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Introduction

ALZET® osmotic pumps are frequently used for the continuous zero-order delivery of compounds in preclinical studies that would eliminate the need for repeated dosing. Such continuous delivery at high concentrations becomes more important for compounds with faster clearance and shorter biological half-life to be able to maintain their desired steady state plasma concentrations. An ALZET® osmotic pump comprises

a collapsible reservoir (drug solution/suspension reservoir) surrounded by a salt layer (osmotic agent) which in turn is enclosed by a semipermeable membrane (outer shell). The operation of the ALZET® osmotic pump is based on the osmotic pressure gradient developed between the salt layer compartment and the tissue environment in which the pump is implanted. The high osmolality of the salt layer causes water influx into the salt layer compartment from the tissue through the

ORIGINAL ARTICLE

Development of ALZET® osmotic pump compatible solvent compositions to solubilize poorly soluble compounds for preclinical studies

Rampurna Gullapalli1,2, Angelina Wong1,3, Elizabeth Brigham1, Grace Kwong1, Angie Wadsworth1, Christopher Willits1, Kevin Quinn1, Erich Goldbach1, and Bhushan Samant1

1Elan Pharmaceuticals, 800 Gateway Blvd., South San Francisco, CA 94080, USA, 2Pharmaceutics International Inc. (Pii), 10819 Gilroy Rd., Hunt Valley, MD 21031 USA, and 3Pharmacyclics Inc., 995 E. Arques Ave.,Sunnyvale, CA 94085 USA

AbstractContext: Hydrophilic, non-aqueous solvents are frequently used to solubilize poorly water soluble compounds for use in ALZET® osmotic pumps used during the discovery and preclinical stages. Though these solvents exhibit the potential to solubilize several poorly soluble compounds, the solubilized compounds are prone to crystallization up on contact with aqueous fluids in vitro and in vivo. Crystallization of a compound can potentially cause pain at the release site, erratic blood levels, and uneven or delayed pharmacokinetic profiles.Objective: In this study, we discussed the development of ALZET® pump compatible hydrophilic, non-aqueous vehicles that solubilized two poorly soluble model compounds (ELND006 and ELN 481594) and prevented their crystallization from solutions in vitro and in vivo.Methods: The selected formulations were filled into the pumps at three concentrations for each model compound and investigated for their compatibility with the pumps and the subcutaneous tissue of mice where the pump was inserted.Results and Discussion: The results showed that the formulations were stable physically with no crystallization and chemically with no degradation and were compatible with the pump and animal tissue. The plasma concentration of ELND006 decreased with time at each dose. The extent of the time-dependent decrease in ELND006 plasma levels increased as the amount of dose delivered increased. This time and dose dependent decrease in ELND006 plasma concentrations was attributed to the known auto-induction of hepatic enzymes by the compound. In contrast, the plasma concentration of ELN 481594 increased significantly at higher dose, likely due to accumulation of the compound.Keywords: ALZET® osmotic pump, poorly soluble compounds, solubilization, crystallization, compatibility, stability

Address for Correspondence: Rampurna Gullapalli, Pharmaceutics International Inc. (Pii), Drug Delivery Technologies, 10819 Gilroy Road, Hunt Valley, 21031 MD, USA. Tel.: +1-914-316-4935. Fax: +1-410-584-0007. E-mail: rampurnaL@gmail.com

(Received 19 March 2012; revised 30 April 2012; accepted 02 May 2012)

Drug Delivery, 2012; 19(5): 239–246© 2012 Informa Healthcare USA, Inc.ISSN 1071-7544 print/ISSN 1521-0464 onlineDOI: 10.3109/10717544.2012.691121

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2012

Development of ALZET® osmotic pump

R. Gullapalli et al.

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semipermeable membrane. As water enters the salt layer compartment, it compresses the flexible drug reservoir and forces its contents through a delivery portal at a relatively constant rate over a period of time, sometimes up to 28 days.1–5

ALZET® osmotic pumps have proven to be extremely useful for the delivery of compounds that are water-sol-uble6–11 as well as those that are poorly water-soluble.2,8 Aqueous vehicles with or without some amount of a cosol-vent have been reported to be compatible with the pump reservoir material,1,12 and these types of vehicles can be conveniently used in the pumps, provided that the inves-tigational compound is soluble. However, compounds that have insufficient solubility in aqueous vehicles may require solubilization in a non-aqueous vehicle. Several non-aqueous vehicles have been investigated for their use in the osmotic pumps.1,13 While non-aqueous vehi-cles such as neat dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and N,N-dimethylacetamide (DMA) are excellent solvents for many poorly soluble compounds, these solvents are incompatible with the reservoir material, and thus cause significant failure of the pumps.2,12 On the other hand, other neat solvents such as glycofurol, polyethylene glycols (PEG 300 and PEG 400), and propylene glycol (PG) are good solvents for many poorly soluble compounds and do not suffer from this disadvantage.2,12 Nevertheless, crystallization of a poorly soluble compound dissolved in these solvents may follow when the compound encounters an aque-ous environment at the site of its release in vitro (during priming of the pump) or in vivo (animal tissue where the pump is implanted).14,15 The potential for the crystalliza-tion of such a compound increases with an increase in its lipophilicity and concentration in the solution.14 In addition, use of a higher viscosity solvent (e.g. neat PEG 400) was shown to impact its release from the pump adversely and may not be ideal for its use in the pumps.12 The potential for such a crystallization of a solubilized compound in the presence of an aqueous environment can be minimized by reducing the concentration of the solubilized compound and/or by incorporating a care-fully chosen emulsifier in the formulation.16–18

During the drug discovery phase, discovery scientists (e.g. medicinal chemists, pharmacologists, toxicologists, and pharmacokinetists) usually encounter a variety of investigational compounds with wide ranging solubilities both in aqueous and non-aqueous vehicles. Discovery scientists with limited experience in the areas of prefor-mulations and formulations are required frequently to identify suitable ALZET® pump formulations for these compounds during the early preclinical studies. In addi-tion, these scientists are further handcuffed by limited supplies of the available investigational compounds (usually less than 10 mg) and time to evaluate several formulations.15 Thus, it is of tremendous interest to have a generic vehicle composition for ALZET® osmotic pumps that can be used as it is or with some modifications across a variety of compounds during preclinical studies. Use of

such a common composition will also eliminate or mini-mize any vehicle related effects during the comparison of pharmacokinetic and/or pharmacological effects of several compounds in a therapeutic class. The primary aim of the current study was to formulate ALZET® pump compatible vehicle compositions that can solubilize two poorly water soluble model compounds and prevent their crystallization from solutions upon contact with aqueous fluids in vitro and in vivo. Other objectives of the study were to evaluate the compatibility of these for-mulations with the subcutaneous tissue where the pump was inserted and to investigate the corresponding phar-macokinetic profiles in mice.

Materials and methods

MaterialsALZET® osmotic pumps (Model # 1003D, 3-day deliv-ery at 1.0 µL/h rate and Model # 1004, 28-day delivery at 0.11 µL/h rate) were obtained from Durect Corporation, Cupertino, CA. The materials of construction are identical for both pumps used in the studies. Investigational com-pounds (ELND006 and ELN 481594) were obtained from Elan Process Chemistry Group, South San Francisco, CA. PEG 300 (Spectrum Chemicals), Cremophor ELP (Fluka), glycofurol (Sigma), dimethyl sulfoxide (Sigma), ethanol (Acros, 200 proof), and propylene glycol (EMD) were used in the preparation of formulations. Sterile saline solution (0.9% w/v sodium chloride solution) was obtained from Baxter Healthcare Corporation (Deerfield, IL, USA). All the materials were used as received.

MethodsPreparation of formulationsInitially, a poorly soluble investigational compound, ELND006 was used to develop a solubilized formulation composition and to evaluate its applicability to ALZET® osmotic pumps for subcutaneous implantation in mice. ELND006 has poor aqueous solubility across the entire physiological pH range (~3 µg/mL at 37°C), moderate to high permeability [76 ± 23 nm/s on Madin-Darby canine kidney strain II (MDCK-II) cells], and high lipophilicity (Log D 3.91) and the compound was predicted to belong to class II of the biopharmaceutics classification system (BCS). The chemical structure, physicochemical and biopharmaceutical properties of the compound were presented in detail in an earlier publication.19 Phase 1 clinical studies have shown that the compound has an extended half-life in human (220–306 h).20 To be more effectively model the extended human half-life observed in Phase 1 clinical studies, the compound required continuous infusion in preclinical models for extended periods. ALZET® osmotic pumps were investigated as the infusion device to attain and maintain steady state plasma concentrations for the compound for extended periods in preclinical models.

Among various approaches evaluated in the development of formulations for use in ALZET® osmotic

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pumps, use of hydrophilic, non-aqueous solvents (water miscible organic solvents) proved to be promising in meeting the high delivery dose requirements for ELND006 for the preclinical studies. ELND006 exhibited high solubility in PEG 300 (>60 mg/g). However, its solubility in PEG 300 decreased sharply upon dilution with increasing water content; for example, 11.1, 4.0, and 2.4 µg/mL at 20, 10, and 5% PEG 300 concentration, respectively, in the aqueous medium.19 Similar findings have also been reported for other poorly water soluble compounds solubilized in hydrophilic, non-aqueous vehicles.17,21–23 Crystallization of a compound can have the potential to cause pain at implanted site, erratic blood levels, and uneven or delayed pharmacokinetic profiles.15,18 The expected crystallization of the compound in the aqueous environment at the animal tissue where the pump is implanted precluded the use of neat PEG 300 as a vehicle for ELND006. In addition, the higher viscosity of neat PEG 300 (80–105 centipoises at 25°C)24 was also thought to have adverse impact on the rate of release of the solubilized compound from the pump.12 Thus the first step in the development of a pump compatible ELND006 solution formulation was to evaluate alternate hydrophilic, non-aqueous solvents with a lower viscosity that can be used alone or in combination with PEG 300. Among various solvents with lower viscosities investigated, propylene glycol, glycofurol, and dimethyl sulfoxide (DMSO) were shown to have solubility for ELND006 comparable to PEG 300. However, the compound solubilized in these solvents as neat or in combinations exhibited crystallization upon contact with aqueous environment. To circumvent the crystallization process in the current formulations, three non-ionic surfactants, PEG-35-castor oil, purified (Cremophor® ELP), Polysorbate 80, and PEG-660-hydroxystearate (Solutol® HS 15), and ethanol as a cosolvent were chosen

to evaluate their ability to prevent the crystallization of the compound solubilized in the hydrophilic, non-aqueous solvents in the presence of aqueous environment. The in vitro potential for the crystallization (precipitation) of the solubilized compound was assessed using one-to-one dilution procedure with normal saline solution as the diluent.

Some examples of formulations studied for ELND006 are presented in Table 1. Based on miscibility, clarity, and ability to solubilize ELND006 and keep it in solution against crystallization upon dilution with saline solu-tion, a vehicle containing 25% w/w PEG 300, 25% w/w Cremophor ELP, 25% w/w glycofurol, 15% w/w ethanol, and 10% w/w propylene glycol was chosen to prepare formulations for ELND006 and to evaluate its applicabil-ity to the ALZET® osmotic pumps for preclinical studies (Table 1: Vehicle # 7). The vehicle was prepared by weigh-ing each solvent into a suitable glass container and mixing the weighed solvents thoroughly to obtain a clear solution. To prepare the ALZET® osmotic pump infusion formula-tions for ELND006, the required amount of the compound was transferred into a 25-mL volumetric flask, and volume of the flask was made up to the mark with the vehicle. The contents of the flask were mixed thoroughly to obtain a clear solution. The infusion formulations were prepared at 2, 10, and 50 mg/mL concentrations for ELND006 to meet the requirements set forth by our preclinical scientists. These infusion formulations were also diluted with sterile saline solution appropriately to prepare subcutaneous bolus solution formulations for administering loading doses at the time of pump implantation. All formulations were stored at ambient conditions in closed glass contain-ers to prevent any loss of ethanol and migration of moisture from the surroundings. The concentration and stability of ELND006 in the formulations were confirmed using a suitable HPLC analytical procedure. The ELND006 dosing

Table 1. Compositions and properties of investigated ELND006 formulations.

IngredientVehicle composition, % w/w

1 2 3 4 5 6 7a

PEG 300 – – – 70 60 25 25Glycofurol 70 60 – – – – 25DMSO – – – – – 45 –Cremophor ELP 20 20 20 20 25 5 25Ethanol, 200 Proof 10 10 10 10 15 15 15Propylene glycol – 10 70 – – 10 10Properties and physical stability of formulationsb

Physical appearance Slightly yellowish solution

Slightly yellowish solution

Slightly yellowish solution

Slightly yellowish solution

Slightly yellowish solution

Immiscible Slightly yellowish solution

Dilution with saline solution (µL to µL ratio) Slightly cloudy

Slightly cloudy

Slightly cloudy

Slightly cloudy

Slightly cloudy

Cloudy Clear

DMSO, dimethyl sulfoxide; PEG 300, polyethylene glycol 300.aVehicle composition # 7 was selected to prepare ALZET® pump infusion formulations for the investigational compounds. These infusion

formulations were also used to prepare subcutaneous (SC) bolus solution formulations after suitable dilutions with sterile saline solution for administering the loading doses at the time of pump implantation.

bEach formulation was prepared by dissolving a known amount of an investigational compound into a known volume of the vehicle in a volumetric flask to yield the desired mg/mL concentration. The properties and physical stability summarized herein correspond to 60 mg/mL ELND006 concentrations in the various vehicles investigated.

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formulations used for the preclinical studies are summa-rized in Table 2.

The suitability of the selected vehicle composition # 7 (Table 1) for preclinical studies was further evaluated using another poorly soluble investigational compound ELN 481594. ELN 481594 exhibited low aqueous solubility (~43 µg/mL at 25°C), high permeability, and high lipophilicity (Log D 3.40) and was predicted to belong to class II of the BCS. The infusion formulations were prepared at 5, 15, and 60 mg/mL concentrations for compound ELN 481594 to meet the requirements for the preclinical studies. The ELN 481594 dosing formulations used for the preclinical studies are summarized in Table 2.

Compatibility between formulations and ALZET® pumpsThe selected infusion formulations for ELND006 and ELN 481594 were evaluated for their compatibility with the pump reservoir material using the ALZAID® chemical compatibility test kit.25 The test kit consists of polymer spheres with identical composition as the polymer (thermoplastic hydrocarbon elastomer) used in the reservoir of the two types of ALZET® pumps used in the current studies (i.e. Model # 1003D and Model # 1004). In brief, to determine the compatibility of a for-mulation with the pump reservoir material, 33 polymer spheres were weighed into an 8-mL screw-cap test tube and incubated in 5 mL of the formulation at 37°C for 4 days. The spheres were collected from the test tube after the incubation period, dried gently with an absorbent tissue, and reweighed accurately. Compatibility of the formulation with the spheres was determined using the following equation recommended by the ALZET® pump manufacturer25:

Percent weight change of spheres

Final weight Initial weig=

− hht

Initial weight100

( )( ) ×

(1)

A formulation is judged to be compatible with the spheres, and thus with the pump reservoir material, when the calculated percent weight change value is within the range of 0–7% (either negative or positive), and incom-patible when the value is outside this range and/or when the spheres are deformed, i.e. become swollen, brittle, or change color.

The chemical stability and compatibility of the for-mulations with the ALZAID® polymer spheres were evaluated using a reverse phase HPLC analytical proce-dure. Formulations were stored at 2–8°C (control), 37°C (representing stability at physiological temperature), and incubated with ALZAID® polymer spheres at 37°C (representing compatibility with ALZET® infusion pump) for 4 days. Quantification of the concentration of ELND006 in the formulations was achieved using reverse phase HPLC analysis conducted on an Agilent 1100 series instrument (Agilent Technologies, Santa Clara, CA USA). Separations were achieved using a Phenomenex (Phenomenex Inc, Torrance, CA USA), Luna C18 col-umn (100 Å, 3 µm, 100 mm × 4.6 mm) with a mobile phase of 0.1% v/v trifluoroacetic acid (TFA) in a mixture of 65% v/v acetonitrile and 35% v/v water. The column temperature was set at 50 ± 2 °C. The mobile phase flow rate was 1.0 mL/min and the detection wavelength was 250 nm. ELND006 standard and sample preparations were made in a diluent consisting of 40% acetonitrile with 0.1% TFA and 60% deionized water with 0.1% TFA at a target concentration of 0.05 mg/mL. The injection volume was 20 µL. The composition and flow rate of the

Table 2. Summary of dosing parameters and corresponding plasma concentrations in mice.

Investigational Compound

ALZET® infusion parameters SC bolus parametersPlasma concentration,

ng/mL, N = 5 (SD)

ALZET® pump type

Conc. in infu-sion solution,

mg/mLa µg delivered/h

Conc. in SC bolus solution,

mg/mLb

SC bolus loading dose, mg/kg 6 h 24 h 48 h 72 h

ELND006 Model # 1003D, 3 day, 1.0 μL/h or 24 µL/day

2 2 0.3 0.59 41.0 (11.1)

42.2 (3.50)

35.9 (5.70)

24.5 (12.9)

10 10 1.5 2.90 373 (82)

250 (81)

203 (42)

104 (48)

50 50 7.4 14.7 1928 (396)

1146 (333)

535 (147)

604 (165)

1 day 14 day 28 dayELN 481594 Model # 1004, 28 day, 0.11

μL/h or 2.64 µL/day5 0.55 0.028 0.056 1.10

(0.21)1.95

(1.19)2.39

(0.29)15 1.65 0.068 0.136 7.69

(7.32)6.80

(4.45)8.81

(0.81)60 6.60 0.274 0.548 17.3

(10.1)16.0c

(4.62)36.9

(6.70)aInfusion solutions were prepared in a vehicle containing 25% PEG 300/25% Cremophor ELP/25% glycofurol/15% ethanol/10% propylene

glycol (all on w/w basis).bSC bolus solutions were prepared by diluting the infusion solutions with sterile saline solution to yield the desired concentrations.cN = 4; Measurements from one animal were discarded due to anomalous plasma concentration reading.

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aforementioned mobile phase were adjusted to effect the quantitation of ELN 481594 in the formulations.

Animal studiesAnimal experiments were conducted in accordance with the National Institutes of Health, Guide for the Care and Use of Laboratory Animals, and Institutional Animal Care and Use Committee.

ALZET® pumps were filled with the infusion formula-tions by aseptic technique by means of a syringe attached with a blunt end needle. Pumps were weighed before and after filling with the formulations to ensure adequate fill volume. Filled pumps were primed by incubating in ster-ile saline solutions at 37°C for 18 h prior to implantation into the mice. The priming of the pumps is essential to deliver their contents at the expected rates immediately upon implantation. The delivery portal of the pumps and incubated saline solutions were checked visually and microscopically for any crystallization of the solubilized compound.

Pumps were implanted under the subcutaneous tissue of 11- to 12-week-old female FVB mice (Taconic Laboratories, body weight 20–25 g) by aseptic surgical technique under isoflurane anesthesia. A loading dose of the compound was administered by subcutaneous bolus route immediately after implantation of the pumps to obtain steady state blood levels quickly (Table 2). Animals were provided with certified rodent diet (Harlan Labs Diet # 2018; 18% protein diet) and water ad libitum. Animals were acclimated for at least 5 days prior to the study start and monitored for mortality, abnormalities, and signs of pain and distress throughout the study. Animals were terminated at 6-, 24-, 48-, and 72-hour time points (each N = 5) for ELND006 and at 1-, 14-, and 28-day time points (each N = 5) for ELN 481594, respectively, post initiation of infusion. Animals were initially anesthetized by isoflurane and a blood sample (200 µL) was taken via retro-orbital bleed, collected in potassium EDTA anticoagulant tubes, and stored on ice until centrifugation. Plasma samples were collected by centrifugation of the blood samples and analyzed using an appropriate LC–MS/MS bioanalytical method. The implantation sites were checked for any abnormalities, such as compound crystallization, local inflammation, or infection after animals were sacrificed.

Results and discussion

Selection of formulationsInvestigational compounds ELND006 and ELN 481594 have poor aqueous solubility, high lipophilicity, and moderate to high permeability, suggesting that these compounds belong to class II of the BCS. A series of hydrophilic, non-aqueous solvents as neat and in com-bination were evaluated in terms of miscibility, clarity, and their ability to solubilize ELND006 and keep it in solution against crystallization upon dilution with saline

solution. Though many of the tested solvents showed sufficient solubility for the compound, use of these sol-vents as neat or in combination resulted in its crystal-lization upon dilution with saline solution. Presence of an appropriately chosen surfactant in a formulation is known to interfere with crystallization process and thus delay, minimize, and/or even eliminate the pro-cess.16–18 The selected surfactants for the current stud-ies, Cremophor ELP, Polysorbate 80, and Solutol HS 15 have been used widely in several commercial injectable products and preclinical formulations.15,18,26 Among the surfactants evaluated, Cremophor ELP has shown the potential to eliminate crystallization of the compound from the formulations upon contact with saline solution. Visual and microscopic observations revealed that the delivery portals of the pumps and the saline solutions in which these pumps were incubated were free from any crystallization of the solubilized ELND006. In addition, the results from the one-to-one dilution studies using normal saline solution indicated no crystallization of the solubilized compounds for more than 1 h (Table 1), suggesting the solubilized compounds were less likely to crystallize in vivo due to physiological dilution by tis-sue fluids. Another one of the criteria was the selection of ingredients and their levels in the formulations at or below acceptable limits reported earlier that would not have any adverse effects in the study animals.15,18,26 The amount of each of the ingredients used in the selected formulations (Table 1: Vehicle # 7) was within the recom-mendations proposed earlier (Table 3).

The selected vehicle composition # 7 (Table 1) was also evaluated for its applicability to ELN 481594 and found to be appropriate in terms of solubilization and resistance with regard to crystallization of the compound upon con-tact with saline solution (results not shown). The delivery portals of the pumps and the saline solutions in which the pumps were incubated were also found to be free from any crystallization of the solubilized ELN 481594.

Compatibility studiesThe calculated values of percent weight change of poly-mer spheres (Equation 1) incubated in the selected for-mulations were 3.1% or less which was significantly lower than the maximum limit of 7% specified by the ALZET® pump manufacturer.25 In addition, the spheres showed no observable change in color and physical appearance at the end of the incubation period. The incubated formu-lations were clear with a slight yellowish tint as were the original formulations. HPLC analysis indicated no detect-able loss of the solubilized compounds in the formula-tions stored at 2–8 and 37°C, and when incubated with the polymer spheres at 37°C. No additional impurity (deg-radation) peaks were identified in the chromatograms of the formulations on stability, as illustrated in Figure 1 for ELND006 (results not shown for ELN 481594). These results suggested that the formulations and the reservoir material of the ALZET® osmotic pumps were mutually

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compatible with each other and the formulations were chemically stable throughout the incubation period.

Gross observations of the sites of pump implantation in mice revealed no compound crystallization, local inflammation, or infection at all time points, suggesting that the formulations were compatible with the subcutaneous tissue of the animals.

Pharmacokinetic studiesThe plasma concentration versus time profiles following the subcutaneous infusion of ELND006 in mice using the investigative formulations filled into ALZET® osmotic pumps are shown in Figure 2. Increased ELND006 delivery rate from the osmotic pumps had a profound impact on its increased plasma concentration in the study animals, for example, the calculated p values were <0.05 between the 10 and 2 µg/h delivery rate groups and the 50 and 10 µg/h delivery rate groups at all sampling time points. The plasma concentration of ELND006 reached a peak state within 6 h and then decreased with time thereafter for the delivery rates studied (Table 2). There were no statisti-cally significant differences among the ELND006 plasma concentrations with time for the 2 µg/h delivery rate (p = 0.05). By contrast, there was a significant decrease in the plasma concentration from 6 to 48 h time point (p = 0.021) for the 10 µg/h delivery rate and from 6 h to

an earlier 24 h time point (p = 0.012) for the 50 µg/h deliv-ery rate. Thus the results suggested that the extent of the time-dependent decrease in the ELND006 plasma levels increased as the amount of dose delivered to the animals increased. For example, the ELND006 plasma concentra-tions at the 48-hour point were about 88, 54, and 28% of those measured at the 6-hour time point for 2, 10, and 50 µg/h delivery rates, respectively. The delivery rates of some compounds from ALZET® osmotic pumps were reported to decrease with time, even from highly compat-ible, low-viscosity, simple aqueous solutions.2 It is possible that those reported decreased delivery rates might be due to non-specific adsorption of the compounds to the pump reservoir lining. However, we believe similar adsorption effects are unlikely from the currently investigated formu-lations for two reasons. First, our studies on compatibility of the formulations with the ALZAID® polymer spheres (representing the pump reservoir lining) suggested no loss or degradation of the solubilized compounds. Second, the adsorption effects, though present, however unlikely, were usually eliminated or minimized during the 18 h of pump priming period that we used in our experiments. The most likely cause for the decrease in ELND006 plasma concentrations over time was due to auto-induction of cytochrome P450 3A (CYP3A) enzymes in these animals. ELND006 was shown from the previous in vitro and in vivo

Table 3. Physical and toxicological profiles of ingredients used in the formulations.Property PEG 300 Glycofurol Ethanol Propylene glycol Cremophor ELPDensity, gm/mL 1.120a,f 1.070–1.090b,g 0.7904–0.7935b,c,h 1.038b,i 1.05–1.06a,j,k

Viscosity, cP 80–105a,f 8–18b,g 1.22b,h 58.1b,i 600–750a,j

Reported amount compatible with

ALZET® pumps, %

Up to 100l N/A Up to 15l Up to 100l Up to 25l

Amount used in current study, % 25 25 15 10 25Reported LD

50 Toxicity, per kg 7.1 mLd 3.5 mLe 1.97 gme 6.63 gme 2.5 gme

Maximum daily amount delivered in current study, per kg

240 mg 240 mg 144 mg 96 mg 240 mg

N/A, Not available.a25°C; b20°C; cSpecific gravity; drat IV; emouse IV.fPrice, 200624; gWeller, 200627; hOwen, 200628; iOwen and Weller, 200629; jSingh, 200630; kCremophor® EL Technical Leaflet31; lAlzet® Technical Information Manual 1.

Figure 1. Representative chromatograms of ELND006 60 mg/mL infusion solution formulations stored at 2–8°C (top, control), 37°C (middle, representing formulation stability at physiological temperature), and incubated with ALZAID® polymer spheres at 37°C (bottom, representing compatibility with ALZET® infusion pump) for 4 days. Infusion solution formulations were prepared in a vehicle containing 25% PEG 300/25% Cremophor ELP/25% glycofurol/15% ethanol/10% propylene glycol (all on w/w basis).

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studies to induce CYP3A enzymes in the rodent models (unpublished reports) and the time- and dose-dependent reduction in the plasma ELND006 concentrations in the current studies is consistent with those observations.

For ELN 481594 solution-filled pumps, the corresponding plasma concentration of ELN 481594 reached a steady state within 24 h for all three delivery rates studied (Table 2). There was no significant change in ELN 481594 plasma concentration with time for the 0.55 and 1.65 µg/h delivery rates (p > 0.05). By contrast, the plasma concentration of ELN 481594 increased significantly from day 1 and 14 to day 28 day time point (p = 0.007) for the 6.60 µg/h delivery rate, suggesting accumulation of the compound with time. Increased ELN 481594 delivery rate from the osmotic pumps had a clear impact on its increased plasma concentration only at the 28 day time point.

Conclusions

From these studies, we concluded that the selected vehi-cle composition was an appropriate solubilizer for the poorly soluble investigational compounds studied with no or minimal potential for crystallization of the solubi-lized compounds up on contact with aqueous fluids in vitro and in vivo. The formulations were also found to be compatible with the ALZET® osmotic pumps and with the subcutaneous tissue of mice where the pump was implanted. Even though the selected vehicle composition was investigated with a limited number of poorly soluble compounds in the current study, the data generated offers scientists with a newer ALZET® osmotic pump compat-ible vehicle composition that can be used as is or with some modification to solubilize a variety of other poorly soluble compounds during their preclinical studies.

Acknowledgements

The authors thank Drs. Thomas Tarnowski, Gene Kinney, Daniel Ness, Ted Yednock, and Mark Hoch for their valu-able comments during the preparation of the manuscript.

Declaration of interest

The authors declare that they have no conflicts of interest to disclose.

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2008. http://www.alzet.com/resources/documents/TIM2008.pdf. Accessed on December 2011.

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Figure 2. Mean (±SD, N = 5) plasma concentration versus time profiles of ELND006 in female mice following subcutaneous implantation of ALZET® osmotic pumps. Refer to Table 2 for dosing parameters.

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