The Effect Of Diabetic Rats' Skin On The Percutaneous Permeability And Absorption Of Different Dexamethasone Esters

Background and Objetive:Diabetes mellitus(DM) that threatens the human lives is a group of metabolic diseases characterized by high blood glucose levels, due to defects in insulin secretion, or action, or both.Startlingly, there are about 2,900,000 people dying of the Diabetes mellitus per year all over the world, which accounts for 5.2% of deaths in total ratio. And there are about 1,205,000 people joining the ranks of diabetes mellitus every year just in china, that is, about 3,000 people suffers diabetes every day. The complications of diabetes is the main reason leading to the death and physical disability of diabetic, hence the effective prevention and cure to various complications of diabetes is the important mean to improve the living quality and lengthen life of diabetic. It's necessary for diabetic to use glucocorticoids locally to ease symptoms in their symptomatic treatment of skin disease whose incidence rate is very high in all complications induced by diabetes..As antibiotic, the abuse of Glucocorticoids, a kind of hormone most widely used in clinical, has caught extensive attentions from the whole society. The step-up of blood glucose could be induced by Glucocorticoids, and to people with diabetes, the various complications would be aggravated, the toxic and side-effect would be increased and the treatment effectiveness would be deceased if the glucocorticoids were used inappropriately. There is a conclusion that people suffering from diabetes should be avoided using glucocorticoids theoretically. Nevertheless, a great many diabetics still use glucocorticoids frequently, especially the reasonable application of glucocorticoids because of the high incidence rate of dermatoses which is one of the various complications of diabetes. According to investigation statistics,25%-30% diabetics would suffer from dermatoses including Acanthosis Nigricans, Necrobiosis Lipoidica, scleredema, granuloma annulare, erythrasma, Necrotizing Fasciitis, and mucormycosis, so they usually need reasonable application of glucocorticoids to match the treatment in the symptomatic treatment of skin disease so as to relieve symptoms Therefore, the question about how to make the logical application of glucocorticoids more reasonably to diabetics was elicited from above-mentioned situation.On the other side, the previous study discovered that almost all the diabetics suffer from skin damage if only the microcirculation dysmetabolism and/or their affect to skin collagen protein being considered. From all above it is very natural to consider whether there is a difference of permeation and absorption between normal skin and diabetic skin when glucocorticoids transferred through them. Unfortunately, no study has been carried out in this field so far.Among the numerous existing artificial glucocorticoids, dexamethasone remains to be used most commonly, mainly throughout esterified forms such as acetate (dexamethasone acetate, DA)or phosphate (dexamethansone sodium phosphate, DSP) in the market. Therefore, what can be happened if dexamethasone esters are administrated via skin with its characteristics being changed due to the diabetes? The purpose of this article is provide some experimental datas for answering this question.Microdialysis is a unique sampling technique in monitoring the local drug concentration of the extracellular space in living tissue. Its biggest advantage is can comply in-vivo, real-time and on-line sampling in the body during normal course of life with no interference,In this study, we introduced improved Franz diffusion cells and Microdialysis to evaluate the influence of diabetes on the percataneous permeation and absorption of fat-soluble DA and water-soluble DSP in rats, evaluate the percutaneous transport rule of different fat-soluble cortical hormone when they transport via the skin of control or diabetes rat. In the field of diabetes research, this should be the first time to investigate the effect of diabetic skin on the drugs permeation and absorption.The fingdings was aimed to provide evidence for the clinical pharmacist consultation to give reasonable guidence of topical use of DA and DSP preparations to patients with diabetes, and also provide experimental reference for clinicians and nurses in taking care of diabetics when the patients topically use DA and DSP preparation.Moreover, in order to research the relevance between the oil/water partition coefficients and in vitro transdermal speed of fat-soluble dexamethasone acetate and water-soluble dexamethansone sodium phosphate, their oil/water partition coefficients in different pH environment were determined by reversed phase high performance liquid chromatography, and their in vitro transdermal speed were obtained via transdermal-absorption test with improved Franz diffusing cells. The findings was aimed to provide experimental data dexamethasone for the design of trans-dermal drug preparation of dexamethasone and using them more reasonably and scientifically in clinical.Method and ResultIn order to determine the concentrations of DA and DSP in different samples, the article established stable and reliable detection method for these two drugs. The HPLC conditions for DA were as follows:Mobile phase used was methanol:water (65:35; v/v), at flow rate of 1.0 ml/min, and the UV detector was operated at1=243nm. On the other side, the mobile phase used for DSP assay was comprised of potassium dihydrogen phosphate buffer (3.4%) with the pH adjusted to 3.0 using phosphoric acid/methanol (40:60, v/v), The flow rate was 1.0 ml/min and the detection was performed at 245 nm. In all HPLC studies, the instrument was operated at the temperature of 30℃and the volume of the inject samples maintains 20μl all the time. In the method of LC-MS/MS, 10μL of dialysis sample with DA was injected into an Agilent, ZORBAX SB-C18column with an isocratic mobile phase comprised of methanol-water containing 0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The column was maintained at 25℃. Ionization was achieved using electrospray in the positive and quantification was performed using multiple reaction monitoring (MRM) mode with a dwell time of 200 ms at m/z 435→415 for DA, The detector multiplier voltages were set to 400. Nitrogen was used as nebulizer gas and nebulizer pressure was set at 50 psi. Desolvation gas was heated to 350℃and delivered at a flow rate of 7L/min. The fragmentor energies of MS1 for DA was set at 110V. The optimized collision energies of 5eV were used.Our study employed DA Cream and DSP Cream made in our laboratory, blank matrix materials and preparative method of cream which is a transdermal drug delivery commonly used were selected according to the regulation of《New hospital preparations technology》. Examining methods of creams also came from the PPRC indicating that the drug content in cream made in our laboratory met the specification which specifies that the actual content should be 90%～110% of the indicator (DA cream:103.00%; DSP cream:103.17%).In order to evaluate the influence of diabetes on the percataneous permeability and absorption of drugs in rats, we need to establish a diabetes animal model. In our study, rats were chosen as experiment subject, DM rat model was prepared with an easy and highly applicable method of streptozocin (STZ, 55mg·㎏-1)single intraperitoneal injection. Animals were kept for 30 days before the experiments. During this period, fasting blood glucose levels were measured from the tail vein, using a ONE TOUCH glucometer to confirm diabetes. Animals having a blood glucose of>14mmol/L were considered diabetic; animals with less than 14mmol/L blood glucose were excluded from the study. Meanwhile, abdominal skins of control and DM rat made model more than 30 days were HE stained and made for tissue slices to find if any histological changes occurred with DM skin. The results indicated that the photomicrographs of control rat skin showed normal skin layers, when compared to the normal rats, the epidermal tissue of rats with diabetes induced by STZ more than one month was much thinner, the epidermic cell layer was less clear, the Stratified arrangement of epidemic cell almost disappeared and progressive atrophy was developed on the subcutaneous fat.To study the influence of diabetic skin's change on the transdermal character of different liposolubility dexamethasone, we introduced improved Franz diffusing cells and microdialysis for in vitro transdermal diffusion experiment and in vivo transdermal permeation experiment.In the in vitro transdermal diffusion experiment, abdominal skins whose hair had been removed of control and DM rats made model more than 30 days were mounted in improved Franz diffusion cells for the experiment. When DA and DSP cream were given respectively, the solution containing ethonal:saline(20:80) was put in the receiver chamber used as the receptor medium, The drug concentration in collected samples was determined by HPLC and then the transdermal permeably fluxs can be calculated. In order to determine the amount of DA or DSP in full-thickness skin, the rat skin was removed from the cell, then the drugs were extracted from skins and concentrations of drugs were determined by HPLC.The results showed that for DA, the cumulative amount (m) penetrated the skin on each time point in Group CTL and Group DM were almost the same, the transdermal permeably fluxs and the depositional amount at 24 h remained in the whole skin of two groups which were determined to be 0.7947±0.3630,0.7801±0.4480μg/h*cm-2 and 2.5451±1.4717, 2.5133±1.7766μg/g, respectively showed no significant deviation analyzed by independent-sample t test(t=0.113, P=0.911; t=0.062, P=0.951), and for DSP, Obvious differences could be seen in the release profiles of two groups(Group Control and Group DM) from results which showed that the cumulative amount of DSP penetrated the skin on each time point of Group DM were higher compared to the Group Control. Statistical analysis was applied to the mean flux values((μg/cm2/h)and the mean depositional amount at 24 h remained in the whole skin using independent-sample t test indicating that both of two parameters of Group DM (68.3435±15.1866μg/h*cm-2 and 83.0609±30.0689μg/g) were increased significantly compared to those of Group Control which were determined to be 42.6156±10.6145μg/h*cm-2 and 36.0671±13.3097μg/g (t=-6.210, P<0.001;t=-6.391,P<0.001).In the in vivo transdermal permeation experiment, microdialysis probe was planted into the rat abdominal dermis when the rat was anesthetized, subcutaneous dialysis probes were perfused with physiological saline for DA and DSP, at a constant flow-rate of 0.5μl/min. After approximately 2 h which was found to be optimal time period to minimize the needle-induced trauma for a postsurgical stabilization of the dialysate levels, DA or DSP Cream was administered transcutaneously via the abdominal skin of the rat where the linear probe was placed. Then a dialysate sample of 30μl was collected every 1h.12 samples were collected for every rat and assayed by LC-MS or HPLC directly. Then pharmacokinetic parameters (AUCall:Area Under concentration-versus-time Curve, Cmax:maximum concentration, tmax:time to maximum concentration) of DA and DSP in subcutaneous site of rats in Group CTL and Group DM were obtained by Phoenix WinNonlin 2.0. Statistical analysis indicated that there was no significant difference between Group CTL and Group DM for all the pharmacokinetic parameters of DA. Likewise, the tmax of DSP between the two groups were identical. In contrast, the subcutaneous AUCall values and the Cmax of DSP in the rats with diabetes were significantly increased compared to the control. These indicated that there surely are changes in the skin of diabetic rats and increase the topical drug absorption of DSP.This paper also research the relevance between the oil/water partition coefficients and in vitro transdermal speed of fat-soluble dexamethasone acetate and water-soluble dexamethansone sodium phosphate. In this study, oil/water partition coefficients of dexamethasone acetate and dexamethansone sodium phosphate in different pH environment were determined by reversed phase high performance liquid chromatography, their in vitro transdermal speed were obtained via transdermal-absorption test with improved Franz diffusing cells. Then the relevance of above-mentioned two index of dexamethasone acetate and dexamethansone sodium phosphate were examinationed. The results we obtained showed that in the fat-soluble and pH range of our test, the relationship between oil/water partition coefficients and in vitro transdermal speed of dexamethasone acetate and dexamethasone sodium phosphate followed a linear relationship, whose equation were Y=4.682-0.03X, r2=0.906 and Y= 7.923+3.393X, r2=0.939 respectively.Conclusion and discussionIn this study, a feasible diabetic rat modelling methods was established, and HPLC methods for DA and DSP, a highly selective LC-MS/MS method for DA were developed for the quantification, furthermore, we improved the methods of microdialysis experiments. The results of pathological section showing the changes of abdominal skin of DM rat might indirectly indicate that the skin of diabetic patients may have a similar pathological change.In conclusion, diabetic rat's skin significantly increased the percataneous permeation and absorption of DSP but had no effect on that of DA.Based on previous studies, it was well known that intact skin is composed of stratum corneum, epidermis, dermis and subcutaneous fats and has well-woven structures. The main approach of absorption for almost all drugs is penetrating into the stratum corneum cells through its cellular film and accessing to other layers through the epidermis. Thus we speculated that the amount of almost all drugs penetrating through the diabetic skin should be increased in consequence of the characteristic pathological changes of diabetic skin showed above. But the result we obtained was that the amount of DA penetrating through the normal and diabetic skin has no significant difference but the mount of DSP penetrating through the diabetic skin were increased significantly compared to that through the normal skin. As is well known, solubility and oil-water partition coefficient of drugs would directly affect the percutaneous absorption of drugs. Generally speaking, The ability to penetrate the skin is:oil-soluble drugs> water-soluble drugs, but actually, the drugs which can dissolve in both oil and water solution have higher penetrability. The drugs with highest penetrability has almost the same solubility in both oil and water solution, the high oil-soluble drugs may aggregate and be detained in corneum, so it is hard for them to be absorbed further. Thus, we considered that one of the reasons causing above results might be related to the different solubility and oil-water partition coefficient of DA and DSP. So this study also determined oil-water partition coefficient of two model drugs after getting relevant data of two drugs' transdermal behavior. The results indicated that the oil-water partition coefficient of DA and DSP were 391.3079±38.3824(n=5) and 0.7563±0.2606(n=5)respectively. the lipid solubility of DA was so great that DA may aggregate and be detained in corneum, so it is hard for it to be absorbed further and the changes of diabetic skin could not increase the amount of DA penetrating though the diabetic skin.In addition, as we know, the transfer of drugs contained in transdermal drug delivery systems through the skin should include two main processes:1) the diffusion of drugs from matrix to skin surface; 2) the permeation of drugs which has reached the skin surface through skin. So we speculated that there was another factor inducing above state, that is the limiting step of DA is the diffusion from matrix to skin surface but not the permeation through skin, by contrast, the limiting step of DSP is its permeation through skin. So the pathological changes in horny layer and cuticular layer of diabetes skin only had a significant effect on the permeability process of DSP but not DA. Therefore, we concluded that there would be a similar situation when patients with diabetes use DA and DSP Cream. Thus, these results indicated that it may be necessary for patients with diabetes to consider the dose of medication when they need to use DA, DSP or other glucocorticoids topically.In addition, by means of the discussion of the Relationship between the Oil/Water Partition Coefficient and Transdermal Action of dexamethasone acetate and dexamethansone sodium phosphate, we concluded that the oil/water partition coefficients of dexamethasone acetate increases along with the raise of its pH value, but its in vitro transdermal speed descend. On the other hand, when pH value increases, the oil/water partition coefficients and in vitro transdermal speed of dexamethasone sodium phosphate are all under a descent. Thus, we conclude that the pH value and oil/water partition coefficients of external preparation made with different liposolubility Dexamethasone may play a different role on the medicine curative effect. In this research, for the reasons of limited time, energy and other factors, there were still many deficiencies. For example, we can not concluded that whether the increase of the transdermal permeation of drug through DM skin is related to the drug's Oil/Water Partition Coefficient or its limiting step only by the data of DA and DSP, the conclusion need more study. In the in vivo experiment, the drug concentration in plasma did not been detected because of the low level of drugs and complicated enzyme metabolic system.etc. It remains need further studies. Also the calculation of recovery of probe needs a futher improvement. etc.. These disappintments hoped to be performed in the future research.