Assessment and Development of the Antifungal Agent Caspofungin for Aerosolized Pulmonary Delivery

Invasive Pulmonary Aspergillosis (IPA) and Pneumocystis jiroveci Pneumonia (PCP) are serious fungal pulmonary diseases for immunocompromised patients. The brand name drug CANCIDAS® (Caspofungin acetate for injection) is FDA approved to treat IPA, but is only 40% effective. Efficacious drug levels at the lung infection site are not achieved by systemic administration. Increasing the dose leads to toxicity. The objective, here, is to reformulate caspofungin for aerosolization to high drug concentration by lung targeted delivery and avoid systemic distribution. Described in this paper is a new, room temperature-stable formulation that meets these goals. The in vitro antifungal activity, solid state and reconstituted stability, and aerosol properties of the new formulation are presented. In addition, pharmacokinetic parameters and tissue distribution data are determined from nose-only inhalation studies in rats. Plasma and tissue samples were analyzed by High Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS-MS). Inhaled drug concentrations for caspofungin Active Pharmaceutical Ingredient (API), and the new formulation, were compared at the same dose. In the lungs, the parameters Cmax and Area Under Curve (AUC) showed a 70%, and 60%, respective increase in drug deposition for the new formulation without significant systemic distribution. Moreover, the calculated pharmacodynamic indices suggest an improvement in efficacy. These findings warrant further animal toxicology studies and human clinical trials, with inhaled caspofungin, for treating IPA.


S2.1. HPLC Analysis
HPLC analysis was carried out on a Waters 2695 separations module equipped with an autosampler and a Waters 996 photodiode array detector. The HPLC method is described below: Column: Waters symmetry C18 3.5 μm, 4.6 x 75 mm Flow rate: 1.0 mL/min Detection: 220 nm Column Temperature: 30 °C Autosampler temperature: 4 °C Injection volume: 50 μL Run time: 70 min Mobile phase: A: Add 1.0 mL of perchloric acid and 0.75 g of sodium chloride in 1000 mL HPLC grade water; B: Acetonitrile.
Gradient: As shown in Table S1 The liquid formulations to be lyophilized were prepared according to Table S2. Aliquots of 0.5 mL of each liquid formulation were placed in 3 mL glass vials, frozen on dry ice for 40 min. The vials were partially stoppered with lyo septa and lyophilized for 5 days. The vials were placed on stability at 5 °C and 25 °C (dark). Caspofungin acetate (100 mg, 97.6% assay) was dissolved in DI water (0.525 g) to obtain 0.625 g caspofungin acetate stock solution at a nominal concentration of 160 mg/mL (assuming density = 1). PVP K30 (400 mg) was dissolved in PBS buffer (3.6 g), adjust pH to 6 with 25 μL of 1.0 N HCl to obtain 4.0 g of PVP stock solution with a nominal concentration of 100 mg/mL (assuming density = 1). Caspofungin acetate stock solution of step 1 was combined with the PVP solution of step 2. An additional 0.375 g of PBS buffer was added to obtain 5.0 g of the pre-lyo solution with a nominal caspofungin acetate concentration of 20 mg/mL (assuming density = 1). The theoretical wt% caspofungin acetate concentration is 100 × (100 × 97.6%) / 5000 = 1.952% S2.3.2. Preparation of Caspofungin Acetate Lyo Formulation Based on Weight of the Pre-Lyo Solution (0.5 g/vial) 0.5 g of the pre-lyo solution was weighed into a lyo vial. The vial was frozen on dry ice for 1 hr and lyophilized for 5 days as described above.
S2.3.3. Preparation of Caspofungin Acetate Lyo Formulation Based on Volume of the Pre-Lyo Solution (0.5 mL/vial) 0.5 mL of the pre-lyo solution was pipetted into a lyo vial. The vial was frozen on dry ice for 1 hr and lyophilized for 5 days as described above. Mobile phase B: Acetonitrile Gradient program: As shown in Table S3. Caspofungin acetate lyo cake (50 mg) was reconstituted by adding 1.0 mL of DI water via a pipette to obtain stock solution. The solution was vortexed for 20-30 seconds and visually checked to ensure complete dissolution. 55 μL of the stock solution was pipetted and added into a HPLC vial containing 1.0 ml of DI water. The solution was vortexed for a few seconds to mix.

. Preparation of Liquid Formulations for Lyophilization
Caspofungin diacetate/PVP K30 formulations: The caspofungin diacetate stock solution of Part A (1.25 mL), the PVP K30 stock solution (8.0 g) and the corresponding diluent in the PVP K30 solution (0.75 mL) of Part B was combined to obtain 10 mL of the liquid formulations ready for lyophilization. The ratio of caspofungin diacetate/PVP K30 is 1:4 (w/w).
Caspofungin diacetate only (control): Caspofungin diacetate (0.2 g) was dissolved in deionized water (9.8 mL) to obtain a clear solution with caspofungin diacetate concentration of 20 mg/mL. Compositions of each formulation were shown as Table S6. Fourteen 0.5 mL aliquots of each liquid formulation prepared in part C were placed in 14 3-mL glass vials. The vials were frozen on dry ice for about 1 h and lyophilized for 5 days to obtain white cake. The vials were stoppered, crimp sealed and placed on stability at −20 °C, 5 °C, and ambient (dark) conditions. Lyophilization was accomplished using a VirTis benchtop manifold lyophilizer under vacuum of <50 mTorr.

S2.4.5. HPLC Analysis
One vial of the lyophilized cake was reconstituted in 1 mL of deionized water to obtain a 10 mg/mL caspofungin diacetate solution. An aliquot (55 μL) was removed and diluted with 1 mL of deionized water for HPLC analysis.

S3. Aerosol Characterization
The stock solutions of caspofungin diacetate (100 mg/mL) and polyvinylpyrrolidone (PVP K30, 100 mg/mL) were prepared. In addition, 0.9% saline was used to generate aerosols for the purpose of comparing particle size.

S3.1. Preparation of Caspofungin Diacetate Stock Solution:
500 mg of caspofungin diacetate was dissolved in 5.0 mL of 0.9% saline to obtain 5 mL of caspofungin diacetate stock solution. The concentration is 100 mg/mL (assuming the density of the stock solution was close to 1).

S3.2. Preparation of PVP K30 Stock Solutions:
In a 25 mL volumetric flask 2.5 g of PVP K30 was dissolved in 20 mL 0.9% saline. Adjusted the pH to 6 with 1N NaOH solution. Normal saline was added to the mark and mixed well. This provided 100 mg/mL PVP K30 stock solution.
Temperature, Relative Humidity and Airflow Rate: Inhalation exposure chamber temperature, relative humidity and airflow rate (liters per minute; LPM) were measured and recorded once during the exposure. The chamber temperature and relative humidity were monitored with a hand-held thermohygrometer (35612 series, Oakton Instruments, Vernon Hills, IL).

S4. Inhalation Pharmacokinetics
Caspofungin was administered to rats at a target dose of 2 mg/kg by nose only inhalation (bydeposition) or intravenously (IV) to determine the plasma and tissue concentrations and pharmacokinetics.

S4.1. Animal Preparation
Sprague-Dawley derived rats [Crl:CD®(CD)Br] were obtained from Charles River Laboratories, Inc., Wilmington, MA, for use in this study. The rats were 52 days of age (approximately 7.5 weeks) upon arrival. The animals were held in quarantine for 7 days prior to administration of the test article. The test animals were approximately 8.5 weeks old at the start of the first exposure to the test article. To condition the animals to placement and restraint in the nose-only exposure system and reduce stress during the exposure phase, the animals were acclimated to the holding tubes by placing each rat in a noseonly holding tube for approximately 45 minutes one working day prior to exposure.

S4.2. Test Article Preparation for API
For inhalation exposure, a 10 mg/mL dosing solution was prepared by dissolving 800 mg of caspofungin diacetate powder in 80 mL of 0.9% saline solution. The resulting solution was aseptically filtered and kept refrigerated between 2−8 °C until used. The formulation was aerosolized for inhalation administration.

S4.3. Test Article Preparation for TTI-016
S4.3.1. Preparation of Caspofungin diacetate stock solution 800 mg of caspofungin diacetate, which was warmed from storage as previously described, was dissolved in in 8.0 mL of 0.9% saline to obtain 8 mL of caspofungin diacetate stock solution. The concentration should have been close to 100 mg/mL (assuming the density of the stock solution was close to 1).

S4.3.2. Preparation of PVP K30 Stock Solution
In a 50 mL volumetric flask, 5.0 g of PVP K30 was dissolved in 45 mL of 0.9% saline. The pH was adjusted to 6 with 1N NaOH solution dropwise. Normal saline was added to the mark, approx 5 mL, and was mixed well to give a total volume of 50 mL. This provided 100 mg/mL PVP K30 stock solution.
S4.3.3. Preparation of Test Article: Caspofungin Diacetate (10 mg/mL), PVP K30 (40 mg/mL) In a 100 mL flask or glass bottle, 8.0 mL of caspofungin diacetate stock solution and 32 mL of PVP stock solution were added. 40 mL of normal saline was added in the flask and gently well mixed. The solution was sterile filtered through a 0.2-micron filter using a slight vacuum into a sterile 100 mL flask or bottle. The flask was stoppered. The test article was stored at 4 °C until use.

S4.4. CANCIDAS ® Solution for IV Administration (IV)
A 2 mg/mL dosing solution from commercially obtained CANCIDAS ® (containing 54.6mg of caspofungin diacetate) was prepared by adding 10.8 mL of 0.9% saline into the CANCIDAS ® vial and swirling gently until the powder dissolved. 10.0 mL of this solution was extracted and added a 25 mL volumetric flask which was diluted to the mark with 0.9% saline and mixed well. The resulting solution was aseptically filtered and kept refrigerated between 2-8 °C until used.

S4.5. Test Article Dosing
The animals were randomized into 15 animals per group based on body weight. Each group was dosed as shown in Table S7 below.

S4.6. Inhalation Exposure Methods
The inhalation exposure part of the study was conducted in an inhalation facility. The supply air to the laboratories was preconditioned and automatically controlled with a thermostat and humidistat. Each flow-past nose-only inhalation exposure chamber (Lab Products Inc., Seaford, DE) is comprised of 52 ports. The chambers were encased in an acrylic enclosure to isolate the exposure chamber and protect laboratory personnel. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure. After flowing out of the supply port, any excess test atmosphere, along with exhaled air, is drawn into the chamber exhaust manifold without entering other ports.
During the inhalation exposure, the animals were restrained in nose-only holding tubes (CH Technologies, USA, Westwood, NJ). Following confirmation of the correct animal number, each tube was placed in a pre-designated port of the inhalation exposure chamber. Chamber ports were rotated for each exposure; placement for each exposure is documented in the study records. Animal tube loading and unloading and tube insertion and removal from the chamber manifold processes were performed according to laboratory standard operating procedures that are designed to minimize stress to the rats. The rats were observed frequently while restrained to ensure that they remained in the tubes and were not in danger of injury. At the end of each exposure, when the chamber was purged of the test substance (less than one minute), the tubes with the animals were removed. The rats were removed from the tubes and returned to their home cages. The holding tubes were sanitized after each use.

S4.6.1. Test Atmosphere Generation
Test atmosphere at the desired concentrations was generated by aerosolizing the test substance and mixing it with compressed filtered air to produce a continuous supply of test atmosphere. Test atmospheres were generated by aerosolizing the test formulation with a commercially available nebulizer using compressed air of breathable quality and which is filtered with a compressed air filter and a carbon adsorber. Exhaust from the exposure chambers was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotometers.

S4.6.2. Test Atmosphere Monitoring
Gravimetric Analysis: The test atmosphere concentration in the exposure chamber was determined gravimetrically each exposure by collecting test atmosphere samples on filters placed in closed-face filter holders in the breathing zone of the animals. The gravimetric sampling train consisted of a pre-weighed filter in series with a dry-gas meter connected to a constant flow vacuum pump. Samples were collected at a constant flow rate equal to the port flow of the delivery tube. The filter samples were weighed to determine the aerosol mass collected. The dry-gas meter measured the corresponding volume of chamber air sampled and the weight-to-volume ratio was determined to obtain the aerosol mass concentration.

S4.7. Intravenous Administration
Animals in the IV dosing group received a single injection via the tail vein at a dosing volume of 1 mL/kg.

S4.8. Toxicology Methods
Moribundity/Mortality Observations and Physical Examinations/Clinical Observations: Prior to initiation of dosing (exposure), animals were observed at least once daily for mortality or evidence of moribundity. A detailed, hand-held physical examination was conducted on all animals once during the quarantine period (prior to randomization). During the treatment period, the animals were observed daily for mortality or evidence of moribundity; these checks were separated by a minimum of four hours. Daily cage-side clinical observations were conducted during exposure, and daily hand-held clinical observations were conducted before and after exposure. Observations included, but were not limited to the following: changes in the skin and fur, eyes, and mucous membranes; effects on the respiratory, circulatory, autonomic and central nervous systems; and effects on somatomotor activity and behavior pattern.
Body Weights and Body Weight Changes: Body weights were determined one day after animal receipt; at randomization; and prior to exposure on Study Day 1, 2, 3 and 7 (as applicable based on scheduled euthanization).
Plasma and Tissue Samples/Necropsy: Whole blood samples were collected from three animals per time-point at approximately 0.5, 1, 2, 4, 8, 12, 24 and 48 hours and 7 days after dose administration for plasma drug level determination. Rats were anesthetized with 70% CO2/30% air and blood was collected from the retro-orbital plexus and placed into tubes containing anticoagulant (EDTA). Blood samples were placed on ice immediately following collection and processed (i.e., centrifuged) to plasma. The samples were then stored frozen (at approximately −70 °C) until analyzed. All study animals surviving to scheduled necropsy were euthanized by an overdose of an intraperitoneal injection of sodium pentobarbital at 35-45 mg/kg. Tissue specimens (lung, liver and kidney) were collected from three animals per time point at 0.5, 2, 24 and 48 hours and 7 days after dose administration. All tissue specimens were stored frozen at approximately −70 °C until analyzed.

S4.9. Bioanalytical Method and Analysis
Calibration and Internal Standards: The reference standard, caspofungin acetate (lot number 02220902; Chunghwa Chemical Synthesis & Biotech, Taiwan), was stored at approximately −70 °C; and used without further purification for the preparation of calibration standards and quality control (QC) samples for the determination of caspofungin in plasma and tissue samples collected from this study. The internal standard (caspofungin acetate-d4; lot number 10-GJF-162-1) was stored at −20 °C.
Sample Preparation: For the determination of caspofungin in plasma, a 100 μL aliquot from each sample (in a 2 mL centrifuge tube) was mixed with 0.3 mL of acetonitrile (ACN; Spectrum, New Brunswick, NJ) containing 150 ng of internal standard. After shaking for five minutes, the sample was centrifuged at 4 ºC for 10 minutes to remove precipitated proteins and supernatant was transferred to an autosampler tube, diluted with 0.5 mL of water, and vortex-mixed for instrumental analysis.
For the determination of caspofungin in tissue, samples (lung -entire organ; liver -1 gram; kidneys -one organ) were finely cut and extracted for analysis by adding 2.5 mL of ASTM Type I water and shaking for approximately 0.5 hour, after which 2.5 mL of acetonitrile (ACN; Spectrum, New Brunswick, NJ) were added following by shaking for another 0.5 hour. Subsequently, 100 μL of the supernatant was transferred to a 2 mL centrifuge tube and processed for analysis using the same procedure as for plasma.
Freshly prepared caspofungin standard curves and quality control (QC) samples were analyzed along with the study samples. Instrument calibrators and QC samples were prepared by adding 10 μL of a stock caspofungin solution in ACN/water (v/v 50/50) to 100 μL of blank rat plasma (for both plasma and tissue samples). Calibrator concentrations for plasma specimen analysis were approximately 0.050, 0.10, 0.20, 0.50, 1.0, 2.5, 5.0 and 10 μg/mL; QC samples were prepared at approximately 0.12, 4.0 and 8.0 μg/mL. Calibrator concentrations for tissue specimen analysis were approximately 1, 2, 5, 10, 20, 50 and 100 ng/sample; QC samples were prepared at approximately 2.4, 40 and 80 ng/sample. Calibrators and QC samples were processed for analysis following the procedure described above.
Analytical Equipment and Conditions: Calibrator, QC and study samples were analyzed under LC-MS-MS instrument conditions as detailed in Table S8. Data System: Analyst® 1.6.3 (Applied Biosystems/MDS Sciex, Foster City, CA) The retention time of caspofungin was approximately 2.3 minutes. Calibration curves were calculated from the linear regression (weighting factor of 1/x 2 ) of the caspofungin peak area to internal standard peak area ratios versus caspofungin concentration. Concentration of caspofungin in the samples was determined using the peak area ratio and the regression parameters of the calibration curve. Tissue results in ng were converted to μg/g using the amount of tissue extracted and the final extract volume.