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`RESEARCH ARTICLE
`
`Pharmaceutical Development of a Parenteral Lyophilized Formulation
`of the Novel Antitumor Agent Aplidine
`
`B. Nuijen1*, M. Bouma1, R. E. C. Henrar 2, P. Floriano 3, J. M. Jimeno 3, H. Talsma 4,
`J. J. Kettenes-van den Bosch 5, A. J. R .Heck 5, A. Bult 4 and J. H. Beijnen1,4
`
`1Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Amsterdam,
`The Netherlands; 2NDDO-Oncology, Amsterdam, The Netherlands; 3Pharma Mar s.a., Tres Cantos, Madrid, Spain;
`4Faculty of Pharmacy, Utrecht University, Utrecht, The Netherlands; 5Biomolecular Mass Spectrometry, Utrecht
`University, Utrecht, The Netherlands
`
`ABSTRACT: Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate
`Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid
`human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to
`develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was
`characterized by using several chromatographic and spectrometric techniques. These experiments showed that
`aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed
`that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation
`under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-
`drying was carried out with a 500 m g/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection
`(WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to
`determine the optimal freeze-drying cycle parameters. The prototype, containing 500 m g aplidine and 25 mg
`D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization
`cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried
`formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal
`reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both recon-
`stituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be
`stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formu-
`lation is stable for at least 1 year when stored at +2–8(cid:176) C in the dark.
`
`Introduction
`
`Aplidine (dehydrodidemnin B (DDB), MW 1109,
`Fig. 1) is a novel representative of an evolving group
`of anticancer agents derived from marine sources
`
`* Author to whom correspondence should be
`addressed: Slotervaart Hospital/The Netherlands
`Cancer Institute, Department of Pharmacy &
`Pharmacology, Louwesweg 6, 1066EC Amsterdam,
`The Netherlands, Tel: (31) 20 512 4737, Fax: (31) 20
`512 4753/4824, e-mail: apbnu@slz.nl
`
`(1, 2). This naturally occurring cyclic depsipeptide
`is isolated from the Mediterranean tunicate Aplidium
`albicans and belongs to the didemnin family, a class
`of marine-derived compounds which exhibit
`antiviral, antitumor, and immunosuppressive
`activity. All didemnins share a common macrocy-
`clic peptide structure and differ only in the side-
`chain attached to the backbone by the amino group
`of threonine (3). Aplidine exists in two conformers
`or rotamers referring to the cis and trans isomers of
`the pyruvoyl-proline amide bond (Fig. 2) (4).
`
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`AGILA ET AL - EXHIBIT 1009
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`Didemnin B (DB), the most potent representative
`of the didemnin class up to now, was the first marine-
`derived anticancer compound to enter clinical trials
`in the early 1980’s. Although interesting results were
`seen in this phase I/II programme sponsored by the
`National Cancer Institute (NCI), the occurrence of
`dose-limiting neuromuscular and cardiac toxicity
`hindered further dose-escalation and repeated cycles
`of therapy (2). The dehydro-derivative of DB, dehy-
`drodidemnin B (DDB) or aplidine, displays even
`more potent in vitro and in vivo antitumor activity
`against various solid human tumor xenografts. As
`its parent compound, the antitumor effect of aplidine
`is believed to be primarily mediated through inhibiti-
`on of the cell cycle progression in the G1 phase by
`binding to elongation factor 1a
` in the presence of
`GTP, thus interfering with protein synthesis (5, 6).
`In vitro experiments revealed that aplidine exerts
`less neurotoxicity and cardiotoxicity at antitumor
`concentrations than didemnin B. On the basis of
`these results, aplidine has been identified as a new
`generation of the didemnin class, with a possibly
`significantly higher therapeutic index (2, 7, 8, 9).
`
`In animal toxicology studies a maximum tolerated
`dose (MTD) of 1250 m g/kg body weight in mice
`and a MTD of 570 m g/kg body weight in rats were
`determined, respectively. Taking the 1/10 MTD
`mouse-equivalent, the initial dosage level in Phase
`I clinical trials was therefore defined at 350 m g/m2
`(10, 11). A suitable parenteral formulation contai-
`ning 500 m g aplidine per dosage unit was required
`to start early clinical studies. Because of its very
`poor aqueous solubility (< 0.1 mg/mL), an adequa-
`te vehicle had to be found to administer aplidine to
`the patient. The work described here was directed
`towards developing a suitable parenteral formulati-
`on for toxicological and clinical evaluation
`according to the EORTC/CRC/NCI Joint
`Formulation Working Party (JFWP) guidelines (12,
`13, 14). Aplidine bulk drug substance was fully
`structurally and analytically characterized. The
`formulation approach involved the use of a
`cosolvent-surfactant system to enhance the
`solubility, and lyophilization to improve the stability
`of the compound.
`
`Materials and Methods
`
`Chemicals and Materials
`
`Aplidine was obtained from natural didemnin A by
`a three-step synthesis under the responsibility of
`Pharma Mar s.a. (Tres Cantos, Madrid, Spain) and
`provided through the New Drug Development
`Office-Oncology (NDDO-Oncology, Amsterdam,
`The Netherlands). All chemicals used were of
`analytical grade and were used without further
`purification. Distilled water was used throughout.
`Excipients, including tert-butanol, and primary
`packaging materials used in the manufacturing of
`aplidine lyophilized product and reconstitution
`solution were of European Pharmacopeia III (Ph.
`Eur. III) or United States Pharmacopeia 24 (USP24)
`grade and provided by the supplier with a Certificate
`of Analysis. Substances were approved on the basis
`of in-house quality controls carried out according
`to monographs in the mentioned pharmacopeias.
`Normal saline (0.9% w/v sodium chloride in WfI)
`was manufactured in-house at the Department of
`Pharmacy of the Slotervaart Hospital (Amsterdam,
`The Netherlands).
`
`Characterization of Aplidine Bulk Drug
`
`An interim reference standard of the aplidine bulk
`drug material, i.e., batch of highest purity available,
`was defined (Lot APL-297) and structurally
`characterized by fast-atom bombardment mass
`spectrometry (FAB-MS), nuclear magnetic
`resonance (NMR), and infra-red (IR) spectroscopy,
`and analytically characterized by high performance
`liquid chromatography (HPLC) and ultraviolet/
`visible (UV/VIS) spectrophotometry. On the basis
`of these results, specifications were drawn up for
`the aplidine raw material.
`
`FAB-MS: The FAB mass spectrum was obtained with
`a Model JMS-SX/SX 102A tandem mass
`spectrometer (BEBE; JEOL, Tokyo, Japan). The
`acceleration voltage was 10 kV. A xenon source with
`an energy of approximately 6 keV was used, and
`the matrix was glycerol. Positive ion spectra were
`recorded over a mass range of 10-1500 D.
`
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`NMR-spectroscopy: 1H NMR spectra were recorded
`with a Gemini 300 BB instrument (Varian Assoc.,
`Palo Alto, CA USA) at 300.1 MHz. The sample
`(1 mg) was dissolved in deuterochloroform (CDCl3)
`or hexadeuterodimethylsulfoxide (DMSO-d 6). In
`CDCl3, TMS was used as an internal reference; in
`DMSO-d 6 the central DMSO line was set at 2.50
`ppm.
`
`Infra-red spectroscopy: Infra-red (IR) spectra
`(4000–600 cm-1) were recorded on a Model PU 9706
`IR spectrophotometer (Philips Nederland B.V.,
`Eindhoven, The Netherlands) with the potassium
`bromide (KBr) pellet technique. The pellet consisted
`of 1 mg of aplidine bulk drug and 200 mg of KBr.
`The ratio recording mode was set on autosmooth
`and the scan time was 8 minutes.
`
`UV/VIS spectroscopy: UV/VIS spectra (800–200
`nm) of aplidine bulk drug (50 m g/mL in 79% (v/v)
`
`methanol/H2O) were recorded with a Model UV/VIS
`918 spectrophotometer (GBC Scientific Equipment,
`Victoria, Australia) equipped with an LEO personal
`computer and an Epson LX-400 plotter.
`
`Formulation of Aplidine
`
`Solubility studies: The solubility of aplidine in
`various solvents at ambient temperature (20–25(cid:176) C)
`was examined by accurately weighing
`approximately 1 mg of aplidine in a glass test tube
`and adding subsequent solvent volumina of 100 m L,
`1 mL, and 10 mL to the bulk drug. After each
`addition the mixture was vigorously shaken for 30
`seconds, placed in an ultrasonic bath for 15 minutes
`and examined visually under polarized light for
`complete dissolution of the aplidine drug substance.
`In this way, the solubilities of aplidine in the various
`solvents were selectively distributed over four
`solubility ranges (s < 0.1 mg/mL, 0.1 mg/mL £
` s <
`
`Figure 1: Chemical structure of aplidine (Hip: hydroxyisovalerylproprionyl; Ist: isostatine; Leu: leucine; Pro:
`proline; Pyr: pyruvoyl; Thr: threonine; Tyr: tyrosine).
`
`[3S, 4R, 5S-Ist]
`
`[2S, 4S-Hip]
`
`O
`
`O
`
`NH
`
`N
`
`HO
`
`O
`
`NH
`[1S, 2R-Thr]
`O
`O
`
`O
`
`O
`
`O
`
`O
`
`N
`
`O
`
`NH
`
`N
`
`[(S)-Leu]
`
`[(S)-Pro]
`
`[(S)-Pro]
`
`O
`
`[(S)-Me -Tyr]
`2
`
`[(R)-N(Me)-Leu]
`
`O
`
`O
`
`N
`
`[Pyr]
`
`O
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`1 mg/mL, 1 mg/mL £
` s < 10 mg/mL and s > 10 mg/
`mL, respectively). In addition, solvents in which
`aplidine dissolved were examined by diluting the
`solutions 1:1, 1:5, 1:10, 1:50, and 1:100 (v/v) with
`normal saline for infusion in glass test tubes. After
`gentle agitation, each of the dilutions was examined
`visually under polarized light over a one day period
`for any sign of precipitation.
`
`Reconstitution of aplidine lyophilized product with
`solutions composed of 40/10/50% (v/v/v) propylene
`glycol 400/ethanol/polysorbate 80 (PET), 40/10/5-
`0% (v/v/v) propylene glycol 400/ethanol/WfI
`(PEW), and 5/5/90% (v/v/v), 15/15/70% (v/v/v), and
`30/30/40% (v/v/v) Cremophor EL/ethanol/WfI was
`studied by adding increasing volumes of the
`reconstitution solutions to 500 m g aplidine lyophili-
`zed cake. After each addition, the resulting mixture
`was agitated and subsequently examined visually
`under polarized light. Quantitative analysis was
`carried out by diluting 50 m L samples of reconstitu-
`ted product with 950 m L of acetonitrile and
`subsequent injection onto the HPLC system.
`Furthermore, in case of complete dissolution, the
`reconstituted solution was diluted 1:10 or 1:100
`(v/v) with normal saline for infusion in glass test
`tubes. Stability of the reconstituted solution and the
`infusion solutions were examined visually under
`polarized light and by HPLC without further
`dilution, over a 24-hour period at room tempera-
`ture (20–25(cid:176) C) and a normal day-night light cycle.
`
`Differential scanning calorimetry: Transition
`temperatures and freezing characteristics of tert-
`butanol, D-mannitol, and aplidine solutions were
`examined by differential scanning calorimetry
`(DSC). The DSC experiments were carried out with
`a TA Instruments DSC 2920 (TA Instruments, New
`Castle (DE), USA) equipped with an LNCA for low
`temperatures. Samples were sealed in a closed
`aluminium pan with an empty pan as reference.
`Temperature scale and heat flux were calibrated with
`indium. Samples were cooled to -50(cid:176) C at a rate of
`5(cid:176) C/min. The DSC heating rate was 2.5(cid:176) C/min.
`Analyses were performed under a helium purge.
`
`Formulation process: Aplidine lyophilized product
`was aseptically prepared from a 500 m g/mL aplidine
`solution in 40% (v/v) tert-butanol in WfI contai-
`ning 25 mg/mL D-mannitol as a bulking agent. The
`formulation solution was prepared by weighing
`aplidine and D-mannitol and dissolving the
`substances by magnetic stirring in 40% (v/v) tert-
`butanol in WfI. To make a final concentration of
`500 m g/mL aplidine, 40% (v/v) tert-butanol in WfI
`was added. The formulation solution was sterile
`filtered through a 0.2 m m Midisart 2000 filter
`(Sartorius, Nieuwegein, The Netherlands). Subse-
`quently, 1 mL aliquots of the formulation solution
`were filled into 10 mL type 1 glass vials with a
`Model 501Dz peristaltic pump (Watson Marlow,
`UK). After filling, vials were partially closed with
`grey butyl rubber stoppers, placed in a Model
`Lyovac GT4 freeze-dryer (AMSCO/FinnAqua,
`Germany), and lyophilized. The freeze-dryer was
`equipped with a cold trap filled with liquid nitrogen
`attached to the condensor to ensure complete
`condensation of tert-butanol. After completion of
`the freeze-drying cycle, sterile filtered medical grade
`nitrogen gas was leaked into the freeze-drying
`chamber to reach a final vacuum of 100 mbar. Subse-
`quently, the vials were pneumatically closed,
`capped, and labeled. In-process-controls consisted
`of integrity testing of the filter unit, weight variation
`of the filling volume, and determination of the
`aplidine concentration of the formulation solution
`before and after filtration. Only clean, sterile inert
`materials and glassware were used throughout the
`manufacturing process. All critical manipulations
`took place under a class 100 (A) down-flow conditi-
`on with a class 100 (B) background (Interflow,
`Wieringerwerf, The Netherlands). Air particle
`counts in the critical areas as well as microbiological
`contamination of the area and personnel were
`monitored at operating state.
`
`Reconstitution solution 15/15/70% (v/v/v)
`Cremophor EL/ethanol/WfI (CEW) was prepared
`by mixing the appropriate volumes of excipients by
`magnetic stirring. The solution obtained was filtered
`through a 0.2 m m Midisart 2000 filter unit (Sartorius,
`Goettingen, Germany) and subsequently 2 mL
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`aliquots were filled into type 1 glass ampoules with
`a Model R910 ampoule fill-and-seal machine (Rota
`den Boer B.V., The Netherlands). After filling, the
`solution was sterilized by autoclaving (Model 6.6.15
`autoclave, Koninklijke Ad Linden B.V., The
`Netherlands) for 20 minutes at 120(cid:176) C. All
`manipulations involving an “open” solution or
`excipient were conducted under class 100 (A)
`conditions. The manufacturing of both aplidine
`lyophilized product and its reconstituted solution
`were performed according to the Good Manufac-
`turing Practice (GMP) guidelines (15).
`
`Figure 2: cis/trans isomerism of the pyruvoyl-proline
`amide bond.
`
`O
`
`-
`
`O
`
`N
`
`R
`
`N+
`
`N
`
`R
`
`O
`
`O
`
`N
`
`O
`
`O
`
`O O
`
`N
`
`N+
`
`O-
`
`O O
`
`N
`
`N
`
`O
`
`R
`
`R
`
`Quality Control of Aplidine Lyophilized Product
`
`Quality control of aplidine lyophilized product
`consisted of identification by visual inspection of
`appearance and colour of the pharmaceutical
`product; determination of
`reconstitution
`characteristics and pH of the reconstituted product;
`content, content uniformity, and purity
`determination by HPLC analysis; residual moisture
`determination with the Karl-Fischer titration
`method; and residual tert-butanol content by gas
`chromatographic (GC) analysis. Furthermore,
`sterility of the pharmaceutical product was checked
`by the filtration method and the presence of bacterial
`endotoxins with the limulus amoebocyte lysate
`(LAL) test, both carried out according to the
`European Pharmacopeia III.
`
`HPLC analysis: Aplidine was assayed by a
`validated, stability-indicating reversed phase-HPLC
`method. The HPLC system consisted of a Model
`SP8800 pump (Thermo Separation Products, USA),
`a Model Spectra 200 UV-VIS detector (Spectra-
`Physics, San Jose, USA), and a Model SP8880
`autosampler (Thermo Separation Products, USA).
`Analyses were carried out with a Zorbax SB-C18
`analytical column (4.6 mm ID x 150 mm, particle
`size 3.5 mm, Waters, USA) held at a constant
`temperature of 80(cid:176) C with a Model 7971 column
`heater (Jones Chromatography, USA). The mobile
`phase at a flow of 0.6 mL/min consisted of a linear
`gradient of acetonitrile (ACN) containing 0.04%
`trifluoroacetic acid (TFA) 35% to 70% in 15 minutes
`and water containing 0.04% TFA. An injection
`volume of 20 m L and a total run time of 30 minutes
`were used. UV detection was performed at 225 nm.
`Under these conditions the chromatogram of
`aplidine consisted of a single peak eluting at 21
`minutes. A series of standard solutions of aplidine
`in ACN in the concentration range of 5 m g/mL to
`300 m g/mL were prepared in duplicate from a stock
`solution of 1 mg/mL aplidine in ACN and injected
`into the HPLC system. Quality control samples at
`concentrations of 7.5, 100, 250, and 275 m g/mL of
`aplidine in ACN were prepared in quadruplicate
`from another stock solution with separate weighing
`of aplidine and injected into the HPLC system.
`Aplidine lyophilized product was diluted to a test
`concentration of 250 m g/mL by dilution with a
`solution of 1:1 (v/v) ACN/water. Least squares
`regression analysis was used to calculate the slope
`and intercept for the standard calibration curve from
`measured peak areas versus concentration. Sample
`concentrations were calculated from
`the
`corresponding peak areas using the regression
`equation. All aplidine chromatograms were
`electronically stored in the computer system
`LABNET (Spectra-Physics, San Jose, USA).
`Reprocessing of aplidine chromatograms was
`performed using PC1000 software (Thermo
`Separation Products, USA).
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`Residual moisture content: Residual moisture levels
`in aplidine lyophilized product were determined
`with the Karl-Fischer titration method. The content
`of a vial was quantitatively transferred to the titration
`unit of a Model 658KF titrino apparatus (Metrohm
`Herisau, Switzerland) with previously dried metha-
`nol, and subsequently titrated using Hydranal®
`Titrant 2.0 mg H2O/mL (Riedel-de Haen, The
`Netherlands). The end-point of the titration was
`determined biamperometrically.
`
`Residual tert-butanol content: Residual tert-butanol
`levels in aplidine lyophilized product were
`determined by gas chromatographic (GC) analysis.
`The GC system consisted of a Model 5890 gas
`chromatograph (Hewlett Packard, USA), equipped
`with a flame ionization detector (FID), a HP-5 cross-
`linked phenyl-methyl silicone capillary column
`(25 m x 0.32 mm x 0.52 mm film thickness, Hewlett
`Packard, USA) and a Model 6890 series autosampler
`(Hewlett Packard, USA). The column temperature
`was held at 60(cid:176) C for 2 minutes and was subsequently
`linearly ramped to 180(cid:176) C at a heating rate of
`30(cid:176) C/min and kept at this temperature for 2 minu-
`tes. The injector temperature was kept at 275(cid:176) C and
`the detector temperature at 250(cid:176) C. An injection
`volume of 1 m L and a run time of 12 minutes were
`employed. tert-Butanol standard solutions were
`prepared from a 2 mg/mL stock solution in 40% (v/
`v) propylene glycol in water in a concentration range
`of 20–100 m g/mL. Least square regression analysis
`was used to calculate the slope and intercept of the
`standard calibration curve from the measured peak
`areas versus tert-butanol concentration. Sample
`solutions were prepared by reconstituting a vial of
`aplidine lyophilized product with 3 mL of 40% (v/
`v) propylene glycol in water and injecting 1 m L onto
`the GC system. The tert-butanol concentrations in
`the sample solutions were calculated from the
`corresponding peak areas using the regression
`equation. All tert-butanol chromatograms were
`electronically stored in the computer system
`LABNET (Spectra-Physics, San Jose, USA).
`Reprocessing of tert-butanol chromatograms was
`performed using PC1000 software (Thermo
`Separation Products, USA).
`
`Quality Control of Aplidine Reconstitution Solution
`
`Quality control of 15/15/70% (v/v/v) Cremophor
`EL/ethanol/WfI reconstitution solution consisted of
`visual inspection of the appearance of the
`pharmaceutical solution and presence of particulate
`matter, determination of the pH, refractive index,
`relative density, and ethanol content. Also, as
`aplidine lyophilized product, sterility and the
`presence of bacterial endotoxins were examined
`according to the European Pharmacopeia III using
`the filtration method and LAL test, respectively.
`
`The specificity of the tests for the refractive index,
`relative density, and ethanol content were examined
`by comparing the results of the tests performed on
`solutions composed of varying ratios of Cremophor
`EL, ethanol and WfI. The refractive indices and
`relative densities of solutions composed of 5/5/90%,
`10/10/80%, 15/15/70%, 20/20/60%, 25/25/50%
`(v/v/v) Cremophor EL/ethanol/WfI were measured
`and the ethanol content of 10/10/80%, 15/15/70%,
`20/20/60% (v/v/v) Cremophor EL/ethanol/WfI
`solutions was determined. Also, to simulate
`erroneous compounding of the excipients, the
`refractive index and relative density of solutions
`composed of 15/70/15% and 70/15/15% (v/v/v)
`Cremophor EL/ethanol/WfI were examined.
`
`Refractive index: The refractive indices of the
`various Cremophor EL/ethanol/WfI solutions were
`determined on a Model Abbé 302 refractometer
`(Atago, Japan) at 20(cid:176) C.
`
`Relative density: The relative densities of the various
`Cremophor EL/ethanol/WfI solutions were
`determined by accurately weighing 1.00 mL of the
`respective solutions on a Model 440 balance
`(Mettler Toledo) at 20(cid:176) C.
`
`Ethanol content: Ethanol content of Cremophor EL/
`ethanol/WfI solutions was determined by GC
`analysis. The GC system consisted of a Model
`HP5710A gas chromatograph (Hewlett Packard,
`USA) equipped with a flame ionization detector
`(FID) and a Tenax GC 60–80 Mesh 1 m x 2 mm
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`column. The column temperature was held at 105(cid:176) C.
`The injector and detector temperatures were kept at
`200(cid:176) C. An injection volume of 1 m L and a run-time
`of 5 minutes were employed. Standard solutions
`containing approximately 0.75‰, 2.5‰ and 4‰
`ethanol were prepared by weighing accurately
`approximately 75 mg, 250 mg and 400 mg of
`absolute ethanol, respectively, into a 100 mL
`volumetric flask and adding distilled water to reach
`a final volume of 100.0 mL. As an internal standard,
`a solution containing approximately 3.5‰
`2-propanol was prepared by weighing
`approximately 350 mg of 2-propanol into a 100 mL
`volumetric flask and adding distilled water to reach
`a final volume of 100.0 mL. Before injection into
`the GC system, each ethanol standard was diluted
`1:1 (v/v) with the internal standard solution.
`Subsequently, 1 m L of the resulting solution was
`injected into the GC system. From the obtained
`ethanol/2-propanol peak ratios, a calibration curve
`was calculated using least squares regression
`
`analysis. 10/10/80%, 15/15/70%, 20/20/60%
`(v/v/v) Cremophor EL/ethanol/WfI solutions were
`diluted to a theoretical ethanol concentration of
`1.6‰ with distilled water. Before injection onto the
`GC system, each sample was diluted 1:1 (v/v) with
`the internal standard solution. Subsequently, 1 m L
`of the resulting solution was injected into the GC
`system. From the obtained ethanol/2-propanol peak
`ratios, the ethanol concentration (‰) was calculated
`with the standard calibration curve. The ethanol
`content of the Cremophor EL/ethanol/WfI solution
`was calculated by dividing the obtained ethanol
`concentration (‰) by the theoretical ethanol
`concentration (‰), times 100%.
`
`Shelf-life Studies
`
`Long-term stability of aplidine lyophilized product
`was studied at +2–8(cid:176) C in the dark. Content and
`purity of the lyophilized product were determined
`by HPLC analysis.
`
`Figure 3: Chromatogram obtained with an isocratic HPLC system showing the double-peak for aplidine due to
`cis/trans isomerism of the pyruvoyl-proline amide bond (concentration of 60m g/mL aplidine in ACN).
`
`0.025
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`0.02
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`0.015
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`0.01
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`mAU
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`0.005
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`0
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`0 1 2 3 4 5 6 7 8 9
`Time (minutes)
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`Figure 4: Representative chromatogram of aplidine (R t of 21 minutes) obtained with stability-indicating gradi-
`ent HPLC system (concentration of 250 m g/mL aplidine in ACN).
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`0.2
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`0.18
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`0.16
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`0.14
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`0.12
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`0.1
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`0.08
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`0.06
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`0.04
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`0.02
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`mAU
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`0
`0.00 5.00 10.00 15.00 20.00 25.00 30.00
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`Time (minutes)
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`Figure 5: DSC thermogram of aplidine formulation solution (500m g/mL aplidine and 25 mg/mL D-mannitol in
`40% v/v tert-butanol/water).
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`0.0
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`-0.5
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`-1.0
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`-1.5
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`Heat Flow (w/g)
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`-2.0
`-60 -50 -40 -30 -20 -10 0 10
`>
`exo
`Temperature (°C)
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`Results and Discussion
`
`Characterization of Aplidine
`
`FAB-MS: The FAB-MS shows the protonated
`molecular ion [M+H]+ at m/z 1110.7. The major
`fragmentations are represented in Table 1 and are
`indicative of the structure of aplidine.
`
`1H-NMR: Table 1 gives the partial assignments for
`the 1H-NMR spectrum in CDCL3, based on 1D
`proton and 2D HH-COSY spectra. Two conformers
`appear to be present in an approximately 1:1 ratio
`(NH protons and methyl protons of the pyruvoyl
`group). The existence of two conformers relating to
`cis/trans isomerism of the pyruvoyl-proline amide
`bond was also shown with 13C NMR (4). The
`spectrum in DMSO-d6 shows the presence of even
`four conformations in approximately a 35:35:15:15
`ratio (methyl protons of the pyruvoyl group). The
`additional signals probably originate from cis/trans
`isomerism of the second proline amide bond. This
`hypothesis was examined by carrying out
`temperature experiments in DMSO-d6 at 105(cid:176) C
`which showed that only the signals for the pyruvoyl
`methyl group are still present at this elevated
`temperature. In the CDCL3 spectrum, the threonine
`NH signal of one conformer is more shielded than
`that of the other conformer. This indicates that the
`two conformers arise, indeed, as a result of cis/trans
`isomerism at the pyruvoylproline residue (Fig. 2).
`From the literature the occurrence of more than one
`conformation in proline-containing peptides is well-
`known (16,17). The proline residue and cis/trans
`isomerization is extremely important in the folding,
`denaturation, and renaturation of polypeptides and
`proteins, and might also be a factor in the biological
`activity of aplidine.
`
`UV/VIS spectrophotometry: The UV spectrum of
`aplidine shows absorption maxima at 212 and 273
`nm, respectively (Table 1).
`
`Infra-red spectroscopy: Major assignments are
`presented in Table 1.
`
`HPLC analysis: The existence of multiple
`conformations of aplidine as described in the NMR
`section complicated the development of a stability-
`indicating HPLC method. The initial assays, both
`normal phase and isocratic and gradient reverse
`phase high-performance liquid chromatographic
`(HPLC) methods, showed the presence of two
`aplidine peaks in the chromatograms referring to the
`equilibrium between the cis and trans isomers of
`the pyruvoyl-proline amide bond (18). At ambient
`temperature (20–25(cid:176) C) this equilibrium appears too
`slow to elute both isomers as one peak but also too
`fast to elute them as two baseline-separated peaks
`(Fig. 3). By lowering the column temperature this
`equilibrium is frozen, thus resulting in improved
`resolution. Collection of one of the individual
`conformations at the detector outlet and reinjection
`of the component results again in a double-peak,
`indicating a continuous interconversion of both
`conformers. Although quantification of aplidine
`concentrations on the total area of both isomer peaks
`was satisfactory, the stability-indicating capability
`of the assay was not. It was proven that upon stress-
`testing of aplidine solutions degradation products
`co-eluted with the aplidine isomer peaks. Therefore,
`at present a gradient HPLC method with a column
`temperature of 80(cid:176) C is employed. Due to this high
`column temperature aplidine elutes as a single peak.
`Furthermore, this method shows good selectivity for
`impurities and degradation products. A represen-
`tative chromatogram of aplidine is given in Fig. 4.
`Aplidine elutes at a retention time (Rt) of approxi-
`mately 21 minutes. The peaks eluting at
`approximately 14 and 20 minutes, respectively, are
`impurities of the aplidine bulk drug. Identification
`of these impurities is ongoing.
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`Table 1: Identification and characterization of aplidine bulk drug (Lot APL-297)
`
`Analytical Method Results
`
`Appearance
`
`FAB-MS
`
`1H NMR (CDCl3)
`
`UV/VIS
`spectrophotometry
`
`IR spectroscopy
`
`Pale beige, amorphous powder
`
`Molecular formula: C57H87N7O15; Protonated molecular ion [M+H]+
`1110.7; 1040.7 [M+H]+-70, CH2COCO; 985.6 [M+H]+-125, Pro-CO;
`943.6 [M+H]+-167, pyruvoyl-Pro; 859.5 [M+H]+-251, Leu-diMe-keto-
`OH-hexanoic acid-H2O; 816.0 [M+H]+-294, pyruvoyl-Pro-N-Me-Leu;
`701.4 [M+H]+-409, Leu-diMe-keto-OH-hexanoic acid-NH2-OH-Me-
`heptanoic acid-NH; 295.2, pyruvoyl-Pro-N-Me-Leu; 177.1, Tyr-CH2
`(N-CH3); 169.1, pyruvoyl-Pro
` Proposed assignments:
`1; 1.95 H-b
`; 2.10 H-b
`Amino acid residue: Proline (1): d 4.70 H-a
`2;
`3.66 H-d
`1; 3.84 H-d
`2; 2.54/2.52 CH3CO; N-Me-Leucine: d 3.15/3.11
`2; 1.28 H-g ; 0.90 CH3; Threonine:
`N-CH3; 4.62 H-a
`; 2.15 H-b
`1; 1.78 H-b
`7.61/ 7.05 NH4; 4.57/4.65 H-a
`; 5.29/5.17 H-b ; 1.41/1.40 g -CH3;
`4-Amino-3-hydroxy-5-methylheptanoic acid: 7.20/7.19 NH4; 3.25 H-a
`1;
`2; 4.08 H-b ; 4.08 H-g ; 1.80 H-d ; 1.64 d -CH3; 0.90 H-e ; 0.90 e-
`2.62 H-a
`CH3; 2,5-diMe-hydroxy-3-ketohexanoic acid: 4.23/4.19 H-a
`; 1.33/1.32
`a -CH3; 5.40 H-g ; 1.70 H-d ; 0.90 d -CH3; Leucine: 7.86/7.81 NH4; 4.80
`; 2.90 H-b
`1; 1.20 H-b
`2; 0.90 CH3; Proline (2): 5.10 H-a
`H-a
`; 1.60 H-b
`1;
`1.90 H-b
`2; N,O-diMe-tyrosine: 3.80 O-CH3; 2.54 N-CH3; 3.57 H-a
`; 3.36
`H-b
`1; 3.16 H-b
`2; 7.08 H-1,4; 6.85 H-2,3
`Absorption maxima at 212 and 273 nm
`
`Characteristic absorption bands (approximately):
`3340 cm-1: N-H stretching (amide); 2960/2880 cm-1: C-H stretching
`(aliphatic); 1740 cm-1: C=O stretching; 1640 cm-1: N-C=O stretching
`(amide); 1520 cm-1: C=C stretching (aromatic); 1450 cm-1: C-H
`deformation; 1250 cm-1: phenolic C-O; 1170/1070 cm-1: C-O
`stretching;
`820 cm-1: C-H bending of paradisubstituted benzene
`
`Solubility Studies
`
`Table 2 gives the approximate solubilities of aplidine
`in various solvent systems. Solvents were chosen
`on the basis of current use and experience in clinical
`practice (14, 19). Aplidine is very poorly soluble in
`water, but dissolves well in a number of organic
`solvents. Furthermore, aplidine has high solubilities
`in 60/40/10% (v/v/v) polyethylene glycol 400/
`ethanol/polysorbate 80 (PET) and 50/50% (v/v) Cre-
`mophor EL/ethanol cosolvent/surfactant systems.
`However, upon dilution with normal saline up to 1:100
`(v/v), only aplidine solubilized in