Effect of 2-Hydroxypropyl-β-cyclodextrin on Percutaneous Absorption of Methyl Paraben

A potential use of 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) to solubilize methyl paraben and to suppress its percutaneous absorption was examined, and compared with nonionic surfactant HCO-60. HP-β-CyD significantly increased the solubility of methyl paraben in water, where the apparent 1:1 stability constant of the soluble complex was determined to be 2150 M^?1. The in-vitro cutaneous permeability of methyl paraben through an isolated skin of hairless mouse was suppressed by HP-β-CyD, thus promoting the bioconversion of methyl paraben to the less toxic metabolite, p-hydroxybenzoic acid (p-HBA) in the epidermis. These effects of HP-β-CyD were greater than those of HCO-60. HP-β-CyD (2% w/v) reduced the in-vivo percutaneous absorption of methyl paraben by 66% 24 h after the topical application of a solution containing [¹⁴C]methyl paraben to hairless mouse skin. Additionally, the percutaneous absorption of [¹⁴C]HP-β-CyD was confirmed to be extremely low. These results suggest that HP-β-CyD is useful in liquid preparations of methyl paraben for topical application.     

Inhibitory effect of prostaglandin E1 on laurate-induced peripheral vascular occlusive sequelae in rabbits: optimized topical formulation with beta-cyclodextrin derivative and penetration enhancer HPE-101.

Prostaglandin E1 (PGE1) and its inclusion complexes with beta-cyclodextrin (beta-CyD) and O-carboxymethyl-O-ethyl-beta-cyclodextrin (CME-beta-CyD) were made as topical preparations. The PGE1 preparations, when applied with a penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentane-2-one (HPE-101), markedly increased the regional blood flow in the ear of rabbits and were longer acting than when administered by the intravenous route. Topical application of the PGE1 preparations significantly protected rabbits against laurate-induced peripheral vascular occlusive sequelae; the protective potency increased in the order of PGE1 alone = beta-CyD complex < CME-beta-CyD complex preparation. The PGE1 preparations elicited skin reactions such as erythema and oedema depending on their vasodilating actions. These reactions disappeared gradually after removal of the preparations, and hence may not be serious obstacles for their safe use. These results suggest that combinations of CME-beta-CyD and HPE-101 work synergistically to facilitate the entry of PGE1 into the skin, and consequently enhance the therapeutic potential of PGE1 in the topical preparation tested.     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.     

Further Evaluation of a New Penetration Enhancer,HPE-101

Abstract— The penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101), significantly enhanced the excretion of topically applied [¹⁴C]indomethacin when dissolved in dipropylene glycol, triethylene glycol, diethylene glycol, 1,3-butylene glycol, trimethylene glycol, glycerin, water, silicone or triethanolamine, but not when dissolved in ethanol, isopropyl alcohol, oleyl alcohol, olive oil, peppermint oil, isopropyl myristate or hexylene glycol. HPE-101 significantly enhanced the excretion of [¹⁴C]indomethacin, [¹⁴C]nicotinic acid, [¹⁴C]5-fluorouracil, [³H]oestradiol and [³H]triamcinolone acetonide, but not that of [³H]testosterone. HPE-101 also significantly enhanced the excretion of [¹⁴C]indomethacin applied to intact skin of rabbit, guinea-pig and rat, and to tape-stripped skin of guinea-pig, but did not enhance the excretion of [¹⁴C]indomethacin applied to tape-stripped skin of rat or rabbit.     

Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-α generation

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNFα) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists.
2. In human monocytes that were adherent to plastic, neither prostaglandin D₂ (PGD₂), prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNFα-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells.
3. Exposure of human monocytes to LPS (3 ng ml⁻¹, ∼ EC₈₄) resulted in a time-dependent elaboration of TNFα which was suppressed in cells pretreated with prostaglandin E₁ (PGE₁), PGE₂ and cicaprost. This effect was concentration-dependent with mean pIC₅₀ values of 7.14, 7.34 and 8.00 for PGE₁, PGE₂ and cicaprost, respectively. PGD₂, PGF_2α and U-46619 failed to inhibit the generation of TNFα at concentrations up to 10 μM.
4. With respect to PGE₂, the EP-receptor agonists, 16,16-dimethyl PGE₂ (non-selective), misoprostol (EP₂/EP₃-selective), 11-deoxy PGE₁ (EP₂-selective) and butaprost (EP₂-selective) were essentially full agonists as inhibitors of LPS-induced TNFα generation with mean pIC₅₀ values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE₁, another EP₂-selective agonist, AH 13205, inhibited TNFα generation by only 21% at the highest concentration (10 μM) examined. EP-receptor agonists which have selectivity for the EP₁- (17-phenyl-ω-trinor PGE₂) and EP₃-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNFα generation.
5. Pretreatment of human monocytes with the TP/EP₄-receptor antagonist, AH 23848B, at 10, 30 and 100 μM suppressed LPS-induced TNFα generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves.
6. Given that AH 13205 was a poor inhibitor of TNFα generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 and 30 μM AH 13205 inhibited the generation of TNFα by 31% and 53%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves at either concentration examined.
7. Since PGD₂ and 17-phenyl-ω-trinor PGE₂ (EP₁-agonist) did not suppress TNFα generation, the EP₁/EP₂/DP-receptor antagonist, AH 6809, was employed to assess if EP₂-receptors mediated the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 μM AH 6809 did not affect LPS-induced TNFα generation but produced a parallel 3.5 fold rightwards shift of the PGE₂ concentration-response curve.
8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNFα generation. One of these probably represents IP receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank order of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP₂- and/or EP₄- and/or IP) or novel EP-receptors mediate the inhibitory effect of PGE₂ on TNFα generation.

     

Development of a topical ointment of betamethasone dipropionate loaded nanostructured lipid carrier

The purpose of this study was to design an innovative topical ointment containing betamethasone dipropionate loaded nanostructured lipid carrier (BD-NLC) for the treatment of atopic dermatitis (AD). BD-loaded NLC was produced with precirol ATO 5 and oleic oil (OA) by melt emulsification method. Effects of surfactant concentration, amount of solid lipid and liquid lipid on skin retention and skin penetration were investigated by in vitro percutaneous permeation experiment. The optimized BD-NLC showed a homogeneous particle size of 169.1 nm (with PI = 0.195), negatively charged surface (−23.4 mV) and high encapsulation efficiency (85%). Particle morphology assessed by TEM revealed a spherical shape. In vitro skin permeation study was carried out to investigate the percutaneous behaviors of W/O ointment with BD-NLC and Carbopol emulgel ointment with BD-NLC. W/O ointment with BD-NLC showed high skin retention (35.43 µg/g) and low penetration (0.87 µg/ml). In vitro drug release studies were carried out to demonstrate the drug releasing properties of the two ointments. W/O ointment with BD-NLC showed an advantage for skin retention as it was better for drug release. The tissue distribution test suggested that BD distribution was skin > muscle > blood. Self-made topical ointment in mice showed no skin irritation. The animal experiments indicated that BD-loaded NLC ointment was effective and safe for topical use.     

An exploratory microdialysis study investigating the effect of repeated application of a diclofenac epolamine medicated plaster on prostaglandin concentrations in skeletal muscle after standardized physical exercise

Aim

Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac.

Methods

Microdialysis was used to determine the local interstitial concentration of PGE₂ and 8-iso-PGF_2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days.

Results

At baseline PGE₂ concentrations were 1169 ± 780 pg ml⁻¹ at rest and 1287 ± 459 pg ml⁻¹ during dynamic exercise and increased to 2005 ± 1126 pg ml⁻¹ during recovery. After treatment average PGE₂ concentrations were 997 ± 588 pg ml⁻¹ at rest and 1339 ± 892 pg ml⁻¹ during exercise. In contrast with the baseline phase no increase in PGE₂ concentrations was recorded during the recovery period after treatment (PGE₂ 1134 ± 874 pg ml⁻¹). 8-iso-PGF_2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies.

Conclusions

We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE₂ induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF_2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases.     

Eicosapentaenoic acid suppression of systemic inflammatory responses and inverse up-regulation of 15-deoxyΔ12,14 Prostaglandin J2 production

Background and Purpose

Eicosapentaenoic acid (EPA) has been shown to suppress immune cell responses, such as cytokine production and downstream PG production in vitro. Studies in vivo, however, have used EPA as a minor constituent of fish oil with variable results. We investigated the effects of EPA on systemic inflammatory responses as pure EPA has not been evaluated on immune/inflammatory responses in vivo.

Experimental Approach

Rabbits were administered polyinosinic: polycytidylic acid (poly I:C) i.v. before and after oral treatment with EPA for 42 days (given daily). The responses to IL-1β and TNF-α were also studied. Immediately following administration of poly I:C, body temperature was continuously monitored and blood samples were taken. Plasma levels of IL-1β, PGE₂ (PGE₂), and 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) were measured by enzyme immunoassay.

Key Results

Following EPA treatment, the fever response to poly I:C was markedly suppressed compared with pretreatment responses. This was accompanied by a parallel reduction in the poly I:C-stimulated elevation in plasma levels of IL-1β and PGE₂. Paradoxically, the levels of 15d-PGJ₂ were higher following EPA treatment. EPA treatment did not significantly alter the fever response or plasma levels of PGE₂ in response to either IL-1β or TNF-α.

Conclusion and Implications

Oral treatment with EPA can suppress immune/inflammatory responses in vivo via a suppression of upstream cytokine production resulting in a decreased fever response and indirectly reducing circulating levels of PGE₂. EPA also enhances the production of the cytoprotective prostanoid 15d-PGJ₂ indicating the therapeutic benefit of EPA.     

Characterization of biosynthesis and modes of action of prostaglandin E2 and prostacyclin in guinea pig mesenteric lymphatic vessels

Background and purpose:

Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E₂ (PGE₂) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels.

Experimental approach:

Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE₂ and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed.

Key results:

Prostaglandin E₂ and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP₄ receptor antagonists, GW627368x (1 µM) and AH23848B (30 µM) and the IP receptor antagonist CAY10441 (0.1 µM) suggest that PGE₂ predominantly activates EP₄, whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 µM) or glibenclamide (1 µM) suggested a role for the activation of protein kinase A and ATP-sensitive K⁺ channels.

Conclusions and implications:

Our findings characterized the inhibition of lymphatic pumping induced by PGE₂ or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.     

Transdermal Iontophoretic Delivery of Hydrocortisone from Cyclodextrin Solutions

Enhanced skin penetration of hydrocortisone can be desirable for treatment of several diseases. Transdermal iontophoretic delivery of hydrocortisone solubilized in an aqueous solution of hydroxypropyl-β-cyclodextrin (HP-β-CyD) was investigated and compared with chemical enhancement of co-solvent formulations. The passive permeation of hydrocortisone through human cadaver skin was higher when delivered from propylene glycol than when delivered after solubilization in an aqueous solution of HP-β-CyD. However, the iontophoretic delivery of the 1% hydrocortisone-9% HP-β-CyD solution was higher than the amount delivered passively by the 1% hydrocortisone-propylene glycol formulation, even if oleic acid was used as a chemical enhancer. Iontophoretic delivery of 1% hydrocortisone with 3% or 15% HP-β-CyD was lower than that of the 9% HP-β-CyD solution. These data suggest that free hydrocortisone rather than complexes is predominantly delivered iontophoretically through the skin and the HP-β-CyD complex serves as a carrier to replenish depletion of hydrocortisone. HP-β-CyD prevents hydrocortisone from forming a skin reservoir. Iontophoresis provides better enhancement of transdermal delivery of hydrocortisone than the chemical approach when just sufficient HP-β-CyD is added to solubilize the hydrocortisone.     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H₂ receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H₂ receptor were prepared and were used to infect insect Sf9 cells.
2. The His-tagged H₂ receptors expressed in insect Sf9 cells showed typical H₂ receptor characteristics as determined with [¹²⁵I]-aminopotentidine (APT) binding studies.
3. In Sf9 cells expressing the His-tagged H₂ receptor histamine was able to stimulate cyclic AMP production 9 fold (EC₅₀=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with K_i values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).
4. The expression of the His-tagged H₂ receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg⁻¹ protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×10⁶ copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [¹²⁵I]-APT binding up to 169±5% (mean±s.e.mean, n=3).
5. The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC₅₀ value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/10⁶ cells in the absence and 81±11 pmol/10⁶ cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3).
6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H₂ receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.
7. These experiments demonstrate the successful expression of His-tagged histamine H₂ receptors in insect Sf9 cells. The H₂ receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H₂ receptor protein is functional. These functional receptors are targeted to the plasma membrane.

     

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder

1. Cumulative concentration-response curves (CRC) to prostaglandin E₁ (PGE₁), PGE₂, PGD₂ and PGF_2α (0.01–30 μM) and to the thromboxane A₂ (TXA₂) receptor agonist U-46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation.
2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF_2α>U-46619>PGE₂ whereas weak contractile responses were obtained with PGD₂ and PGE₁. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM.
3. The selective TXA₂ antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK_B value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF_2α and it was a non-surmountable antagonist of PGE₂ (apparent pK_B of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U-46619 (apparent pK_B value of 5.88±0.04; n=4).
4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U-46619 (0.01–3 μM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK_B of 8.54±0.14 (n=4 for each antagonist concentration). PGF_2α in the range 0.01–10 μM (n=7), but not PGE₂ and PGE₁ (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5).
5. Cumulative additions of U-46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U-46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2).
6. We concluded that TXA₂ can induce direct contraction of human isolated urinary bladder through the classical TXA₂ receptor. Prostanoid receptors, fully activated by PGE₂ and PGF_2α are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE₂ seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA₂ and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

     

Cardiac and microcirculatory effects of different doses of prostaglandin E1 in man

Summary A cumulative dose response to intravenous PGE₁ was established in 12 healthy volunteers. Systolic time intervals, including pre-ejection period (PEP), the ventricular ejection time (VET) and the RR-interval, were continuously determined, and transcutaneous oxygen pressure (tcpO₂) was recorded.RR-intervals fell in a dose dependent manner, reaching a significantly lower level at 128 ng·kg^–1·min^–1 of PGE₁ (basal value 842 ms falling to 756 ms). PEP decreased from 89 ms to 74 ms and the ratio PEP/VET decreased from 35% to 30%, indicating increased myocardial contractility. The maximal increase in tcpO₂ was 125% on the calf and 60% on the foot. The peak tcpO₂ was observed at an infusion rate of 16 ng·kg^–1·min^–1 PGE₁. A decline in tcpO₂ was seen at infusion rates >64 ng·kg^–1·min^–1 PGE₁, indicating a decrease in skin perfusion.The results indicate that the effects of intravenous PGE₁ on skin perfusion occur at a lower threshold than the increase in myocardial contractility. A maximal increase in skin perfusion can be achieved with doses of PGE₁ devoid of systemic haemodynamic effects.     

Progress in the prostaglandin E1-therapy of the intermittent claudication by means of bolus injections of LIPO-prostaglandin E1 (LIPO-PGE1)

Objective: We compared the efficacy of a bolus injection (5?min) of LIPO-PGE₁ (Prostaglandin E₁ in lipid emulsion) with conventional PGE₁-cyclodextrin (PGE₁-cyclodextrin) infusions (2?h) in patients with intermittent claudication. The quantitative blood-flow in the common femoral artery was measured using a computerized ultrasound Doppler system (MAVIS^®). we also monitored the transcutaneous oxygen pressure, the skin temperature on the foot, and the reactive change in blood pressure and pulse as well as side effects. Results : Dose finding of LIPO-PGE₁: After bolus injection of 30, 50, and 80?μg LIPO-PGE₁ a significant dose-dependent increase of the blood flow in the leg (+?96.9%, 80?μg) with a peak 3?h after injection was seen. After LIPO-PGE₁ we observed an enhanced microcirculation (significant rise in the transcutaneous oxygen pressure and the skin temperature on the foot). We noted longer lasting pharmacodynamic properties with LIPO-PGE₁ (50?μg) compared to PGE₁-cyclodextrin (60?μg). Comparison to PGE₁-cyclodextrin: In a cross-over, placebo-controlled study, 20 patients with intermittent claudication received 4 weeks therapy with a bolus of 50?μg LIPO-PGE₁ or a 2?h infusion of 60?μg PGE₁-cyclodextrin per day. A significant increase in the blood flow was measured at the end of 4 weeks therapy compared to the initial values before treatment. This rise correlates significantly with the increase in the patient’s maximal walking distance (+112%, LIPO-PGE₁). Compared to conventional PGE₁-cyclodextrin infusions given over 2?h, a clearly prolonged increase in perfusion of the affected limb after LIPO-PGE₁ was demonstrated. No serious adverse effects were observed.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

Effects of β-cyclodextrins on skin: implications for the transdermal delivery of piribedil and a novel cognition enhancing-drug, S-9977

The effects of β-cyclodextrin (β-CD), randomly methylated β-cyclodextrin (RAMEB) and 2-hydroxypropyl β-cyclodextrin (HPβ-CD) on skin were investigated. The three cyclodextrins (CDs) were able to destabilize model liposomes and to extract significant amounts of cholesterol from isolated stratum corneum (SC). However, only RAMEB extracted all the major lipid classes from isolated SC, as shown by thin layer chromatography. Both RAMEB and HPβ-CD could release 5–10% of the extractable cholesterol as well as proteins from hairless rat skin. Nevertheless, CDs did not induce any major modification of the differential scanning calorimetry (DSC) profile or the Fourier-transformed infrared (FTIR) spectrum of SC. This was explained by the low percutaneous penetration of CDs. Furthermore, the influence of RAMEB on the transdermal diffusion through hairless rat skin of piribedil, a central dopaminergic agonist and of S-9977, a novel cognition enhancing drug, was studied. RAMEB was found to decrease the transdermal flux of piribedil, with which it forms an inclusion complex, as shown by NMR. Conversely, RAMEB increased by 2-fold the percutaneous absorption of the S-9977 hydrochloride, which does not interact with CD. Finally, a combination of oleic acid and RAMEB greatly increased by about 30-fold the flux of S-9977 hydrochloride.     

Biopharmaceutics: Effect of Ointment Bases on Topical and Transdermal Delivery of Salicylic Acid in Rats: Evaluation by Skin Microdialysis

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water-soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o-type emulsion containing water). The ointments (0.1 g) containing 25 μmol salicylic acid were applied for 2 h to the surface of rat skin (1 cm²) with (intact) or without the stratum corneum. For intact skin, the extent of topical delivery from different ointments, evaluated by the area under the concentration-time curve (AUC) of salicylic acid in the skin tissue (AUCskin), increased in the order solbase. white petrolatum, poloid, hydrophilic poloid. absorptive ointment. The ratio of AUC_skin (topical delivery) to the AUC of salicylic acid in plasma (AUC_plasma, transdermal delivery) varied remarkably among the different bases, the greatest ratio being observed for absorptive ointment. When the ointments were applied to skin surface without stratum corneum, AUC_skin for solbase was much higher (about 45 times that for intact skin), whereas only a small (two-fold) increase was observed for poloid and hydrophilic poloid and the increase was negligible for white petrolatum and absorptive ointment. For skin without the stratum corneum, the ratio AUC_skin/AUC_plasma for the different ointments was comparable, although the magnitudes of AUC_skin and AUC_plasma still varied substantially. The variance of AUC values arises as a result of the different rates of release of salicylic acid from the bases. These results indicate that: the topical and transdermal delivery of salicylic acid in intact skin varies substantially among different ointment bases, and the greatest topical delivery is observed for absorptive ointment; use of absorptive ointment increases the retention of salicylic acid in the stratum corneum; and the stratum corneum functions strongly as a penetration barrier for solbase, moderately for poloid and hydrophilic poloid, and less for absorptive ointment and white petrolatum.     

Effect of Different Carriers on in vitro Permeation of Meloxicam through Rat Skin

The ability of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, polyvinyl pyrrolidone and urea to influence the percutaneous absorption of meloxicam through isolated rat skin was evaluated. Carrier complex were prepared by kneading method in 1:1 and 1:2 in molar ratios for β-cyclodextrin and hydroxypropyl-β-cyclodextrin and in 1:1, 1:3 and 1:5 in weight ratios for polyvinyl pyrrolidone and urea. The complexes were characterized by IR, DSC and evaluated for solubility, dissolution and skin permeability. The solubility, dissolution and permeability of meloxicam were enhanced by using the carriers. The influence of cyclodextrins, polyvinyl pyrrolidone and urea on in vitro permeation of meloxicam through rat skin was investigated by incorporation of prepared carrier complex in 1% carbopol gel. The prepared gel was evaluated for drug content, pH and viscosity and in vitro permeation. All the percutaneous parameters like flux (Jss), amount permeated (Q(6)), diffusivity (D), permeability coefficient (K(p)), partition coefficient (K) and release rate constant (k) were calculated statistically. In vitro permeation study showed the trend that the penetration flux and enhancement factor increases with increasing concentration of β-cyclodextrin and hydroxypropyl-β-cyclodextrin and then decrease dramatically in case of hydroxypropyl-β-cyclodextrin gel formulation with the increase to 1:2 ratio. Similar changes in pattern of permeation were also observed with polyvinyl pyrrolidone and urea carrier complex. These findings concluded that the carriers cyclodextrins, polyvinyl pyrrolidone and urea could be used as transdermal permeation enhancer in topical preparation of meloxicam.