1. Micro-Formulation for Intravenous Injection
oBJECTIVES
•To formulate new chemical entities (NCEs) for IV injection using only 2 -3 mg of the
NCE.
•To develop standardized methods for rapidly carrying out “micro-formulation of NCEs.
•To test the methods using a series of model compounds.
ABSTRACT
PURPOSE: The purpose of this work was to develop methodologies for formulating new
chemical entities (NCEs) for intravenous injection using only a few milligrams of the
NCE. This “micro-formulation” approach was developed to facilitate in vivo screening of
drug candidates at an early stage of development, when many NCEs may be under
consideration and very little of each may be available. METHODS: A series of
formulation bases, representing various intravenous formulation approaches, were
prepared. The model compounds were distributed among several small tubes to which the
different formulation bases were added. Solubilities were determined
spectrophotometrically after processing by shaking and/or sonication. Several model
compounds were formulated in these studies. In some cases, the results obtained on the
small scale were compared to results obtained using conventional formulation
approaches. RESULTS: Suitable intravenous formulations were developed for several
water-insoluble model compounds using 2-3 mg of each compound. Micro-formulation
results were reproducible and generally agreed with results obtained using conventional
formulation approaches with the same compounds. The micro- formulation approach was
found to be time efficient and could be applied to compounds for which no solubility or
other chemical characterization data was available. CONCLUSION: Formulations of new
chemical entities suitable for intravenous injection in animals can be developed using as
little as 2-3 mg of each chemical entity.
INTRODUCTION
High-throughput in vitro screening of large numbers of molecules for their agonist or
antagonist activity is an important mechanism of drug discovery. However, because
receptor binding is only one factor impacting on the overall effectiveness of a drug in the
body, early screening in an animal model, usually by IV injection, may be a necessary
supplement to the in vitro evaluation. This can become a daunting task if an in vitro
screening campaign identifies dozens of strong binders of which only very low milligram
quantities are available to formulate for animal testing.
A typical formulation strategy is to first measure the solubility of the drug in various
solvents and as a function of pH and then to identify one or more formulation approaches

2. to pursue. The goal is to maximize drug solubility within the constraints of acceptable
toxicity, pH, tonicity, and sterility. For drugs with poor water solubility, formulation
approaches include high or low pH solutions, micellar dispersions, lipid emulsions,
liposomes, and inclusion complexes.
Although the amount of drug required for a formulation campaign can be minimized
through careful experimental design, gram quantities, or at least high milligram
quantities, are typically needed. Our goal was to develop methodology that could be
applied to the very low milligram quantities available at the early stages of drug discover
METHODS
•Drug Stock Solution. For drugs provide as powders, 2 - 3 mg was accurately weighed
into a small vial. The drug was dissolved to 1.00 mL volume. Methodology was
established to test solubility of the same drug aliquot in successive solvents.
•Standard Curve. Dilutions of the stock solution were used to establish a standard curve
based on the highest wavelength absorption peak available.
•Formulation Bases. A series of formulation bases was prepared to represent high and
low pH, micellar dispersions, lipid emulsions, and inclusion colmplexes.
•Micro-Formulation. 100 µL of stock drug solution was placed in each of 7-8 micro-
tubes, and the solvent was evaporated under a nitrogen stream. Formulation bases were
added at 50 µL per tube. Tubes were sonicated 60 min. at 40°C and vortexed for 24 hours
at room temperature. Contents were then filtered, and the filtrate was assayed against the
standard curve.
•Standard Formulation. About 20 mg of powder was placed into each of 7-8 small vials.
Formulation bases (1.00 mL) were added, and the vials were shaken on a platform shaker
for 24 hours before the contents were filtered and assayed.
RESULTS
The goal of the described research was to develop methodologies to rapidly formulate
new chemical entities (NCEs) for intravenous injection using a minimal amount of the
NCE. To do this we simplified our formulation space to a series of 7-8 formulation bases
representing different types of formulations. Through optimization of technique, we
reduced the working volume per formulation to 50 µL, bringing the total amount of NCE
required to prepare a standard curve and test up to 8 formulation bases down to 2-3 mg.
Table 1 compares this “micro-formulation” approach to a standard formulation campaign.
Some challenges of working at the 50-µL scale were:
•Distributing the NCE among test containers

3. •Overcoming surface tension during agitation
•Filtration of this small a volume
We distribute the NCE among the test containers by dissolving it in a suitable solvent,
pipetting the solution into microtubes, and then evaporating off the solvent. This
approach can also be used when the molecule is supplied in a solvent. We have combined
bath sonication and vortexing to maximize dispersion of the NCE in the liquid since
mixing of such small volumes is inhibited by surface tension. By centrifuging
formulations through 3 mm syringe-tip filters, we found that we could limit filtration loss
to under 5 µL for all formulation bases.
We tested our methodology on a series of molecules with a range of physicochemical
properties. When feasible, we compared the “micro- formulation” results with results
obtained using more standard formulation methodology. Results of these comparisons are
given in Tables 2 through 4 and are shown graphically in Figure 1 through 3.
Using our methodology, reproducible solubility data was obtained at a 50-µL scale using
100 - 150 µg of compound per formulation tested. In general, results on the “micro-
formulation” scale were comparable with results obtained using a standard formulation
approach, except where the solubility of a compound in a formulation was higher than the
amount of the compound available to dissolve. In all cases, we were able to identify at
least one formulation in which the compound of interest could be formulated for IV
injection at a level of 4 mg/mL or higher.
Conventional
Formulation:
•
•
•
•
•
•
Micro-
Formulation:
Goal: Product
Development
• Goal: Animal Screening
Extensive pre-
formulation
•
Little or no pre-
formulation
Optimized formulation • Adequate formulation
Infinite possible
formulations
•
5-10 formulations
considered
Process development
work
• No process development
HPLC method
development
• Spectrophotometric assay

4. • Short and long-term stability • No stability evaluation
• Requires grams of drug • Requires 2 - 3 mg of drug
Table 1. Conventional formulation compared to “micro-formulation
50 µL (micro) Scale
(mg/mL)
1 mL (std) Scale (mg/mL)
Rep. #1 Rep. #2 Rep. #3 Rep. #1 Rep. #2
water 0.34 0.40 0.42 0.23 0.21
pH 4.5 buffer 6.1 6.1 6.0 12.1 12.3
pH 8.5 buffer 0.41 0.42 0.33 0.23 0.23
cyclodextrin 5.4 5.5 6.1 10.0 10.6
micelles 6.1 6.2 6.4 8.1 7.4
lipid emulsion1.9 2.1 2.9 2.8 2.9
PEG-based 3.9 3.8 4.0 >20 >20
Table 2. Solubility of quinine in different formulation bases. Results obtained working at
a 50-µL scale are compared with those obtained working at a 1-mL scale.
50 µL (micro) Scale
(mg/mL)
1 mL (standard) Scale (mg/mL)
Rep. #1 Rep. #2 Rep. #3 Rep. #1 Rep. #2 Rep. #3
water 0.027 0.016 0.019 0.003 0.002 0.004
pH 4.5 buffer 0.015 0.016 0.016 0.004 0.002 0.002
pH 8.5 buffer 0.017 0.016 0.022 0.002 0.002 0.002

5. cyclodextrin 0.27 0.30 0.25 0.26 0.27 0.27
micelles 1.8 1.8 1.5 1.7 1.7 1.6
lipid emulsion2.9 2.8 3.1 8.4 7.8 7.5
PEG-based 3.8 3.7 4.4 8.1 8.1 8.1
Table 3. Solubility of acenaphthene in different formulation bases. Results obtained
working at a 50-µL scale are compared with those obtained working at a 1-mL scale.
50 µL scale 50 µL scale
1 mL scale 1 mL scale1 mL scale
vortexed andvortexed and
shaken onlysonicated sonicated
sonicated sonicated
water 0.7 0.5 0.0 0.3 0.2
pH 4.5 buffer 0.6 0.5 0.1 0.6 0.1
pH 8.5 buffer 0.5 0.4 0.0 0.5 0.2
cyclodextrin 6.3 6.1 9.2
micelles 1.3 1.4 1.2
lipid emulsion2.0 1.9 2.2
PEG based 5.4 4.7 11.7
Table 4. Solubility of estradiol in different formulation bases. Results obtained working
at a 50-µL scale are compared with those obtained working at a 1-mL scale. Higher
solubility in water and buffers at the 50-µL scale may be due to sonication of the micro-
tubes. Sonication of 1-mL vials also resulted in higher solubilities in water and buffers.

6. cyclodextrin 0.27 0.30 0.25 0.26 0.27 0.27
micelles 1.8 1.8 1.5 1.7 1.7 1.6
lipid emulsion2.9 2.8 3.1 8.4 7.8 7.5
PEG-based 3.8 3.7 4.4 8.1 8.1 8.1
Table 3. Solubility of acenaphthene in different formulation bases. Results obtained
working at a 50-µL scale are compared with those obtained working at a 1-mL scale.
50 µL scale 50 µL scale
1 mL scale 1 mL scale1 mL scale
vortexed andvortexed and
shaken onlysonicated sonicated
sonicated sonicated
water 0.7 0.5 0.0 0.3 0.2
pH 4.5 buffer 0.6 0.5 0.1 0.6 0.1
pH 8.5 buffer 0.5 0.4 0.0 0.5 0.2
cyclodextrin 6.3 6.1 9.2
micelles 1.3 1.4 1.2
lipid emulsion2.0 1.9 2.2
PEG based 5.4 4.7 11.7
Table 4. Solubility of estradiol in different formulation bases. Results obtained working
at a 50-µL scale are compared with those obtained working at a 1-mL scale. Higher
solubility in water and buffers at the 50-µL scale may be due to sonication of the micro-
tubes. Sonication of 1-mL vials also resulted in higher solubilities in water and buffers.