Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment

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Abstract

The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50–100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2–3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

Introduction

After 75 years of development, insulin formulations have not yet attained the physiological goal of providing distinct insulin peaks during meals and a constant, low insulin level to meet the basal need between meals and during the night. Because of too slow absorption of regular insulin and variable absorption of prolonged-acting insulin after subcutaneous (s.c.) injection, diabetics often experience large excursions in blood glucose and risk hypoglycemic attacks and the long-term complications caused by high glucose levels. The recent DCCT study showed that strict glycemic control significantly reduced diabetic complications [1].

Since insulin was first isolated, there has been a continuous effort to replace the discomfort and inconvenience of repeated s.c. injection with non-invasive delivery. Indeed, it would be fair to say that virtually no body orifice or surface has escaped attention in this regard [2]. Even the conventional route of administration, however, fails to normalize plasma glucose levels as efficiently as desired: absorption in response to the rapid increase in glucose at meal-times is too slow, and the physiological, nearly constant, basal insulin secretion of about 1 IU/h is not provided at all. The former problem has been addressed most recently by recombinant DNA technology and the introduction of monomeric insulin analogues [3]which are absorbed three times more rapidly than hexameric human insulin [4].

Transdermal drug delivery by iontophoresis has attracted considerable attention recently. The potential control of drug input (either pulsatile or zero-order, or some combination thereof) is a seductive advantage for compounds of complex pharmacokinetic and/or pharmacodynamic behaviour. Over the last 15 years or so, the transdermal iontophoresis of insulin has been examined in several animal experiments 5, 6, 7, 8, 9, 10, 11, 12. A careful and thorough review of this work [13]concludes that insulin can be transported across animal skin by iontophoresis. However, while the quantity delivered may be sufficient to treat a diabetic rat or rabbit [10]a simple calculation shows that scaling-up to man demands a surface area of contact that is well beyond practical usefulness. Only the transport rate of a highly charged, monomeric, sulfated insulin (3.9 μg/cm2/h) [12], which would correspond to delivery of 1 IU/h of a fully potent insulin from a 10-cm2 device, was sufficient to meet the basal, daily demand of an adult diabetic.

Given that the new insulin analogues are themselves charged (more so than the parent insulin) and monomeric, it was therefore hypothesized that their iontophoretic delivery would be possible and, if only on the basis of size (see Fig. 1), considerably better than that of the native hexamer. The availability of several analogues having different associative states and charges (Table 1) allowed the influence of two important physicochemical parameters to be examined. In addition, the effect of selected skin pretreatments on the efficiency of iontophoretic transport was carefully considered.

Section snippets

Chemicals

The insulins tested are shown in Table 1. Human insulin and human insulin analogues were prepared as previously reported [3]. The zinc content was <0.01 mol per mol insulin. Purity was at least 97% determined by RP-HPLC. Sulfated insulin was obtained from Connaught-Novo Lab. (Toronto, Canada). Carrier free (porcine) 125I-insulin 10 μCi/ml was the same as used for the insulin assay [14]. Ethanol 100% (ethyl alcohol 200 proof, dehydrated alcohol, USP, Punctilious) was obtained from Quantum

Passive versus iontophoretic transport (intact skin)

Without electrical enhancement (passive delivery) no detectable transport of any of the investigated insulins could be detected (results not shown). Initial experiments using cathodal iontophoresis revealed insignificant flux of hexameric human insulin; based on the immunoreactive insulin (IRI) detection limit it can be deduced that the normalized flux was less than 0.2×10−6 cm/h. This was also the case for some of the analogues with reduced association state; however, low and variable fluxes

Discussion

The literature reports that the normalized iontophoretic flux of native insulin across animal skin is in the order of 10−5 cm/h 7, 11. Here, though, we have found that, through intact (i.e. untreated) hairless mouse skin, the value is less than 0.2×10−6 cm/h, a result in agreement with that obtained in the isolated, perfused porcine skin flap model [12].

Unlike certain previous investigations 7, 11we had no success when attempting anodal insulin iontophoresis. The problem of precipitation, which

Acknowledgements

The authors wish to thank Mrs. Mary-Ann Jones for performing the insulin assays and our colleagues in the Skin Bioscience Group at UCSF for stimulating discussions. The work was supported in part by a grant (HD-27839) from the US National Institutes of Health.

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      A corresponding increase in plasma insulin levels was also detected (~1.4 ng/mL 18 h post-treatment) that exceeded levels detected in rats treated with intraperitoneally injected insulin. Pretreatment of skin such as stripping skin [72,73], using penetration enhancers [74–77] and depilatory cream [75,78,79] has been reported to enhance the transport of insulin through the skin under iontophoresis. An investigation on the effect of diverse chemical enhancers including ethanol (EtOH), propylene glycol (PG), dimethylacetamide (DMA), ethyl acetate (EtOAc) and IPM demonstrated that skin permeability was severely improved with DMA, followed by EtOH and EtOAc, while IPM and PG exhibited relatively insignificant skin barrier altering potential [77].

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