Evaluation of sildenafil pressurized metered dose inhalers as a vasodilator in umbilical blood vessels of chicken egg embryos

Abstract

Sildenafil citrate is a selective phosphodiesterase-5 inhibitor used for the treatment for erectile dysfunc- tion and pulmonary hypertension. The delivery of sildenafil directly to the lung could have several advan- tages over conventional treatments for pulmonary hypertension because of the local delivery, a more rapid onset of response, and reduced side effects. The major problem of sildenafil citrate is its limited solubility in water. Sildenafil citrate was complexed with cyclodextrins (CDs) to enhance its water solubility prior to development as an inhaled preparation. Four sildenafil citrate inhaled formulations were prepared with the aid of HP-b-CD (#1), a-CD (#2) and c-CD (#3) and their effects were compared with the formulations without CDs (#4). The sildenafil citrate pressurized metered dose inhalers (pMDI) used ethanol as a solvent, PEG400 as a stabilizing agent, sorbitan monooleate as a surfactant and HFA-134a as a propellant. All formulations consisted of sildenafil citrate equivalent to a sildenafil content of 20 lg/puff. These products were evaluated according to a standard guideline of inhalation products. Vasodilation testing was performed to investigate the efficacy of sildenafil pMDIs in relieving a vasocon- stricted umbilical blood vessel of the chicken egg embryo. The sildenafil contents of the pMDI formulations #1–#3 were within the acceptance criteria (80–120%). The emitted doses (ED) were 102.3 ± 11.5%, the fine particle fractions (FPF) were 60.5 ± 5.6% and the mass median aerodynamic diameters (MMAD) were 2.3 ± 0.3 lm. The vasodilatory activity of those formulations reduced umbilical blood pressure by 67.1–73.7% after treatment by intravenous injection whereas only a 50.1–58.0% reduced blood pressure was obtained after direct spraying of the sildenafil pMDI containing CDs. With sildenafil formulations of a pMDI without CD the blood pressure was reduced by only 39.0% (P-value < 0.05). The available sildenafil in the blood vessels of chicken egg embryos after spraying sildenafil-CDs pMDIs was within the range of 751–825 ng/mL which was much higher than that of a sildenafil only pMDI.

1. Introduction

Sildenafil citrate or (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3- propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl] sulfo- nyl]-4-methyl-piperazine citrate) (Fig. 1) is a highly selective phosphodiesterase-5 (PDE-5) inhibitor. It was the first oral agent for treatment for erectile dysfunction [1–3] and has recently been used for treating pulmonary hypertension [4–7].

Nowadays sildenafil is normally administered to patients with pulmonary hypertension in an oral tablet dosage form. Sildenafil is rapidly absorbed after oral administration, but provides a relatively low absolute bioavailability of only about 40–41% and because it has a late onset of action [2,8] this has led to a serious concern for treating infants and unconscious persons. It would be preferable if pulmonary hypertension could be treated by direct delivery to the target site of action. Also, adverse drug reactions can occur after administration of high oral doses to treat pulmon- ary hypertension. Delivery of the sildenafil directly to the lung could have several advantages over conventional treatments for pulmonary hypertension because of its immediate access to the target site, a more rapid onset of response, and reduced side ef- fects. In addition, since the receptors are located on the smooth muscle cells of the blood vessel walls in the lung, sildenafil would have more selective hemodynamic effects; this may increase the effectiveness of the delivered dose and reduce the dose needed [6,9,10]. Several studies have successfully developed inhaled silde- nafil as a nebulizer [11,12] or by intratracheal instillation [13] rather than by a conventional pressurize metered dose inhaler modified the process for rapid testing and increased experimental efficiency using umbilical blood vessels from late chicken egg em- bryos as an in vitro model.

Consequently we have now used this model system to help to de- velop sildenafil as a pMDIs using sildenafil complexed with various types of CD as a solubilizing agent (a-cyclodextrin, a-CD; hydroxy-propyl-b-cyclodextrin, HP-b-CD and c-cyclodextrin, c-CD). The vasodilatory activity of the pMDIs formulation was also investigated in blood vessels of chicken egg embryos after sildenafil pMDIs was applied by a direct spraying method onto blood vessels and com- pared to the result from an injectable route.

To date there have been no publications on the preparation and use of sildenafil pMDIs. The pMDIs could provide a more conve- nient way to treat pulmonary hypertension patients. The major problem of sildenafil citrate is its limited solubility in water (4.1 mg/mL in water) [8]. We have now developed sildenafil citrate complexed with cyclodextrins (CDs) suspended in a HFA-134a propellant in order to improve its in vitro deposition efficiency and improve its stability in the pMDI formulations. Sildenafil is well known for its vasodilatory activity through inhibition of the cyclic guanosine monophosphate (cGMP) enzyme and the nitric oxide (NO) degradation pathway [3,10]. In order to investigate the efficacy of sildenafil pMDIs on its vasodilatory activity, we have developed a model to facilitate experimentation.

The specified dose in this study (sildenafil 20 lg/dose) has been previously reported [19]. Briefly, a dose was calculated from the Cmax of sildenafil multiplied by the extravascular lung fluid. Usually pulmonary administration reduces the dose required by from 40 to 100 times in comparison with oral administration when only 20–25% of the delivery dose of pMDI reached the target site.

Animal models of pulmonary hypertension have been used extensively [20]. These models have produced a plethora of scientific information, such as the role of certain molecular mechanisms and genetic contributions during a pulmonary hyper- tension episode. In addition, animal models have been utilized for the discovery and testing of possible therapeutic approaches. Pulmonary hypertension can be induced in animals either through use of pharmacologic/toxic agents, genetic techniques, and envi- ronmental factors or through surgical interventions [20].

The most commonly used animal models used for pulmonary hypertension studies involve the injection of a monocrotaline or by chronic exposure to hypoxia (classical models). There are a variety of other in vivo animal models used for testing pulmonary hypertension that mimic features of different pulmonary hyper- tension groups [20]. Matinez-Lemus et al. [21] reported changes in the vasodilatory activity that occurred in Broiler and Leghorn chickens from late embryonic life to 5 weeks of age. Pulmonary arteries were isolated from 19 to 20 day old embryos, hatchings, and 1, 2, 3, 4 and 5 week old chickens of both groups and subjected them to KCl (45.4 mM) and endothelin-1 (10—7.5 M) induced vaso- constrictions. The vasoconstrictions were greatest at the hatch stage, then rapidly declined. In addition, U46619, thromboxane A2 (9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2a) was of more interest for inducing vasoconstriction in pulmonary arteries than KCl [22]. This method is still rather complicated for the analysis of vasodilation in chicken embryos.

2. Materials and methods

2.1. Materials

Sildenafil citrate and a reference standard (potency 99.4% as is) were obtained from Smilax Laboratories Limited (Andhra Pradesh, India). All types of CD (a-CD, or CAVAMAX® W6 Pharma; HP-b-CD, or CAVASOL® W7 HP Pharma; and c-CD, or CAVAMAX® W8 Pharma) were from ISP Pharmaceuticals (Wayne, NJ, USA). PEG 400 and sorbitan monooleate were from the P.C. Drug Center Co. (Bangkok, Thailand). Dried ethanol (99.9%) was obtained from RCI Labscan (Bangkok, Thailand). The propellant HFA-134a (ZEPHEX® 134a; pharmaceutical grade, purity 99.99%) was from Mexichem Fluor (Runcorn, Cheshire, UK). All solvents used were HPLC grade and supplied by RCI Labscan (Bangkok, Thailand). Isotonic sodium chloride solutions and Lactated Ringer’s solutions were obtained from A.N.B. Laboratories Co., Ltd. (Bangkok, Thailand). KCl solution and U46619 (9,11-Dideoxy-11a,9a-epoxymethanoprostaglandin F2a) solution 10 mg/mL in methyl acetate were obtained from Sigma–Aldrich (St. Louis, MO, Germany). Omeprazole sodium was from Aastrid International Pvt. Ltd. (Mumbai, India).

2.2. Chicken egg embryos

Chicken eggs from Rhode Island Red Cross hens as a hybrid layer strain were obtained from the Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. Fertile eggs were placed in a hatching incubator for 15 days at 37 °C and 55% humidity. They were used within 3 h after the chick- en egg shells first cracked.

2.3. Preparation of sildenafil pMDIs

Sildenafil citrate pMDI formulations were prepared by the pressure filling method. In brief, the required quantity of sildenafil citrate for each type of CD (a-CD, HP-b-CD, c-CD) and dried ethanol were mixed in a vessel and maintained in an ice bath to form the complex solutions. Stabilizing agents (PEG 400 and sorbitan monooleate) were added and then mixed using a Vortex-Genie 2 (Scientific Industries, Bohemia, NY, USA) to obtain product concen- trates. The product concentrates were dispensed by aliquots into glass bottle canisters (Schott AG, Mainz, Germany). Then 50 lL metering valves (Bespak Europe, Ltd., Norfolk, UK) were immedi- ately crimp-sealed onto the canisters using an aerosol crimping machine (model 2016, Pamasol Willi Mäder, Zurich, Switzerland) with a 20 mm neck diameter; the canisters were then filled with a specified amount of HFA-134a propellant with the same machine. Nanocrystals of sildenafil citrate monohydrate were formed in situ after an antisolvent (HFA-134a propellant) was added. Uniformity of the dispersion was guaranteed by sonicating the canisters for 30 min in an ultrasonic bath (Sonorex; Bandelin, Berlin, Germany) to achieve well-dispersed suspensions [23–25]. The details of all formulations are presented in Table 1.

2.4. Preparation of sildenafil citrate for intravenous injection

Sildenafil citrate was administered as an intravenous injection that was similar to those of the sildenafil pMDI #1, #2 and #3 except that the propellant was absent. The concentrated product formulations #1, #2 and #3 were diluted with normal saline solu- tion to obtain a concentration of sildenafil similar to that used for the sildenafil intravenous injection.

2.5. Analysis of sildenafil citrate from sildenafil pMDIs

Sildenafil citrate was analyzed by high performance liquid chro- matography (HPLC). The HPLC system (Waters, Milford, MA, USA) consisted of a solvent delivery pump equipped with an in-line degasser (Waters 1525 binary HPLC pump), a sample loop with an injection volume of 20 lL, and a Waters 2707 autosampler. Data were recorded using Empower 2 software. Separations were performed on a reversed-phase stainless steel column (ACE 5 C18-AR; Advanced Chromatography Technologies, Aberdeen, Scotland) (250 mm long × 4.6 mm internal diameter) filled with a 5 lm octadecylsilane and maintained at 25 °C. The mobile phase consisted of a degassed mixture of 0.2 M ammonium acetate buffer and acetonitrile in a ratio of 40:60 by volume at ambient tempera- ture; the pH was adjusted to 7.0 with 0.1 M NaOH prior to use. The flow rate was maintained at 1.0 mL/min, and the separation was monitored by UV detection (Waters 2998 photodiode array detec- tor) at a wavelength of 240 nm. The method was validated for 8 parameters: specificity, range, linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and robustness. In addition the systems suitability parameter was also calculated.

2.6. Content of sildenafil delivered by actuation of the pMDI valve

The content per spray was determined by discharging the pres- surized container through a central hole of a stainless steel base plate that was placed in a suitable vessel. A specified volume of the mobile phase used for the HPLC was added into the vessel. The inhaler was discharged in an inverted position under the surface of the solvent. The pressurized inhaler was shaken for 30 s prior to each actuation; the first two doses were discharged as waste. Ten deliveries at the beginning (3rd–12th dose), middle (101st–110th doses) and end (191st–200th doses) of the calculated number of doses were analyzed by HPLC for the amount of sildena- fil. The result was calculated as the amount of active ingredient delivered from each actuation of the valve.

2.7. Assessment of the size distribution of the aerodynamic particles and aerosol properties of the pMDIs

The measurement of the aerodynamic particle size distribution was performed on an eight-stage Andersen cascade impactor (ACI); details are provided in the British Pharmacopoeia as Apparatus D [26]. The pressurized inhaler was shaken for 5 s and the first deliv- ery was discharged as waste. The pMDI was connected to the metal inlet of the ACI using a rubber adaptor. Air was drawn through the apparatus and the flow rate was adjusted to 28.3 L/min (±5%). Then, exactly 2 sprays were fired from the inverted inhaler into the apparatus. A shaking time of 5 s was required between each delivery. Eight perforated stages were arranged in ascending order of their diameters, with stage 0 on the top and stage 7 on the bot- tom. The metal inlet and all stages were rinsed with the mobile phase. Each fraction was adjusted to a specified volume, and the amount of drug deposited on each plate was determined by HPLC. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated as described elsewhere
[27,28]. The deposition of the drug from stage 2 (particle size less than 4.7 lm) to the filter was termed the fine particle fraction (FPF) and the amount of sildenafil deposited from metal inlet to all stages of ACI was termed the emitted dose (ED).

2.8. Testing for vasodilatory activity

2.8.1. Testing for intravenous sildenafil
The vasodilatory testing method was developed in our labora- tory. The chicken egg embryo was placed in a Petri dish and soaked with Lactated Ringer’s solutions on the tissue all the time during the experiment. A blood pressure transducer (AD Instruments, Australia) was connected with a Needle No. 26 and the opposite way was connected to a syringe that contained U46619 in an iso- tonic solution. A 3-way valve was connected to the umbilical blood vessel, the syringe and a blood pressure monitor (Fig. 3). The mon- itor terminal was connected to a Bridge Amplifier (AD Instruments, Australia) equipped with PowerLab 4/20 Data Acquisition Systems (AD Instruments, Australia) and connected to a computer to dis- play the results and record blood pressure from the blood vessels. The Needle No. 26 was inserted into an umbilical blood vessel of the chicken egg embryo (see Fig. 2). Details of the circulatory sys- tem of the late chick embryo have been described elsewhere [29]. Fig. 3(A) shows the experimental set up for monitoring the arterial blood pressure. Prior to the experiment, the measurement of the blood pressure was calibrated by a 3-point calibration with a sphygmomanometer. At the beginning, the path to the blood pres- sure transducer was blocked by switching the 3-way valve and the path to the syringe containing U46619 in an isotonic solution was opened. U46619 was then administrated to induce vasoconstric- tion of the chicken umbilical blood vessel. Following that, the 3-way valve was changed to block the syringe and open the way to the blood pressure transducer to measure blood pressure (Fig. 3(A)).

The syringe containing the vasoconstriction solution was chan- ged to a syringe containing the sildenafil formulation for injection. The 3-way valve was again closed for pressure measurements and the way to the blood vessel for the formulation was opened and the sildenafil formulation (20 lg) was injected. The way from this syringe to the blood vessel was then closed and the way to the pressure measurements was opened. The vasodilatory activity was observed from the computer display. This experiment was repeated 12 times for each formulation.

2.8.2. Sildenafil spray testing

The experiments were carried out in a similar way to the intra- venous injection but without an injection. After the U46619 was injected for the vasodilation, the sildenafil pMDI formulations were sprayed onto a blood vessel (Fig. 3(B)). The experimental setup had been readied to record the blood pressure changes on a real time basis. This experiment was repeated 12 times for each formulation. The normal saline solution (NSS, sodium chloride 0.9%) solution was used as a negative control and an intravenous sildenafil solu- tion was used as a positive control.

2.9. Determination of sildenafil in a blood vessel

Liquid chromatography–mass spectrometry–mass spectrome- try (LC–MS/MS) was employed to determine the sildenafil in a blood vessel of the chicken egg embryo. In brief, the sildenafil levels in the blood of the chicken egg embryo were quantified using an Agilent 1260 Infinity HPLC system (Agilent, Singapore). The HPLC system consisted of a solvent delivery pump equipped with an in-line degasser and autosampler. The HPLC was equipped with an ESI probe and a quadrupole mass analyzer (ABSCIEX API 3200™ LC/MS/MS system, Singapore). The control of the LC–MS/ MS system and the data acquisition was performed using the Analyst® version 1.6 software. The carrier flow used was 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid, which was delivered isocratically at a flow rate of 0.2 mL/min. The carrier flow was first filtered and degassed using a 0.22 lm nylon membrane filter before use. The internal standard used in this study was omeprazole as determined from a previous report [30]. The optimized MRM settings for the individual drugs for the ESI operating in the positive mode are shown in Table 2.

The samples were prepared by spiking 25 lL of internal standard (10 lg/mL omeprazole) into 0.5 mL of blood samples. Each sample was mixed for 10 s in a 10-mL glass tube. Diethyl ether (4 mL) was added to each tube and the drugs were extracted by vortexing the tubes for 5 min. The samples were then centrifuged at 3000g for 10 min, the ether layer was transferred to another tube and evaporated to dryness under a gentle stream of nitrogen at a temperature set at 40 °C. The residue was reconstituted in 50 lL of ACN and an aliquot of 25 lL was injected into the column.

Both sildenafil and omeprazole were eluted at room temperature within 1 min in a positive ESI mode with a single-ion recording at m/z 475 and 346, respectively, as these are consistent with the [M+H]+ molecular ions. A calibration curve was prepared by a spik- ing technique in the range of 0.5–2000 ng/mL. Fig. 4 shows the ESI major fragments from sildenafil and omeprazole that were used to determine the sildenafil in the blood sample.

2.10. Statistical analysis

All data were expressed as a mean ± SD. A one-way ANOVA was used to test for differences between multiple groups, and the significance of the differences between means was tested with the Fischer’s protected least significant difference (Fischer’s PLSD).

3. Results and discussion

3.1. Analysis of sildenafil in pMDIs

This method was developed to determine sildenafil in the pMDIs formulation when CDs and other excipients were present. The retention time of sildenafil was about 5.5 min. From the HPLC chromatogram, the CDs that had been complexed with sildenafil did not interfere in the determination of sildenafil in this system. The analytical parameter results were acceptable for the determination of the low concentrations of sildenafil in the pMDIs. The accuracy of the method to assay for sildenafil was 99.5% recov- ery and the precision for both intra-day and inter-day assays were 0.5–1.0% and 1.1%, respectively. There was a linear correlation over the concentration range of 0.5–500 lg/mL (r2 = 1.0). The LOD and LOQ were 1.30 and 6.10 ng/mL, respectively. This method also resolved any degradation products from the sildenafil peak. The details of the analytical validation of the sildenafil pMDIs assay have been previously described [31].

3.2. Sildenafil contents of the pMDIs

The amounts of the active ingredients delivered by actuation of the valve after 1 month of storage are shown in Fig. 5. The delivered dose of formulation #1 at the beginning (first three dose) was 107.9 ± 3.6%, and at the last three dose was 107.8 ± 6.0%. The delivered dose of formulation #2 varied from 104.6 ± 10.8% to 95.8 ± 3.4%. The delivered dose of formulation #3 was similar to those of #1 and #2 (102.8 ± 6.7% to 107.8 ± 3.4%). All formulations generated sildenafil contents close to 100%.

The delivered dose of sildenafil without CD was only 41.6 ± 9.8% to 40.2 ± 3.3%. This confirmed the importance of CDs in the sildena- fil pMDI formulations. In general, the acceptance criteria of pMDI formulations were within 80–120% LA [26,28]. Formulation #4 gave a very low delivered dose of sildenafil compared with the for- mulations containing CDs. The delivered dose of sildenafil from formulation #4 failed to meet the requirement therefore no further experiments were performed with this formulation. Our previous work revealed that a CD was necessary to obtain an acceptable sildenafil delivered dose when sildenafil was formulated into a pMDI [19].

3.3. Aerosolized properties of sildenafil pMDIs

After 1 month and 6 months of storage, formulations prepared with CDs (#1, #2, #3) were assessed for their particle size distributions using an ACI (Table 3). The MMADs of sildenafil were within a suitable size range (1–5 lm) [32] prior to storage. The size distri- bution of all formulations deposited at the different stages of the ACI is shown in Fig. 6. As sildenafil from the pMDIs was deposited at stage 2 (<4.7 lm) to stage 7 (<0.4 lm) of the ACI it was used as an indicator to predict the efficiency of drug being delivered to the lower airways [32,33]. Although the receptors for sildenafil are located in the arterial smooth muscle, any aerosol particles of sildenafil reaching the lower airways will dissolve and diffuse to the arterial smooth muscle cell of the lung as the target organ [3,9,34]. Sildenafil dissolves in the fluid of the alveoli then passes to the peripheral blood vessels that supply the surrounding the alveoli. When the sildenafil diffused to the smooth muscle cell of the arterioles, vasodilation occurs and relieves pulmonary hypertension.

Formulation #1 consisted of HP-b-CD, with an ED of 81.5 ± 4.1%, and FPF of 54.1 ± 1.3% and an MMAD of 2.06 ± 0.3 lm. Formulation #2 consisted of a-CD, the ED was 108.8 ± 10.8%, the FPF was 45.8 ± 6.0% and the MMAD was 2.90 ± 0.4 lm. Formulation #3 con- sisted of c-CD, the ED was 116.6 ± 9.6%, the FPF was 81.6 ± 7.3% and the MMAD was 1.94 ± 0.2 lm. Formulation #3 with c-CD produced the highest ED and FPF of sildenafil perhaps because this
formulation generated more consistent aerosol properties than the other formulations (less variation in the assay content). The high energy of the HFA-134a propellant also had a significant effect on delivering a high ED of sildenafil because it generated a suitable aerosol cloud from the actuator.
After storage for 6 months, all formulations showed that the ED, FPF and MMAD values had not changed from those at 1 month (P-value > 0.05). These results showed all formulations had highly stable aerosol properties.

The interaction of sildenafil with CDs in the pMDI formulations has been well described elsewhere [19,35]. Sildenafil and CDS formed crystal complexes through assistance with ethanol and PEG. The high stability of the sildenafil released from the pMDI can be explained by two factors: (a) the use of CDs to form complex crystals; and (b) the use of PEG 400 as the excipient. The crystal complex of sildenafil occurred by crystallization of the concen- trated product that was precipitated in the HFA-134a propellant [19,35]. CDs are well-known excipients in the pharmaceutical industry, and are used to improve the stability of a drug [36,37]. CDs have been approved for use as pharmaceutical excipients in intravenous [38] and pulmonary formulations [39–41]. Also, in vitro toxicity studies with human lung cell cultures and human clinical experience have indicated the safety of cyclodextrin deriv- atives [35,42]. The use of PEG 400 in the pMDI formulations en- hanced the stability of the suspension, in accordance with previous reports [35].

3.4. Analysis of sildenafil in a biological fluid

The HPLC–MS/MS analysis technology was found to be very useful and has been a most powerful analytical tool in clinical pharmacokinetics because of its selectivity, sensitivity and repro- ducibility [43,44]. Using the chromatographic conditions described in section 2.9, the retention times ranged from 0.25 to 0.60 min for sildenafil and for 0.45 to 0.65 min for the internal standard ome- prazole. The daughter ion (Q3) of sildenafil was selected at m/z 283 because of its abundance. The daughter ion (Q3) of the internal standard was selected at m/z 197.8 fragment ions according to Smet et al. [45]. The ESI fragments are shown in Fig. 4. The major fragment of sildenafil has been reported and proposed by various workers [45–48]. For this work, the major fragments of sildenafil (m/z = 283) were the pyrazolopyrimidine ring because of the 311 fragment. The major fragment of omeprazole benzoimidazole ring had an m/z of 197.8 [45,48].

The assay for the sildenafil content in the biological fluid after spraying into blood vessels of formulation #1, #2 and #3 was 813 ± 98 ng/mL, 825 ± 82 ng/mL and 751 ± 121 ng/mL, respec- tively. The assay for the sildenafil content from the sildenafil solu- tion without CDs as the control was 310 ± 82 ng/mL. A single dose of sildenafil itself (100 mg) in patients with pulmonary hyperten- sion produced 570 ng/mL sildenafil in the plasma [49]. The maximum concentrations of sildenafil in the blood after adminis- tration of the same dose in fed and fasted condition were 364 and 514 ng/mL respectively [2]. The effective dose for pulmonary hypertension in the plasma was not more than 500 ng/mL of blood. The results of the sildenafil levels in the chicken egg embryo blood vessels after injection of the sildenafil pMDIs were within 750–850 ng/mL i.e. much higher than found for the solution of sildenafil without CDs (310 ng/mL). Thus the contribution from the delivery dose of sildenafil without CDs was lower than the sildenafil pMDIs by about 2.5–3 times. In addition, CDs have been reported as absorption enhancers as well as solubility enhancers [36].

This experiment revealed the availability of sildenafil pMDIs in the blood vessel. The next step in the development of sildenafil pMDIs requires pharmacokinetics data and sildenafil levels in the blood vessel and lung fluid.

3.5. Vasodilatory activity of sildenafil after administered as an intravenous injection and spraying directly onto the blood vessel

All formulations (#1, #2 and #3 including HP-b-CD, a-CD and c-CD, respectively) produced vasodilatory activity by intravenous treatment and spraying that directly delivered sildenafil to the blood vessel. The umbilical blood pressures are shown in Fig. 7.After injection of sildenafil itself without CDs, the umbilical blood pressures were reduced by 55.1%. The umbilical blood pres- sures were reduced 67.1%, 68.4% and 73.7% after treatment with the sildenafil – CD injections (#1, #2 and #3, respectively). The results indicated that sildenafil – CD injections were more effective than the sildenafil solution (P-value < 0.05) due to the CDs provid- ing enhanced solubility and promotion of its biological activity. When sildenafil pMDIs were administered by direct spraying onto the blood vessels where the PDE-5 receptor is located, the umbili- cal blood pressures were reduced by 54.1%, 58.0% and 50.2% after spraying with the formulations #1, #2 and #3, respectively. The umbilical blood pressures after treatment with sildenafil spray solution without the CDs were reduced by only 39.0%. The results showed that the sildenafil – CD spray formulations were more effective than the sildenafil solution spray (P-value < 0.05) a simi- lar observation to that found for the intravenous injection.

It is important to note that the sildenafil complexed with CDs and applied by intravenous injection were far more effective in reducing blood pressure than spraying the sildenafil on the outside of the blood vessel. The results of the intravenous injection showed higher vasodilatory activity than sildenafil pMDIs after it was sprayed onto the blood vessels directly (P-value < 0.05). All formu- lations treated by both treatments including sildenafil solutions produced higher vasodilatory activity than the negative control (NSS) by over 50% (P-value < 0.01). Direct injection into the target site provided a better access to the smooth muscle cells of the blood vessel than spraying presumably because there was no need to cross the outer membranes of the blood vessels. In addition, the spraying method of the pMDIs onto the umbilical blood vessels made it more difficult to obtain precise control of the spraying areas. The spread of the aerosol clouds reaching the effective area may have also contributed to the lower vasodilatory activity of the sildenafil compared with direct intravenous injection.

The mechanism of blood vessels vasodilation has been described in several previous publications [34,50,51]. The PDE-5 isozyme metabolizes cGMP, which is the second messenger for NO and a principal mediator of smooth muscle relaxation and vasodilation. By inhibiting the hydrolytic breakdown of cGMP, sildenafil prolongs the action of cGMP. This augmented smooth muscle relaxation in part, by reducing production of endothelial NO [3]. In this study, the concentration of sildenafil in blood vessels showed a correlation with vasodilatory activity. The higher amount of sildenafil reaching the PDE-5 isozyme in blood vessel walls caused better vasodilation and lower blood pressure.

4. Conclusion

In this report we have described the development of sildenafil citrate pMDIs by using a-CD, HP-b-CD and c-CD as complexing agents that increased the aqueous solubility of sildenafil and produced enhanced biological activity. Formulations without CD produced a lower delivered dose of sildenafil. All formulations with CDs showed that their aerosol properties were suitable for inhala- tion. The sildenafil pMDIs spray showed much better vasodilatory activity than the sildenafil spray solution alone (P-value < 0.05).