Transdermal delivery of insulin and Syzygium aromaticum-derived oleanolic acid by dermal patches in streptozotocin-induced diabetic rats : effects on some selected metabolic parameters.
Hadebe, Silindile Innocentia.
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The tight glycaemic control required to attenuate chronic complications in type 1 diabetes mellitus often requires numerous daily injections of bolus insulin. Typically insulin is administered by subcutaneous needle injection, insulin pen and catheters connected to insulin pumps. The routine multiple sc injections of insulin cause discomfort resulting in noncompliance, a major factor negating the quality of life of diabetic patients. Studies suggest that the bolus insulin injections are associated with hyperinsulinaemia, insulin resistance, glucose intolerance, weight gain and accelerated development of cardiovascular complications. These challenges of needle phobia and stress have encouraged investigations of possible administration routes of insulin delivery such as oral, nasal, buccal, pulmonary, rectal, ocular and transdermal systems. The skin has increasingly become a route of the delivery of drugs with a range of compounds generating a great deal of interest in this area of research. Studies in our laboratory are concerned with developing optional insulin delivery routes based on amidated pectin hydrogel matrix gel. Investigations described in this thesis were mainly designed to establish whether pectin insulin (PI)-containing dermal patches of different insulin concentrations sustain controlled release of insulin into the bloodstream of streptozotocin (STZ)-induced diabetic rats with concomitant alleviation of diabetic symptoms in target tissues, most importantly, muscle and liver. This study also focused on the hypoglycaemic effects of oleanolic acid (OA) which has been shown to significantly reduce blood glucose concentrations in both non-diabetic and diabetic rats when administered orally. OA does not dissolve easily in water hence we assessed the hypoglycaemic effects of OA via the transdermal route. Materials and methods Oral glucose test (OGT) responses to application of dermal patches containing different insulin concentrations were evaluated in separate groups of STZ-induced diabetic rats according to the method described previously by Musabayane et al., with slight modifications (Musabayane et al., 2007). Similarly, OGT responses to application of dermal patches containing different OA concentrations were also evaluated. Groups of STZ-induced diabetic rats were fasted overnight (18 h), followed by measuring blood glucose (time 0). The animals were given a glucose load of 0.86 g/kg and then the patches were applied on the shaved skin on the dorsal region of the animals. OGT responses to PI dermal matrix patches (2.47, 3.99, 9.57 and 16.80 μg/kg) prepared by dissolving pectin/insulin in deionised water and solidified with CaCl2 were monitored. Likewise, OGT responses to OA-containing dermal matrix patches (21, 42 and 84 mg/kg) were also monitored. Short-term (5 weeks) metabolic effects were evaluated in separate groups of non-diabetic and STZ-induced diabetic rats housed individually in Makrolon polycarbonate metabolic cages (Tecniplats, Labotec, South Africa) and were allowed water ad libitum and daily given 30 g standard rat chow (Meadow Feeds, Pietermaritzburg, South Africa). These animals were treated thrice daily with dermal matrix patches 8 hours apart. Rats treated with drug-free pectin and insulin (175 μg/kg, sc.) acted as negative and positive controls, respectively. Blood, liver, gastrocnemius muscle, pancreas and skin were collected for measurements of selected biochemical parameters after the experimental period. Plasma insulin concentrations were measured from blood samples collected after 6 hours (acute) and after 5 weeks (chronic) of treatment. Results Neither inflammation nor necrosis was detected in the skin of the rats after five weeks of daily treatment with PI-containing dermal patches. The density of phosphorylated IRS in skin tissues determined by immunohistochemical staining showed widespread localisation of IRS in cell bodies of the dermis, collagen and subcutaneous layer following treatment with PI-containing dermal patches. OGT responses and the area under the glucose curve (AUCglucose 0-360min) of untreated STZ-induced diabetic rats remained significantly elevated in comparison to the nondiabetic control rats. Topical application of PI-containing dermal patches on the skin of STZinduced diabetic rats at various doses showed a statistically significant decrease in blood glucose and AUCglucose 0-360min at the end of the 6h experimental period by comparison to respective control rats. However, there was no dose-dependent effect on the magnitude of blood glucose lowering induced by PI-containing dermal patches. The blood glucose-lowering effects evoked by PI-containing dermal patches were similar to those of the standard drug (sc. insulin). Treatment of STZ-induced diabetic rats with OA-containing dermal patches at various doses induced similar effects on the skin, IRS and blood glucose concentrations. Similar trends were observed chronically. Plasma insulin concentrations of untreated STZ-induced diabetic rats were significantly low compared with control non-diabetic rats. All PI treatments elevated plasma insulin concentrations of diabetic rats after the 6 h period but, the levels induced by low doses (2.47 and 3.99 μg/kg) were smaller than those caused by high doses (9.57 and 16.80 μg/kg). However, these effects on plasma insulin concentrations were comparable to those of sc insulin treated animals. Similarly, 5-week treatment with PI-containing dermal patches elevated plasma insulin concentrations although dose-dependent effects were not observed. Interestingly, the sc treated group remained low within levels that were comparable to those of the untreated STZinduced diabetic group. On the other hand, the plasma insulin concentrations of all OA treated groups remained significantly low at the end of the 6 h and 5-week experimental period in comparison to the non-diabetic control. There was no change in plasma insulin concentrations of STZ-induced diabetic rats following acute and short-term daily treatment with OA-containing dermal patches. Untreated STZ-induced diabetic rats exhibited significant depletion of glycogen and the expressions of glucose transporter-4 (GLUT-4) and glycogen synthase (GS) in liver and muscle tissues at the end of the 5-week study by comparison to non-diabetic rats at the corresponding time periods. Treatment with the PI matrix patch restored the glycogen levels and the expressions of GLUT-4 and GS to levels comparable to those of non-diabetic control animals and sc insulin. Moreover, treatment of STZ-induced diabetic rats with PI-containing dermal patches decreased plasma creatinine concentrations and increased GFR without altering with the plasma urea concentrations. Treatment of STZ-induced diabetic rats with OA-containing dermal patches induced similar effects on glycogen, plasma creatinine, GFR as well as plasma urea concentrations. Discussion Dermal patches delivered relevant amounts of pharmacologically active insulin and OA as evidenced by blood glucose lowering effects in STZ-induced diabetic rats. PI and OA dermal matrix patches will be easy to use and will not require elaborative devices to prevent drug leakage as in solution formulations. The findings are of considerable importance because this would free diabetic patients from daily bolus injections of insulin. Pectin has been used as a carrier of a wide variety of biologically active agents, for sustained release applications and targeting drugs to the colon for either local treatment or systemic action (Krusteva et al., 1990; Musabayane et al., 2000). The non-invasive PI- and OA-containing dermal patches may offer minimally invasive drug delivery in clinical applications to perhaps improve drug bioavailability and patient compliance. Interestingly, comparisons of the effects of PI and OA dermal patches of different insulin and OA concentrations on blood glucose lowering could not be separated statistically. The failure to observe these effects cannot be explained by the present study, but may be attributed to the narrow range of the doses used in the present study. These effects were also not statistically different from those of sc insulin. In summary, the ability of PI and OAcontaining dermal patches to reduce blood glucose with concomitant alleviation of symptoms associated with diabetes could be attributed to the ability of pectin to entrap and release drugs in a sustained and controlled manner. The PI- and OA-containing dermal hydrogel matrix patch would also provide patients with pain-free self-administration of insulin thereby improving compliance. Conclusions The current study has demonstrated that the pectin hydrogel insulin and OA dermal patches have the potential to deliver insulin and OA across the skin and into the blood stream and lower blood glucose concentrations and alleviate some symptoms associated with diabetes. Recommendations The limitations of the study include the absence of lipid profile and liver function assessment. In this regard, it is envisaged to utilize the obese Zucker diabetic rat model in future studies. Furthermore, limitations of this study also include the absence of plasma OA measurements.