Martha A. Hass

Topical Delivery of Lycopene Using Microemulsions: Enhanced Skin Penetration and Tissue Antioxidant Activity

Topical delivery of lycopene is a convenient way to supplement cutaneous levels of antioxidants. In this study, lycopene was incorporated (0.05%, w/w) in two microemulsions containing BRIJ-propylene glycol (2:1, w/w, surfactant blend) but different oil phases: mono/diglycerides of capric and caprylic acids (MG) or triglycerides of the same fatty acids (TG). Microemulsions containing MG and TG were isotropic, fluid, and clear, with internal phase diameters of 27 and 52 nm, respectively. Both MG- or TG-containing microemulsions markedly increased lycopene penetration in the stratum corneum (6- and 3.6-fold, respectively) and in viable layers of porcine ear skin (from undetected to 172.6 +/- 41.1 and 103.1 +/- 7.2 ng/cm(2), respectively) compared to a control solution. To assure that lycopene delivered to the skin was active, the antioxidant activity of skin treated with MG-containing microemulsion was determined by CUPRAC assay, and found to be 10-fold higher than untreated skin. The cytotoxicity of MG-containing microemulsion in cultured fibroblasts was similar to propylene glycol (considered safe) and significantly less than of sodium lauryl sulfate (a moderate-to-severe irritant) at 1-50 microg/mL. These results demonstrate that the MG-containing microemulsion is an efficient and safe system to increase lycopene delivery to the skin and the antioxidant activity in the tissue.

Obstructive bladder dysfunction: Morphological, biochemical and molecular changes

Abstract Obstruction can cause changes in bladder structure and function, which may at a certain point in time become irreversible. Both the rabbit and the rat have proven to be excellent models to study the morphological, biochemical and molecular changes that occur in the bladder following obstruction. Similarities between partial outflow obstruction in animals and obstructive dysfunction in man include increased bladder mass, increased fibrosis, reduced compliance, increased incidence of detrusor instability and decreased contractile ability. Obstructed bladder function can remain relatively normal for prolonged periods of time, even though bladder mass is increased (compensated stage). If the obstruction is not relieved, bladder function destabilizes and then decompensates, with subsequent risk of serious complications. It is hypothesized that the shift from the compensated to the decompensated stage is related to cyclical periods of ischaemia followed by reperfusion (I/R). This initiates degenerative membrane effects, which supports the process of bladder decompensation. It seems that relief of obstruction during the compensated stage, at least in animals, can induce a rapid and full restoration of the bladder function. However, if the obstruction is relieved during the decompensated phase, bladder function only partially recovers. Irreversible bladder decompensation may be prevented by reducing increased bladder mass and/or by reducing ischaemia/increasing blood supply to the bladder. The antioxidant Vitamin E seems to reduce the progression of decompensation in rabbits. Treatment of rats with the α 1 -adrenoceptor antagonist doxazosin prior to partial outlet obstruction increases blood flow to the bladder, significantly decreases the effect of obstruction on bladder weight, and significantly protects the contractile function of the obstructed bladder. Pre-treatment of rabbits with the α 1 -adrenoceptor antagonist tamsulosin partly prevents the development of bladder wall hypertrophy due to obstruction. In conclusion, there is evidence that I/R plays an important role in the pathogenesis of obstructive bladder dysfunction. Therefore, I/R should be prevented or relieved. As I/R is the consequence of increased bladder mass following obstruction, therapies that prevent or reduce increased bladder mass (such as the α 1 -adrenoceptor antagonist tamsulosin) are likely to protect the bladder from (further) dysfunction. Therapies based on both the relief of I/R (increasing blood flow to the bladder, e.g. α 1 -adrenoceptor antagonists) and preventing I/R induced cellular damage (e.g. antioxidant activity) have been shown experimentally to significantly reduce the severity of bladder dysfunction secondary to partial outflow obstruction. Of course, these potentially useful and novel mechanisms of action of therapy should also be investigated in humans.

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder.

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladders smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.

Decylglucoside-based microemulsions for cutaneous localization of lycopene and ascorbic acid

Cutaneous delivery of combinations of antioxidants offers the possibility of enhanced protection against UV-radiation. In this study, we investigated the potential of sugar-based microemulsions containing monoglycerides to promote simultaneous cutaneous delivery of lycopene and ascorbic acid, and increase tissue antioxidant activity. Lycopene and ascorbic acid were incorporated (0.04% and 0.2% (w/w), respectively) in decylglucoside-based microemulsions containing isopropyl myristate mixed with monocaprylin (ME-MC), monolaurin (ME-ML) or monoolein (ME-MO) as oil phase. The microemulsions increased lycopene delivery into porcine ear skin by 3.3- to 8-fold compared to a drug solution. The effect of microemulsions on ascorbic acid cutaneous delivery was more modest (1.5-3-fold), and associated with an approximately 2-fold increase in transdermal delivery. According to their penetration-enhancing ability, the microemulsions were ranked ME-MC>ME-MO>ME-ML. This superiority of ME-MC coincided with a stronger effect in decreasing skin electrical resistance. After 18 h of treatment, the viability of bioengineered skin treated with ME-MC was 2.2-times higher compared to Triton-X100 (moderate irritant), demonstrating that ME-MC is less cytotoxic. Skin treatment with ME-MC containing both antioxidants increased the tissue antioxidant activity by 10.2-fold, but no synergism between the antioxidants was observed.

Fatty acid profiles in normal and obstructed rabbit bladder smooth muscle and mucosa.

Partial bladder outlet obstruction results in progressive loss in contractile and specific cellular and subcellular membrane functions. There is evidence that ischemic activation of proteolytic and lipolytic enzymes play a major role in the etiology of bladder dysfunction secondary to partial outlet obstruction. The specific aims of the current study were to determine the fatty acid profiles in normal rabbit bladder smooth muscle and mucosa and to determine the effect of partial outlet obstruction on the distribution and content of free and total fatty acids. Fatty acids were isolated by extraction from obstructed and normal bladder smooth muscle and mucosal homogenates, and samples were analyzed by gas chromatography. All samples contained palmitic, stearic, oleic, linoleic, and arachidonic acids. A 100% increase in total fatty acid concentration was observed in the obstructed bladder muscle tissue relative to normal bladders, although the concentration of total arachidonic acid remained constant in the two groups. Significantly higher levels of free arachidonic acid were observed in the obstructed bladder muscle group compared to the normal group. No changes were observed in fatty acid concentrations or distributions in bladder mucosa. These data show that fatty acid composition is altered as a result of bladder obstruction and support the idea that obstruction increases the activity of lipase activity and/or decreases acyl transferase activity. Neurourol. Urodynam. 18:697–711, 1999.

Cutaneous delivery of α‐tocopherol and lipoic acid using microemulsions: influence of composition and charge

To assess whether the composition and charge of microemulsions affect their ability to simultaneously deliver α‐tocopherol and lipoic acid into viable skin layers.

Effect of Oral Tadenan Treatment on Rabbit Bladder Structure and Function After Partial Outlet Obstruction

PURPOSE There is increasing evidence that the progressive dysfunction induced by partial outlet obstruction is mediated by ischemia-reperfusion, and bladder decompensation results from ischemia-reperfusion induced damage to the cellular and subcellular organelle membranes of nerve and smooth muscle, mitochondria and sarcoplasmic reticulum. Tadenan, an extract of Pygeum africanum, is a therapeutic prescribed in Europe to relieve symptoms of obstructive bladder dysfunction secondary to benign prostatic hyperplasia. There is excellent experimental evidence that Tadenan treatment of obstructed rabbits reduces and reverses the progression of bladder decompensation. We determined whether Tadenan therapy can reverse the morphological damage associated with obstructive dysfunction. MATERIAL AND METHODS A total of 36 male New Zealand White rabbits were separated into 6 groups of 6 each. Rabbits in groups 1 and 2 underwent sham operation. For 3 weeks beginning 2 weeks after sham operation group 1 was treated with vehicle and group 2 was treated with 30 mg./kg. Tadenan daily. Rabbits in groups 3 to 6 underwent partial outlet obstruction surgery. Two weeks after obstruction each rabbit was treated for 3 weeks with vehicle in group 3, and with 1, 10 and 30 mg./kg. Tadenan in groups 4, 5 and 6, respectively. After the completion of treatment cystometry was performed on each rabbit and isolated bladder strips were evaluated for contractile responses to field stimulation, adenosine triphosphate, carbachol and KCl. Separate strips were fixed for electron microscopy to determine the location and severity of cellular and subcellular membrane damage. RESULTS Partial outlet obstruction resulted in reduced compliance, decreased responses of bladder strips to all forms of stimulation tested, and significant and extensive damage to cellular and subcellular organelle membranes consistent with an ischemia-reperfusion etiology. Daily 1 and 10 mg./kg. Tadenan treatments had little effect on the obstruction induced increase in bladder weight or the deleterious changes in bladder function and structure. However, treating obstructed rabbits with 30 mg./kg. Tadenan daily resulted in reduced bladder hypertrophy, improved compliance, improved contractile responses to nearly normal levels of isolated bladder strips to all stimuli tested and reversal of obstruction induced structural damage to cellular and subcellular organelle membranes. CONCLUSION Tadenan treatment of obstructed rabbits resulted in a dose dependent improvement in bladder ultrastructure in parallel with improved bladder compliance and contractile responses of isolated strips to stimulation, providing support for the hypothesis that damage to cellular and subcellular organelle membranes mediates the contractile dysfunction induced by partial outlet obstruction.

Stability, Cutaneous Delivery, and Antioxidant Potential of a Lipoic Acid and α-Tocopherol Codrug Incorporated in Microemulsions

The aim of this study was to assess the skin penetration, stability, and antioxidant effects of a α-tocopherol-lipoic acid codrug. To enhance penetration, we evaluated three microemulsions varying in water content and composition of the oil phase (isopropyl myristate with either monocaprylin or oleic acid). The codrug was incorporated at 1% (w/w). Codrug hydrolysis in the microemulsion increased with increases in time (up to 48 h) and formulation water content (10%-30%, w/w). Microemulsions increased the codrug delivery into viable layers of porcine ear skin by 2.9-7.8-fold compared with a control formulation (20% monocaprylin in isopropyl myristate) after 24 h. Penetration enhancement was influenced by the oil phase, with the formulation containing monocaprylin displaying the most pronounced effect. Antioxidant activity, assessed in skin bioequivalents using the thiobarbituric acid-reactive substances (TBARS) assay, demonstrated that TBARS levels decreased by 39% after treatment with the codrug-containing microemulsion compared with the unloaded formulation. In addition to the codrug, tocopherol (8.2 ± 0.6 μg/cm(2)) was detected in the viable bioequivalent tissues, suggesting that the codrug was partly hydrolyzed after 12 h. Taken together, these results support the potential of nanodispersed formulations containing a tocopherol-lipoic acid codrug to improve skin antioxidant activity.

The role of lipids and lipid metabolites in urinary bladder dysfunction induced by partial outlet obstruction.

Benign prostate hyperplasia (BPH) frequently progresses to cause partial outlet obstruction of the urinary bladder, resulting in a range of symptoms including urine retention, poor flow, nocturia, and detrusor instability. Over 80% of males 50 to 60 years of age and older have various degrees of bladder outlet obstruction secondary to BPH that results in urinary dysfunction.23,34 The urinary dysfunction in men with BPH is related to functional,47 structural,21,59 and biochemical46 changes that occur in both the smooth muscle and urothelial components of the urinary bladder during urethral obstruction by the enlarged prostate. However, the specific pathophysiology that leads to BPH symptoms remains unknown.86 The ability to develop rational therapeutic interventions for the treatment of bladder dysfunction associated with BPH requires an understanding of the pathological changes that occur in the underlying cellular mechanisms responsible for maintaining normal bladder function

Identification of components of Prunus africana extract that inhibit lipid peroxidation

Extractive and chromatographic separations were performed on V-1326, a chloroform extract from the bark of Prunus africana (also referred to as Pygeum africanum), which is used to treat the symptoms associated with benign prostate hyperplasia (BPH). The relative amounts of eleven identified constituents in crude V-1326 and in separated fractions were determined using gas chromatographic analysis. The ability of V-1326 and its separated fractions to inhibit ferrous ion-induced stimulation of lipid peroxidation in microsomal preparations from rabbit livers was evaluated. The extract, V-1326, and fractions containing high levels of myristic acid potently inhibited lipid peroxidation.