Zealand White rabbits, bullfrogs, dogs, birds like helmeted guineafowl. Among them the dogs, cats and New Zealand Whit rabbits were in highest concentration which were respectively in 15h, 72h and 24h [2,3,[23][24][25]. The results are similar to the New Zealand White rabbits, in 24h. the concentration was in highest and kept a high blood concentration in longer time. The selamectin solution was also applied to exotic pet, like Golden Hamsters, helmeted guineafowl and bullfrogs [2,3,26]. Up to now, there were no reports on acute toxicity and adverse reactions with the addition on dosaged. So as an antiparasitic drug, it could expand its using scope in the future.
In summary, the regular concentration selamectin solution was an ideal insecticide in some parasites, like scabies mite and itch mite. There was no obviously toxic reation through the skin administration of the longissimus dorsi muscle in pets. The normal tissue structure of skin would not be damaged after administration in these sites. After given selamectin solution, the mechanism in distribution of organs and tissues and the skin repair induced need more further study.

Conclusion
The skin appearance changes were insignificant while the skin thickness and accessory organs increasing obviously in microscope after given more selamectin. in 24h, the concentration was in maximum and kept an effective blood concentration all the time. the blood and urine concentration all were in highest and kept a higher concentration until 72h.

Funding
This research was funded by the pharmacokinetics and skin changes in rabbit on selamectin solution (ybzysc18-31).