Enhanced Activity of Topical Hydrocortisone by Competitive Binding of Corticosteroid-Binding Globulin
Atopic dermatitis of sensitive areas such as the face, particularly in children, is a difficult disease to treat as the standard therapeutic, topical steroids, is contraindicated for this application in children. Hydrocortisone (HC) can be used in these instances because it has been shown to be safe, but is often ineffective as it is a relatively weak steroid, especially at over-the-counter concentrations. To enhance the local topical activity of HC, the terminal inactive metabolite of prednisolone, Δ1-cortienic acid (Δ1-CA), is added to HC, as Δ1-CA preferentially binds transcortin, liberating more HC to elicit its therapeutic effect. Skin blanching studies, which are used to evaluate the potency of topical steroids, were employed to assess the ability of Δ1-CA to enhance the activity of HC. The results demonstrate that Δ1-CA, when applied in combination with HC, does indeed potentiate the vasoconstriction effect of topically applied HC, while having no effect alone. Thus, addition of the inert prednisolone metabolite Δ1-CA can increase the therapeutic effect of over-the-counter concentrations of HC when applied topically.
Glucocorticoids, such as cortisol, regulate a variety of physiological processes including cognition, reproduction, immune function, and many others, which can be exploited for therapeutic development.1, 2 For example, topical corticosteroids still represent the most effective treatment for inflammatory and allergic disorders of the skin. However, topical corticosteroids are not free of adverse effects. Some are topical, such as skin thinning, skin atrophy, and telangiectasia, while others relate to suppression of the hypothalamic-pituitary-adrenal axis and the immune system. Some specific side effects are ophthalmic, such as glaucoma and cataract. For this reason, most steroids cannot be used on the face, which represent a problem for treating children who have the highest incidence of atopic dermatitis and tend to have facial lesions.
The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is present in nearly all tissues, and it is their affinity for this receptor that dictates their relative strength of activity. This activity can be determined by using in vitro binding procedures, resulting in potencies being described as relative binding affinity (RBA). On a scale in which dexamethasone has by convention an RBA of 100, the natural hydrocortisone (HC) is considered weak (RBA = 10), while prednisolone (PR) is a stronger steroid, with an RBA of 40.
Another factor that characterizes corticosteroid activity is the binding to transcortin or corticosteroid-binding globulin (CBG). CBG is a 50-60 kDa glycoprotein with a range of glycosylation states3 and is synthesized primarily in the liver. CBG belongs to the serine protease inhibitor (serpin) superfamily despite having no serpin activity. CBG varies in a diurnal manner opposite to that of total plasma cortisol,
4, 5 ranging from 30 to 52 pg/mL. The primary role of CBG is to bind and transport anti-inflammatory steroids, including cortisol and progesterone.6 The degree of corticosteroid binding to this protein determines the bioavailability of the molecule. Under normal circumstances, approximately 80%-90% of cortisol is bound to CBG, 10%-15% is bound with low affinity to albumin, and the remaining 5%-10% of cortisol is unbound or “free.” The binding site is saturable (and molecules with a stronger binding affinity can displace those with weaker affinity); thus, as cortisol levels rise to over 400-500 nmol/L, CBG binding is saturated and free cortisol levels increase rapidly.
7CBG may also actively transport cortisol to inflammatory sites. Activated neutrophils are found in regions of inflammation, where they release high concentrations of elastase, a protease that cleaves CBG between residues 344 and 345, disrupting the binding site for cortisol and reducing the affinity of CBG for cortisol by 10-fold, thus releasing cortisol. 4, 8, 9, 10The conformational transition that CBG undergoes after cleavage by elastase has been revealed by high resolution crystallographic studies. 8, 9 The relative affinities of steroids for the GR and CBG do not run in parallel. Moreover, many of the potent steroids used currently (dexamethasone, betamethasone valerate, ciclesonide, fluticasone propionate) do not bind CBG. However, the simple steroids HC and PR do bind to both sites. It was also found that the acidic inactive metabolite of HC, cortienic acid (CA), or of PR, Δ1-cortienic acid (Δ1-CA), while having no affinity for the GR (1/10,000 that of HC), bind relatively strongly to CBG (Table 1, unpublished data). These differential binding properties were initially exploited with loteprednol etabonate (LE), a non-fluorinated “soft” ophthalmic steroid, which also binds to CBG. 11 In these studies, the local activity of LE was found to be significantly enhanced when mixed with Δ1-CA. The increase in human vasoconstrictor activity was dose dependent and the 24-h area under the curve (AUC) values indicated an up to 3-fold increase in local vasoconstriction, indicative of stronger anti-inflammatory activity.
11Table 1IC50 Values of Selected Steroids for GR and CBG
GR est. IC50 (μM)
CBG est. IC50 (μM)
As shown in Table 1, while intrinsic GR activity of the CA derivatives 3, 4, and 5 is extremely low, their CBG binding is comparable in value to those of HC and PR. As was demonstrated with LE, the local activity of both HC and PR also could be increased by mixing with the inactive metabolites (compounds 3-5, Table 1). For the studies presented here, the Δ1-derivatives (compounds 4 and 5) were selected, as they have higher binding properties and the Δ1-double bond contributes to increased stability of the steroidal structure. HC was chosen as the active steroid as it is readily available as an over-the-counter (OTC) product at concentrations up to 1%.
The generally accepted human vasoconstrictor activity assay (Stoughton–McKenzie vasoconstrictor assay) was used to assess the relative local anti-inflammatory activity of various concentrations of HC and Δ1-CA (or Δ1-cortienic acid methyl ester [Me-Δ1-CA]) combinations. This assay system was established as a means to assess in vivo bioequivalence and demonstrates relative potencies of topical dermatologic corticosteroids and is an accept method by the US Food and Drug Administration. The molecules were combined in an ethanol/propylene glycol (9:1) solvent mixture, loaded on filter paper disks, and then attached to a water impervious adhesive film. After evaporation of the ethanol, the filters loaded with samples were applied to the forearms of volunteers for 4 h. After removal, the vasoconstriction/blanching reaction was judged by the appearance of pallor at various time intervals, as described in Methods.
Following the studies conducted with ethanol/propylene glycol solutions, the encouraging results prompted the testing of transporter enhancement of the marketed HC creams. Δ1-CA was added to the OTC HC creams (either 0.5% or 1.0% HC) and thoroughly mixing to final concentrations of either 0.5% or 1.0%. The blanching induced by the HC/Δ1-CA mixtures was compared directly on the forearm of volunteers.
Test Articles Preparation
CA, Δ1-CA, and Me-Δ1-CA were obtained from Alchem Laboratories Corporation (Alachua, FL). HC was purchased from Sigma Company (St. Louis, MO), and both 0.5% and 1.0% HC creams were Walgreens brand purchased from Walgreens (Deerfield, IL).
The solution-based test articles for human skin blanching studies were prepared by dissolving HC (50 or 100 mM) and either Δ1-CA or Me-Δ1-CA (0, 10, 25, 50, or 100 mM) in vehicle containing absolute ethanol and propylene glycol (9:1). The resulting solutions were mixed in such a way to provide the needed final concentrations of HC, Δ1-CA, and/or Me-Δ1-CA, as shown in results.
The HC creams compounded with Δ1-CA were prepared by quantitatively transferring to a mixing vessel the HC cream obtained from Walgreens, either 0.5% or 1.0%. Δ1-CA was weighed on the basis of quantitative amount of HC to exactly 0.5% or 1.0% of the cream on a weight basis. The cream and the micronized Δ1-CA were kneaded and thoroughly mixed in a suitable glass vessel for about 60 min under suitable dust-free conditions. The finished creams containing the HC/Δ1-CA combination were transferred to plastic jars and weighed.
Skin Blanching With Solutions
For the skin blanching studies conducted with the test articles in solution, a 20 μL sample of each mixture was loaded on a circular paper disc (7 mm diameter) that was attached to a water impervious adhesive film (3M). After evaporation of ethanol (approximately 15 min), the film loaded with samples were applied to the forearms of human volunteers for 4 h. Subsequently, the vasoconstriction/blanching reaction was judged by the appearance of pallor at various time intervals after the removal of the discs and films. The grading scale was as follows: 0, normal skin; 1, slight pallor; 2, pallor with some defining edges; 3, even pallor with a clear outline of the application sites; 4, very intense pallor.
Skin Blanching With Compounded Creams
For the skin blanching studies conducted with the compounded creams, corn cushions were applied to strips of paper tape. The appropriate cream test article was loaded into the space of the corn cushion, then the sample was applied to the volar forearm area of volunteers by adhesion of the paper tape for either 8 or 10 h. Application of the test articles was randomized and blinded to both the administrator and the subject. Following removal of the samples, scanned images of the area were obtained at the indicated time point using an HP scanner. The images were then read and scored by independent readers who were unaware of which test article corresponded to which site.
Standard error calculations were performed by Excel Data Analysis (Microsoft).
Results and Discussion
Initial studies were conducted with the compounds dissolved in an ethanol/propylene glycol (9:1) solution, loaded onto circular paper discs attached to a water impervious adhesive film, and then applied to the forearm of volunteers.
The blanching activity of HC was evaluated in combination with various concentrations of Δ1-CA and Me-Δ1-CA. As shown in Figures 1 and 2, the dose-response curves and resulting AUC data indicate that Δ1-CA and Me-Δ1-CA increased the blanching activity of HC, while neither Δ1-CA nor Me-Δ1-CA alone showed any intrinsic corticosteroid activity. The addition of 50 mM Δ1-CA, however, caused a decrease in the increasing activity, which is likely due to the precipitation of Δ1-CA when an over-saturated mixture is used.
Next, the effect of Δ1-CA on HC was investigated at 3 different concentrations of HC, in the presence or absence of either equal or half concentration Δ1-CA. The results shown in Figure 3 again indicate that Δ1-CA increased the skin blanching effect of HC, which was most dramatic and maximal with equivalent 25 mM concentrations at time 8 h. This effect was not increased significantly with the 2-fold increase of HC concentration relative to Δ1-CA (50 and 25 mM, respectively) (Fig. 3). The increase in the AUC of the blanching activity of HC in the presence of Δ1-CA and Me-Δ1-CA showed an increase of 2 to 3 times that of HC alone and also displayed a maximal effect (Fig. 3).
Following the initial demonstration of HC activity enhancement with Δ1-CA and Me-Δ1-CA as dissolved in ethanol, OTC cream formulation of HC, 0.5% and 1.0%, was compounded with Δ1-CA for a final concentration of either 0.5% or 1.0%. To optimize time of exposure, the 1% HC/1% Δ1-CA combination was applied as occluded samples in duplicate to the volar forearm of a volunteer for either 8 or 10 h and assessed for blanching intensity after 4 h following removal of samples. It was determined that 8-h exposure provided optimal blanching activity (data not shown).
To assess which combination(s) to focus more narrowly on, a time-course blanching study was performed with the 4 combinations of HC/Δ1-CA (0.5%/0.5%, 0.5%/1.0%, 1.0%/0.5%, and 1.0%/1.0%), plus 2 concentrations of HC alone. The samples were again occluded to the volar forearm of a volunteer in triplicate for 8 h and scans of the treated areas were taken at 1.5, 2, 4, and 6 h. The cataloged scanned images allowed for independent readings by multiple individuals following completion of the study, utilizing the same blanching scoring described earlier.
As shown in Figure 4, the most intense blanching was observed at 1.5- and 2-h post-exposure. At every time point, the combination with Δ1-CA demonstrated considerably more blanching than with the equivalent concentration of HC alone. At 1.5 h, the combinations of 0.5% Δ1-CA and 1.0% Δ1-CA with 0.5% HC demonstrated 2.5-fold greater blanching than that observed with 0.5% HC alone. No discernible difference could be seen when comparing the 0.5% and 1.0% Δ1-CA in combination with either concentration of HC. This could be due to maximum enhancement already having been achieved with the lower concentration of enhancer. As expected, the blanching effect diminished over time (Fig. 4).
As the enhancement with Δ1-CA was already optimal with the 0.5% concentration, a more extensive time-course blanching study was conducted comparing only the 0.5% HC/0.5% Δ1-CA and 1.0% HC/0.5% Δ1-CA combinations. Again, following an 8-h occlusion period with the test articles, scanned images were obtained of the treated volar forearm area at 2-h intervals up until 14-h post-treatment. Independent assessments were then conducted on the extent of blanching for the different treatments at different time points. Enhancement of the blanching effect of HC by Δ1-CA appeared to peak at 4 h and persisted for over 12 h (Fig. 5). Different from what was observed previously, 0.5% HC and 1.0% HC alone were comparable, with the enhanced 1.0% HC with 0.5% Δ1-CA showing a marked increase in blanching, nearly 2.5-fold greater blanching in the presence of the enhancer (hour 4; Fig. 5a). In calculating the AUC, both enhanced versions of HC showed a distinct increase in blanching activity when compared to HC alone; again, the most notable enhancement was seen with 1.0% HC/0.5% Δ1-CA (Fig. 5b).
In summary, as was observed previously with LE,11 the local activity of HC can be enhanced through combination with a molecule that has significant affinity for the CBG but does not bind the GR. These terminal metabolites, Δ1-CA or CA, have no pharmacological activity and cannot be metabolized further into molecules that would have unknown physiological effects. (Indeed, an acute oral limit rat toxicology study showed no toxic effect up to 2000 mg/kg, unpublished results). As such, beyond binding CBG in order to enhance the activity of the functional steroids (i.e., HC and LE), the terminal metabolites act strictly as enhancers of activity and are otherwise inert. Previous studies have shown that the pharmacokinetics of LE were unchanged when combined with Δ1-CA, thus the combination does not have an effect on the amount of steroid absorbed.11
This enhancement can be applied to an OTC formulation of HC to produce a stronger locally acting topical steroid while maintaining the safety of low concentrations of HC. This approach provides a means of treating skin conditions (especially in children) with an inherently safe mild topical steroid, such as HC, yet produces more effective treatment outcomes.