Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/47543 PCT/US99/06198
SL:STAI:~1ED-RELEASE COMPOSTTION
INCLUDING A:rIORPHOi:S POLY:HER
F1FLD~ OF THF tNVE~?ION
'Ihe present invention concerns a highly amorphous sustained-release
composition
for susrained release of a pisarmaceutical substance; an antisolvent
precipitation method for
making the composition: products made using the composition: and uses of the
composition.
B aCKC~ROL~1D OF THE tWE_~tTION
Pharmaceutical substances may be introduced into a human or animal host for
therapeutic or curative purposes in a number of ways. In many pharmaceutical
applications,
the pharmaceutical substance is administered in the form of solid particles.
For example, a
micropump may be used in some applications for prolonged treatment by slowly
injecting a
suspea~sion of small particles in a liauid. Also. small particles having both
a phatmaceuticai
substance and a biodegradable polymer may be placed within tissue for
sustained release of
the pharmaceutical substance. with the biodegradable polymer acting to control
the release
of the pharmaceutical substance. Furthermore, in pulmonary delivery
applications, small
particles may be inhaled to lodge in tissue of the lungs, permitting the
pharmaceutical
substance to then eater the circulatory system or to be released for local
treatment.
Often, however, problems are encountered is attempting to make particles
having the
desired properties for a particular pharmaceutical application. For example.
when particles
having a biodegradable polymer and a pharmaceutical substance are prepared,
the
phamraceutical substance oven concentrates near the surface of the particles.
'Ibis effect may
cause a sudden, undesirable release of the pharmaceutical substance when it is
initially
introduced into the host. Also, whcn using a micropump for continuous
injection of a
suspension over a prolonged period, the solid particles tend to settle over
time, which may
cause an ttndesitable variation in the rate of delivery of the pharmaceutical
substance.
With respect to pulmonary delivery applications, current methods for
delivering the
pha:ataceutical substance in small particles typically result in a majority of
the
pharatacetuical substance being wasted. In one method. called nebutization. a
liquid having
the phartaaceutical substance in solution is sprayed at a high velocity and
inhaled.
Alternatively, nebuliiation may involve spraying a powder as fine particles
propelled by a
carrier gas, with the particles being inhaled. Particles administered by both
these
nebulizatioa methods, however. may have a wide distribution of droplet or
particle sins,
resulting in a very low utilization of the pharmaceutical substance.
Particles, or droplets,
which are too large tend to lodge in the throat and mouth during inhalation
and are not,
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CA 02324254 2000-09-15
WO 99147543 PCT/US99l06198
therefore, effective for delivering the pharmaceutical substance to the lungs.
Particles, or
droplets, which are too small tend not to impact on the lung tissue, but
rather tend to be
exhaled. As much as 80 to 90 percent, or more, of the pharmaceutical substance
may,
therefore, be wasted and only a small portion of the pharmaceutical substance
which is
ac°ministered may attuall~ reach the desired target in the lung. '
Many of these problems with delivery of particles of a pharmaceutical
substance
result from limitations on methods used to make the particles. One method for
making
particles of a pharmaceutical substance, called lyophilization, involves rapid
freeing of the
pharmaceutical substance with water, followed by rapid dehydration of the
frozen material
to produce dry particles of the pharmaceutical substance. This technique has
been used with
proteins and other polypeptides, but the low temperanuts involved may reduce
the biological
activity of some polypeptide molecules. Also, the particles produced by
lyophilization tend
to be large and clumping and are often not suitable for pharmaceutical
delivery methods
which require smaller particles. It is possible to grind the lyophilized
particles to produce
smaller particles, but such grinding may damage some pharmaceutical
substances, especially
proteins. Also, even when a substance may be ground without significant damage
to the
activity of the substance, it is difficult to obtain a pharmaceutical powder
having particles of
a narrow size distribution. Therefore, such pharmaceutical powders are prone
to substantial
waste of the pharmaceutical substance, such as described above for pulmonary
delivery
applications.
One method which has been proposed for making small particles of a
pharmaceutical
substance is called gas antisolvent precipitation. In this method, a
pharmaceutical substance
is dissolved in an organic solvent which is then sprayed into an antisolvent
fluid, such as
carbon dioxide, under supercritical conditions. The antisolvent fluid rapidly
invades spray
droplets, causing precipitation of very small pharmaceutical particles.
The gas antisolvent precipitation technique, however, requires that the
pharmaceutical
substance be soluble in the organic solvent. For hydrophobic pharmaceutical
substances, this
generally presents no problem because those substances can readily be
dissolved in relatively
mild, non-polar organic solvents. Hydrophilic pharmaceutical substances,
however, are
substantially insoluble in such relatively mild organic solvents.
It has been proposed that insulin, a hydrophilic protein, may be processed in
a gas
antisolvent precipitation process by dissolving the insulin in
dimethylsulfoxide (DMSO) or
N,N-dimethylformamide (DMF), both of which are strong, highly polar solvents.
One
problem with such a process, however, is that highly polar solvents such as
DMSO and DMF
SUBSTITUTE SHEET (RULE 26)
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tend to unfold protein molecules from their native tertiary structure, or
conformation. These
protein molecules would, therefore, also be precipitated in an unfolded state
for incorporation
into the solid particles. Such unfolding could seriously reduce the biological
activity of a
protein or other polvpeptide, especially if stored as a solid particle in the
unfolded state for
any appreciable time.
There is a need for improved methods for making solid particles of
pharmaceutical
substances. and especially for making particles of hydrophilic substances, to
permit
preparation of particles having an appropriate size and size distribution
without the molecular
unfolding associated with the gas antisolvent ptrcipitation method and without
the low
temperatures and grinding associated with lyophilization.
Despite intense efforts in the field of gene therapy, there is still a lack of
well-defined
delivery vehicles that will allow efficient and effective delivery of an
oligonucleatide-based
therapeutic agent. Much of the work in this area has centered on the use of
cationic lipids.
The ability of cationic lipids to interact with membranes, to increase the
lipophilicity of
polynucleotides, and to mask the significant negative charge on
polynucleotides, appears to
be essential to achieving a high degree of transfection of the targeted cell.
However, there
remains a need in the art for more effective ways of achieving transfection.
It has been reported that cationic surfactants can be used to conjugate
nucleic acids
to enzymes and to purify nucleic acids. See U.S. Patents Nos. 4,873,187 and
5,010,183. In
particular, the latter patent teaches that the cationic surfactants and
nucleic acids form
hydrophobic complexes that can be dissolved or dispersed in polar solvents for
purification
of the nucleic acids.
However. currently existing cationic surfactants tend to be toxic and not
suitable for
pharmaceutical use or other uses where cell survival is important. Therefore,
a need exists
for new cationic surfactants that are less toxic thaw the existing cationic
stufactant$ ~ which
can be used in situations when cell survival is important.
Furthermore, the morphology of the particles may detrimentally effect
performance.
For example, M. Mayajim et al., Effect of Polymer Crystalliniry on Paperverine
Release from
Poly(1-lactic acid) Matrix, describe a problem encountered with amorphous
poly(1-lactic acid)
in sustained-release compositions. The amorphous polymer had an undesirable
tendency to
crystallize during drug release, thereby altering the drug release
characteristics of the
composition.
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WO 99/47543 PCT/US99/06198
There is a need for improved methods for making compositions for sustained-
release
applications and for improved sustained release-compositions with desirable
drug release
characteristics.
SL'V(1~~L4RY (~= 'T'~~ ltv'V NT10N
According to the present invention. a method is provided for placing a
pharmaceutical
substance into solution in an organic solvent in the form of a hydrophobic ion
pair complex
with an amphiphiIic material. The resulting solution may then be subjected to
gas antisolvent
precipitation using a near critical or supercritical fluid to produce a
precipitate of particles
comprising the pharmaceutical substance. Particles may be produced with a
relatively narrow
size distribution in a variety of sizes, thereby permitting flexibility is
preparing particles for
effective utilization in a variety of pharmaceutical applications.
The present invention, therefore, permits pharmaceutical substances which are
ordinarily substantially not soluble in an organic solvent to be solubilized,
which facilitates
further processing to prepare pharntaceutical powders. The method is
particularly preferred
for use with proteins and other polypeptide molecules. Those molecules may be
dissolved
in a relatively mild, relatively non-polar org,m:c solvent, ;hereby decreasing
the potential for
the reduction in biological activity whica ~oui~i r~~,:it tiom use of a
strong, highly polar
organic solvent in which the hydrophilic molecules are directly soluble.
In one embodiment of the present invention, a biodegradable polymer may be co-
dissolved in the organic solvent along with the pharmaceutical substance and
the amphiphilic
material. When processed by gas antisolvent precipitation, the particles
produced comprise
an intimate mixture of the biodegradable polymer with the pharmaceutical
substance and the
amphiphilic material. Problems of compositional variation or concentration of
the
pharmaceutical substance near the surface of the particle are, therefore,
reduced relative to
processes which require processing of a pharmaceutical substance in a
suspension.
In one embodiment, through careful control of the antisolvent precipitation
process
operating conditions, a highly amorphous sustained-release composition may be
made
including a biocompatible polymer, typically also biodegradable, and a
pharmaceutical
substance in the form of a hydrophobic ion pair complex with an amphiphilic
material. The
polymer is highly amorphous, preferably with no greater than about 25%
crystallinity and
more preferably with an even lower crystalIiniry. Furthermore, the composition
typically
includes a very high loading of the pharmaceutical substance and the
amphiphilic material,
which together in the hydrophobic ion pair complex typically comprise greater
than about 15
4
SUBSTITUTE SHEET (RULE 28)
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WO 99/47543 PCTNS99/06198
weight percent of the composition. and preferably even more. The composition
exhibits
desirable release characteristics for sustained release of the pharmaceutical
substance and
does not appear to have a problem with crystallization of the polymer during
drug release.
as has been reported by others. Also. the amorphous character of the polymer
should be less
likely to provoke an immune response that could cause inflammation. Moreover.
the
composition typically includes low or insignificant levels of residual solvent
from the
manufacture process. The composition may be incorporated into a variety of
product forms
for administration to a mammalian patient. Preferred methods for use of the
composition
include inhalation for pulmonary delivery. subcutaneous placement,
iniraperitoneal
placement and intraocular placement.
To make the highly amorphous sustained-release composition, the antisolvent
precipitation process is controlled to provide the desired characteristics in
the composition.
A high ratio of volume:ric flow of aatisolvent fluid to volumetric flow of
liquid feed (in
which the pharmaceutical substance. amphiphilic material and polymer are
codissolved) is
preferred. with a ratio of from about 20 to about 30 being particularly
preferred. Lower flow
ratios tend to increase crystalliniry of the polymer in the composition. Also,
it has been found
that it is preferred to process the arttisolvent and the liquid feed in
concurrent flow to eaha~e
the quality of the composition. Furthermore, the preferred method of operation
of the
process is to contact the liquid feed and antisolveat fluid at subcritical
conditions, but
preferably at a reduced temperature of greater than about 0.5.
In another embodiment of the present invention, a pharmaceutical substance is
provided having particles comprising a phamaaceuticai substance and an
amphiphilic material
in a hydrophobic ion pair cotrplex. in one embec~:r~:a:. the rarticies 1><:ve
a narrow si~a
distribution, with greater than about 90 weight percent of the particles
having a size smaller
than about 10 microns. In another embodiment. the solid particles are hollow
and have a
substantially elongated. fiber-like shape. Zhese elongated particles are
advantageous is that
they should have a longer retention time, compared to substantially spheroidal
particles, in
the stomach of a htuaan or animal host following ingestion. Therefore, the
particles may be
advatuageotuly used for sustained release applications for delivery of a
pharmaceutics)
substance in the stomach region.
In yet a further embodiment of the present invernion. a method is provided for
delivering a pharmaceutical substance for treatment of a human or animal host
in which a
pharmaceutical formulation is administered having solid particles including a
pharmaceutical
substance sad an amphiphilic material. 'Ihe administration may be by
inhalation of the solid
5
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CA 02324254 2002-12-20
74667-173
particles, by injection of a suspension of the solid
particles in a liquid medium or by ingestion of the solid
particles.
The invention also provides cationic surfactants
having the formula:
P-L-C
wherein:
P is a biocompatible hydrophobic moiety;
C is a biocompatible cationic moiety; and
L is a biodegradable linkage linking P and C.
These cationic surfactants are substantially less
toxic than currently existing cationic surfactants and can
be used for administration of pharmaceutical substances to
animals and in other situations where cell survival is
important. In particular, they can be used as the
amphiphilic material in the methods and compositions
described above. In addition, these cationic surfactants
can be used to deliver nucleic acids into cells, making them
useful in genetic engineering techniques, including gene
therapy.
According to one aspect of the present invention,
there is provided a method for producing a biocompatible
polymer particle for sustained release of a pharmaceutical
substance, wherein said particle comprises substantially
amorphous biocompatible polymer having a crystallinity of no
more than about 25%, and wherein said particle comprises a
substantially homogenous mixture of said pharmaceutical
substance and said amorphous polymer; comprising the steps
6
CA 02324254 2004-02-23
74667-173
of: a) providing: i) a solvent, ii) a pharmaceutical
substance, iii) an amphiphilic material, selected from the
group consisting of sulfates, sulfonates, phosphates,
phospholipids, carboxylates, sulfosuccinates, arginine
esters, cholesterol esters, carbamates, carbonates and
ketals, iv) at least one biocompatible polymer selected from
the group consisting of poly(L-lactic) acid, poly(D-lactic)
acid, polyglycolic acid, polyanhydride,
polycarboxyphenoxyhexane, polybutyrate, and cellulose; and
v) an antisolvent fluid, wherein said antisolvent fluid
comprises at least one antisolvent selected from the group
consisting of carbon dioxide, nitrous oxide, ethane
ethylene, chlorotrifluoromethane, monofluoromethane,
acetylene, 1,1-difluoroethylene, hexafluoroethane,
chlorotrifluorosilane and xenon; b) combining said solvent,
pharmaceutical substance and amphiphilic material to form a
solution, wherein said pharmaceutical substance and
amphiphilic material form a hydrophobic ion paired complex
within said solution; and c) contacting at subcritical
conditions said solution, said polymer, and said antisolvent
fluid by concurrent flow to produce a mixture, wherein said
mixture has a reduced temperature in the range of about
0.75-0.995, and wherein a reduced pressure relative to the
antisolvent fluid is in the range of about 0.5-2.0, wherein
the weight percentage of said hydrophobic ion paired complex
to the total weight of said hydrophobic ion paired complex
and said polymer is in the range of about 15% to 70%, and
wherein the volumetric ratio of flow rate of said
antisolvent fluid to the flow rate of said solution is in
the range from about 5 to about 100.
According to another aspect of the present
invention, there is provided a method for producing a
biocompatible polymer particle for sustained release of a
6a
CA 02324254 2004-02-23
74667-173
pharmaceutical substance, wherein said particle comprises
substantially amorphous biocompatible polymer having a
crystallinity of no more than about 25%, and wherein said
particle comprises a substantially homogenous mixture of
said pharmaceutical substance and said amorphous polymer;
comprising the steps of: a) providing: i) a solvent, ii)
a pharmaceutical substance, iii) an amphiphilic material,
selected from the group consisting of sulfates, sulfonates,
phosphates, phospholipids, carboxylates, sulfosuccinates,
arginine esters, cholesterol esters, carbamates, carbonates
and ketals, iv) at least one biocompatible polymer selected
from the group consisting of poly(L-lactic) acid, poly(D-
lactic) acid, polyglycolic acid, polyanhydride,
polycarboxyphenoxyhexane, polybutyrate, and cellulose; and
v) an antisolvent fluid, wherein said antisolvent fluid
comprises at least one antisolvent selected from the group
consisting of carbon dioxide, nitrous oxide, ethane
ethylene, chlorotrifluoromethane, monofluoromethane,
acetylene, 1,1-difluoroethylene, hexafluoroethane,
chlorotrifluorosilane and xenon; b) combining said solvent,
pharmaceutical substance, amphiphilic material and polymer
to form a solution, wherein said pharmaceutical substance
and amphiphilic material form a hydrophobic ion paired
complex within said solution; and c) contacting at
subcritical conditions said solution, and said antisolvent
fluid by concurrent flow to produce a mixture, wherein said
mixture has a reduced temperature relevant to the
antisolvent field in the range of about 0.75-0.995, and a
reduced pressure relative to the antisolvent fluid in the
range of about 0.5-2.0, wherein the weight percentage of
said hydrophobic ion paired complex to the total weight of
said hydrophobic ion paired complex and said polymer is in
the range of about 15% to 70%, and wherein the volumetric
6b
CA 02324254 2004-02-23
74667-173
ratio of flow rate of said antisolvent fluid to the flow
rate of said solution is in the range from about 5 to
about 100.
According to another aspect of the present
invention, there is provided a biocompatible polymer
particle for sustained release of a pharmaceutical
substance, wherein said particle comprises a substantially
amorphous biocompatible polymer having a crystallinity of no
more than about 25%, wherein said pharmaceutical substance
is combined with an amphiphilic material to form a
hydrophobic ion paired complex; wherein said biocompatible
polymer comprises at least one polymer selected from the
group consisting of poly(L-lactic) acid, poly(D-lactic)
acid, polyglycolic acid, polyanhydride,
polycarboxyphenoxyhexane, polybutyrate, and cellulose;
wherein said amphiphilic material is selected from the group
consisting of sodium dodecyl sulfate, bis-(2-ethylhexyl)
sodium sulfosuccinate, cholesterol sulfate and sodium
laurate; wherein said particle comprises a substantially
homogenous mixture of said pharmaceutical substance and said
amorphous biocompatible polymer; wherein the weight
percentage of said hydrophobic ion paired complex to the
total weight of said hydrophobic ion paired complex and said
biocompatible polymer is in the range of about 15% to 70%.
According to another aspect of the present
invention, there is provided a method for making a
composition for sustained release of a pharmaceutical
substance, wherein said method comprises agglomerating a
plurality of particles as described herein, to form an
agglomerate of said particles.
6c
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74667-173
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the log of the apparent partition
coefficient for the dipeptide Gly-Phe-NH2.
Fig. 2 shows the log of the apparent partition
coefficient for 8-Arg-vasopressin (AVP).
Fig. 3 shows the log of the apparent partition
coefficient for insulin.
Fig. 4 shows the CD spectra of a 6:1 SDS-insulin
complex in 1-octanol.
Fig. 5 shows the CD spectra of insulin extracted
from 1-octanol using an aqueous solution of 0.10 M HC1.
Fig. 6 shows the effect of temperature on the
denaturation of insulin dissolved in 1-octanol.
Fig. 7 shows the logarithm of the apparent
partition coefficient of bovine pancreatic trypsin inhibitor
(BPTI) from pH 4 water into 1-octanol.
Fig. 8 shows the UV-visible absorption spectrum of
human serum albumin (HSA) in NMP (50:1 SDS to HSA ratio).
Fig. 9 shows the melting point of the SDS:insulin
HIP complex as a function of the molar ratio of SDS to
insulin.
Fig. 10 shows a CD scan for a 9:1 SDS:insulin
molar ratio at 222 nm as a function of temperature.
6d
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WO 99/47543 PCT/US99/06198
Fig. 11 shows an absorbance scan for a 9:1 SDS:insulin molar ratio at '?'?2 ~
~ a
function of temperature.
Fig. 1? shows a process flow diagram for one embodiment of an antisolvent
precipitation method for producing pharmaceutical powders.
Fig. 13 shows a process flow diagram for batch processing for gas antisolven!
precipitation relating to Examples 19-29.
Fig. 13 is an SEM photomicrograph of a particle of the present invention
comprising
imipramine.
Fig. 15 is another SEM photomicrograph of a particle of the present invention
comprising imipramine.
Fig. 16 is a SEM photomicrograph of a particle of the present invention
comprising
ribonuclease and poly(ethyleneglycol).
Fig. 17 is a SEM photomicrograph of particles of the present invention
comprising
a-chymotrypsin.
Fig. 18 is a SEM photomicrograph of particles of the present invention
comprising
pentamidine.
Fig. 19 shows a process flow diagram for continuous processing for gas
antisolvent
precipitation relating to Examples 30-32.
Figures 20A-G illustrate schemes for the synthesis of arginine esters, CBZ is
phenylmethoxycarbonyl and tBOC is t-buryloxycarbonyl.
Figures 2IA-F illustrate schemes for the synthesis of cholesterol esters and
carbatnates. THF is tetrahydrofuran. Me is methyl. MeI is methyliodide. MEIC
is methyl
ethyl ketone.
Figure 22A is a graph of surface tension versus concentration for arginine
octyl ester.
Figure 22B is a graph of surface tension versus concentration for arginine
dodecyl
ester.
Figure 23A is a graph of OD,~ versus concentration comparing cytotoxiciry of
ar~tte dodecyl ester and tetradecyltrimethylammonium bromide (CTAB) in CCRF-
CEM
cells.
Figure '_'= B is a graph of OD,~ versus concentratio:. comparing
c,rtoto~cic:ry of
argtame dodecyl ester and tetradecyltrimethylammonium bromide (CTAB) in COS-7
cells.
Figure 24A is a graph showing the time dependence of DNA traasfection using
arginine dodecyl ester.
7
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WO 99/x7543 PCT/US99/06198
Figure 34B is a graph of lucifetase intensin~ versus concentration showing the
effect
of arginine dodecyl ester concentration on DhA transfection.
Figure 25A is a graph of OD,~ versus concentration showing lack of
cytotoxiciry of
CC-cholesterol in COS-7 cells.
Figure 25B is a graph of OD,~ versus concentration showing lack of
cytotoxiciry of
CC-cholesterol in JEG-3 cells.
Figure ?6 shows the steroid backbone.
Figure 27 illustrates a scheme for the synthesis of a ketai starting with 4-
cholesten-3-
one. X represents a cationic moiety.
DETAILED DESCRIPTION OF THE
In one aspect, the present invention permits a pharmaceutical substance to be
solubilized in an organic solvent by associating the pharmaceutical substance
with an
amphiphilic material. The pharmaceutical substance is substantially not
directly soluble in
the organic solvent, but becomes soluble in association with the amphiphilic
material. It
should be appreciated that by substantially not soluble it is not meant that
the phatmaoe~c~
substance is utterly insoluble in an organic solvent. Rather, it is meant that
the direct
solubility of the pharmaceutical substance in the organic solvent is iimited
and that it would
.0 be desir~le to dissolve an amount of the pharnaceutical substance over and
above that
amount which is directly soluble. That desired additions! amount is not
soluble in the
organic solvent. This is often the case for a pharmaceutical substance which
is only slightly
soluble in an organic solvent, when it may be desirable to dissolve more of
the
pharmaceutical substance into the organic solvent than is possible by direct
dissolution.
According to the present invention, when the pharmaceutical substance is
combined with the
amphiphilic material, the solubility of the pharmaceutical substance in the
organic solvent
n'taY ~ ~oreased by an order of magnitude or more, and is often increased by
more than two
orders of magnitude relative to direct dissolution of the pharmaceutical
substance into the
organic solvent, in the absence of the amphiphilic material.
With the present invention, the pharmaceutical substance and the amphiphilic
material are in a true, homogeneous solution in the organic solvent. By a
true, homogeneous
solution, it is meant that the pharmaceutical substance, the amphiphilic
material and the
organic solvent form a single liquid phase. The present invention is,
therefore,
distinguishable from the preparation of emulsions, micellar systems and other
colloidal
8
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suspensions which comprise at least two distinct phases, with one phase being
dispersed
within the other phase.
To assist in the understanding of the present invention, but not to be bound
by theory,
it is believed that the phatznaceutical substance and the amphiphilic material
are associated
in the form of a complex between the amphiphiIic material and the
pharmaceutical substance,
with the complex being substantially not soluble in aqueous liquids at a
physiological pH.
Preferably, the amphiphilic material and the pharmaceutical substance have
oppositely
charged ionic portions which associate to form an ion pair complex. Such an
ion pair
complex is referred to as a hydrophobic ion pair (HIP) complex. Thus, the
ph~~etstical
substance may comprise a cationic portion which associates with an anionic
portion of the
amphiphilic material or an anionic portion which associates with a cationic
portion of the
amphiphilic material.
The pham~aceuticat substance may be any substance which may be administered to
a human or animal host for medical purpose, which is normally a curative,
therap~ic,
preventive, or diagnostic purpose. The pharmaceutical substance is preferably
directly
soluble to some meaningful degree in an aqueous liquid at a physiological pH.
As used
herein, a physiological pH is a pH of from about 1 to about 8. Preferably, the
pharmaceutical
substance exhibits a charged character when dissolved in an aqueous liquid at
a physiological
pH. As used herein, a pharmaceutical substance includes variotu salt forms of
a substance
as well as ionic forms and dissociation products, such as may be found in an
aqueous
solution.
The pharmaceutical substance may comprise a protein or other polypeptide. a
nucleic
acid. an analgesic or another material. The following is a non-limiting list
of representative
types of pharmaceutical substances which may be used with the present
invention. with a few
specific examples listed for each type of pharmaceutical substance:
cholinergic agonists
(pilocarpine, metoclapram:de); anticholinesterase ageniJ (neostigmine,
physostigmiae);
antimuscarinic drugs (atropine, scopalamine); antiadrenergics (tolawline,
phentolamine,
proptattolol, atenolol); ganglionic stimulating agents (nicotine,
trimethaphan); neuromuscular
blocking agents (gallamine, succinylcholine); local anesthetics (procaine,
Gdocaine, cocaine);
benzodiazepines (triazolam); antipsychotics (chlorpromazine, triflupromazine);
antidepressants (fluoxetine, imipramine, amitriptyline, pheneIzine);
antiparkinson's drugs
(L-dopa, dopamine); opioids and ana-opoids (morphine, naioxone, naltrexone,
methadone);
CNS stimulants (theophylline, strychnine): autocoids and anti-autocoids
(histamine, betazole,
chlorpheniramine, cimetidine); anti-inflammatories (tolmetin, piroxicam); anti-
hypettensives
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WO 99/47543 PCTNS99/06198
(clonidine, hydralazine, minoxidil); diuretics (metalozone, bumetattude);
polypeptides
(lysopressin, vasopressin. ox:.~ocin, insulin. calcitanin. gene-related
peptide, LhiRH agonies,
ACT'1-i, growth hormone): antifungals (clotrimazole. miconazole);
antimalaria(s (chloroquine,
primaquine); antiprotomals (pentamidine, melarsoprol); anuhelminthics
(piperazine,
oxacnniquine); antimicrobiaIs (streptomycin, erythromycin, cefaclor,
ceftriaxone,
oxytetracycline, rifampicin, isoniazid, dapsone): aminoglycosides (gentamycin,
neomycin,
streptomycin); antineoplastics (mechlorethamine, melphalan, do~corubicin,
cisplatin);
anticoagulants (heparin); nucleic acids (genes, antisense RNAs, ribozymes,
plastnids).
Additionally, the pharmaceutical substance may be a sympathomimetic drug such
as
catecholamines (epinephrine, norepinephrine); noncatecholatnines (amphetamine,
phenylephrine); and p,-adretergics (terbutaline,- albuteml).
Particularly useful with the present invention are macromolecules such as
polymers,
nucleic acids, proteins or polvpeptides. One advantage of the present
invention is that the
pharmaceutical substance. when in solution with the amphiphilic material in
the organic
solvent, retains a substantially native conformation. This is particularly
important for
materials, such as proteins and ribozymes, which are highly susceptible to
loss of activity due
to loss of native conformational structure.
The amphiphilic material may be any material with a hydrophobic portion and a
hydrophilic portion. These materials are typically surfactants. The
hydrophilic portion is
ionic under the conditions of use. The hydrophobic portion may be any
hydrophobic group,
such as an alkyl, aryl or alkylaryl group. The amphiphilic material associates
with the
pharmaceutical substance to form a hydrophobic ion pair which is soluble in
the organic
solvent when the pharmaceutical substance itself is substantially not soluble
in the organic
solvent. As used herein, amphiphilic material includes different salt forms of
a material as
well as ionic forms and dissociation products of a material, such as may be
present is a
solution. Preferred a.~.:~;.:pILI:;, n.,r.;"~;s ~ dose posing lit:lz or
subst.~.tttially no
toxicological problem for a human or animal host.
F"xamples of anionic amphiphilic materials include sulfates, sulfonates,
phosphates
(including phospholipids), carboxylates, and sulfosuccinates. Some specific
anionic
amphiphilic materials useful with the present invention include: sodium
dodecyl sulfate
(SDS), bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), cholesterol sulfate and
sodium
laurate. Particularly preferred anionic amphiphilic materials are SDS and AOT.
SUBSTITUTE SHEET (RULE 2B)
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Preferred cationic amphiphilic materials are the cationic surfactants of the
invention
(see below). Specific cationic amphiphilic materials include the arginine and
cholesterol
esters, carbamates, carbonates and ketals (sec below).
The solution of the pharmaceutical substance and the amphiphilic mat~ri~ in ~e
organic solvent may be prepared in anv suitable manner. In one embodiment of
the present
invention, small amounts of the amphiphilic material may be added to an
aqueous solution,
in which the pharmaceutical substance is initially dissolved, until a
precipitate forms of an
HIP complex of the pharmaceutical substance and the amphiphilic material. The
precipitate
may then be recovered and dissolved in an organic solvent to provide the
desired solution.
I0 For some situations, it may be possible to dissolve the pharmaceutical
substance in an
aqueous liquid and to dissolve the amphiphilic material in an organic solvent.
The aqueous
liquid and the organic solvent may then be contacted to effect a partitioning
of the
pharmaceutical substance into the organic solvent to form an HIP complex with
the
amphiphilic material. In other situations, it may be possible to dissolve both
the
IS pharmaceutical substance and the amphiphilic material is an aqueous liquid.
The aqueous
liquid may then be contacted with an organic solvent to partition into the
organic solvent at
least some of the pharmaceutical substance and the amphiphilic material in the
form of an
HIP complex.
The organic solvent may be any organic liquid in which the pharmaceutical
substance
20 and the amphiphilic material, together, are soluble, such as in the form of
an HIP complex.
The following is a non-limiting, representative list of some organic solvents,
with specific
exemplary solvents listed in parentheses. which may be used with the present
invention:
monohydric alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-
hexanol, I
octanol, trifluoroethanol); polyhydric alcohols (propylene glycol, PEG 400,1,3-
propanediol);
25 ethers (tetrahydrofuraa ~, diethyl ether, diglyme); allcanes (decalin,
isooctane, mineral
oil); aromatics (benzene, toluene, chlorobenzene, pyridine); amides (n-methyl
pvrrolidone
(NMP), N,N-dimethylfonnamide (DI~ff)); esters (ethyl acetate, methyl acetate);
chlorocarbons (CHiCIr CHCI,, CCI" I~-dichloroethane); and others such as
nitromethane,
acetone, ethylene diamine, acetonitrile, and trimethyI phosphate.
30 In one embodiment, the present invention involves the use of amphiphilic
materials
as ion pairing agents to modulate the solubility and partitioning behavior of
pharmaceutical
substances such as polypeptides, proteins, nucleic acids, and drugs. Complexes
are formed
by stoichiometric interaction of an amphiphilic material, such as a detergent
or other
surfactant (e.g., alkyl sulfate, such as sodium dodecyl sulfate (SDS), or
arginine ester), with
11
SUBSTITUTE SHEET (RULE 2B)
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WO 99/47543 PCT/US99/06198
the ionic functional groups of a polypeptide, protein, nucleic acid. or
organic molecule that
are accessible for ion pairing. The basic group may be an amine (as found in
the lysine amino
acid residue or the V-terminal amino group of a polypeptide) or a
guanidinitutt group (as in
argittine). The acidic group may be a carboxyl group or phosphate group. A,n
ion pair is
subsequently formed. referred to as a hydrophobic ion pair (HIP) complex. The
HIP complex
fotmec :viii aave reduced aqueous solubility, but enhanced solubility in
organic solvents.
It has been discovered that an HIP complex may be dissolved in as orgaaic
solvent
to form a true homogeneous solution. Included in the invention is the
discovery that the
native tertiary structure of proteins is retained even when dissolved in
organic solvents such
as 1-octanol. The method of the invention for forming a true homogeneous
solution is
fundamentally different from any other method for placing proteins into
organic solvents,
such as those which use suspensions, micelles, microemulsions, or chemical
modifications
of the protein. This discovery holds important implications in the area of
drug delivery and
release, including delivery to the body by inhalation and dispersion in a
hydrophobic
I S biodegradable matrix. While the decreased aqueous solubility of the HIP
complex has been
observed previously, the use of an HIP complex precipitate for improved drug
delivery is
novel. Measurement of the apparent partition coefficient, defined as the ratio
of the
equilibrium concentration in an organic phase to that in an aqueous phase,
demonstrates that
the solubility of a peptide or protein is an HIP complex in the organic phase
is greater by 2-4
orders of magnitude relative to the chloride salt of the peptide or protein.
Included in the invention is the discovery that the precipitation of the HIP
complex
out of aqueous solution may be controlled for the production of uniform HIP
complex
particles of a desired size. These particles may then be formed into a
suspension. T"nis
invention also includes a method of obtaining HIP complex particles of
specific sizes by'
controlling the conditions of HIP complex precipitation.
The discovery that HIP complex precipitation can be controlled so as to yield
particles
of specific size can be exploited to effect the rate of drug released from
suspensions. In one
embodiment of a method of the invention, the size of h'IP complexes is
controlled by
controlling the rates of the mixing of a protein solution and the addition of
an anionic or
cationic detergent to the protein soiudon. The HIP complex can produce very
fine
suspensions which have limited solubility in water, and the technology can be
used to
produce panicles of varying specific size. The particle size of the HIP
complex which is
formed in water will depend on the degree of agitation of the protein solution
and the rate of
counterion addition. The smallest particles rre produced with high shear being
applied to the
12
SUBSTITUTE SHEET (RULE 26j
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WO 99/47543 PGT/US99/06198
aqueous protein solution and slow addition of detergent. This approach is also
important in
Pulmonary drug delivery, where the particle size is critical to delivery to
certain sites within
the lone. To obtain particles which will be capable of depositing in the
pulmonary region
upon inhalation, a high speed homogenizer can be used to stir the protein
solution and a
surfactant is added dropwise to the agitated solution. Particles in the 2-10
micron range can
be obtained using this procedure. Particles of this size are required to get a
sufficient amount
of protein delivered to the lung to have a beneficial effect. The particles
once formed can be
separated by centrifugation or filtration. Larger particles will be formed
with slow agitation
speeds and more rapid addition of surfactant. One example of a drug which
could benefit
from formation into a fine suspension of HIP complexes is DNase, an rnzyme
currently being
used by cystic fibrosis patients to dissolve viscous fluid build-up in the
lung. Other examples
include protein and peptide enzyme inhibitors currently being tested for the
treatment of
~phY~a. Further examples include antituberculosis drugs (e.g., streptomycin,
isoniazid,
pyrazinamide, ethambutol). Another example is transgenes used to transfect
lung cells for
gene therapy.
The inventicn includes a me:hcd of cont:alIin2 :i:e ~e!e~se of a protein from
a
suspension by contrellir._ ~'~~ size of the I:IIP cot:.r:~:; r,:-.:c!=. T__"
;~l~~e rate of pmtein
into at aqueous seh.~t:c:.:ror.: az FrTP cor.:;.lex will be much slowe~ than
that e: the protein
itself. This rate will be a function of the particle size of the complex and
the solubility of the
complex in water or biological fluid. The solubility is a function of the
amphiphilic material
used and the strength of its association with the protein. Therefore, extended
(controlled)
release of the protein from the suspension can be achieved. This property
permits proteins
to be formulated as a suspension for depot injection.
This invention also includes the discovery that uncomplexed protein released
from
the HIP complex can be extracted back into aqueous medium with retention of iu
native
structure. The native uncomplexed protein can be reclaimed by dissolution in
an aqueous
solution which contains an excess of chloride or other counterion, indicating
that the
complexation is an entirely reversible process. It has been discovered that
the protein of the
HIP complex subsequently extracted back into an aqueous medium retains its
native
structure. This makes HIP methodology useful in the delivery of proteins for
use as
therapeutic agents.
An important and unique aspect of the present invention is the discovery that
HIP
complexes display greatly enhanced thermal stability relative to the native
protein, both with
respect to chemical degradation and denacuration. This suggests that the HIP
complex is
13
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCTNS99/06t98
useful fcr long term storage of the protein. Further, this aspect of the
invention permits high
ter.:pe:~atur (steam) sterilization of proteins without the loss of biological
activity, which
until now, could not be accomplished. Currently, polymer delivery systems for
proteins are
usually sterilized by radiation as proteins are destroyed by heat. The present
invention
5 discloses a method by which proteins may be processed by heating at
sterilizing temperatures.
Further, the enhanced thermal stability of the present invention may be
important for the
formulation of proteins in maintaining alt active enzyme in an organic solvent
and for long
term storage of sensitive proteins.
Included in this invention is a method of uniformly distributing a drug
throughout a
10 hydrophobic polymer comprisine adding a sufficient amount of a detergent to
an organic
molecule to form a precipitate. isolating the precipitate, and co-dissolving
the precipitate and
a hydrophobic polymer in an organic solvent to form a homogeneous distribution
of the
organic molecule within the polymer.
Many of the current systems for the controlled release of proteins make use of
I 5 biodegradable polymers. There are at least two major problems with such
systems. Under the
prior art, a protein can only be suspended during the incorporation process,
and because of
its polar surface does not suspend well. The term "suspension" refers to the
dispersion of a
substance or substances in another where the boundaries between them are well
defined. A
material is dispersed is a solvent where the material has limited solubility
in that solvent.
20 This leads to an uneven distribution of the drug and irnproducible drug
release profiles.
Secondly, the wattr-soluble drug is leached out of the polymer by biological
fluids (rather
than its controlled release as the polymer is slowly degraded).
The invention provides a new method for distributing a drug uniformly through
a
hydrophobic polymer. HIP complex formation permits both proteins and
hydrophobic
25 polymers to possess similar solubility parameters, thus facilitating
incorporation of the
protein into the polymer matrix. The inventors have discovered that HIP
complexes may be
uniformly distributed in biodegradable polymers as they possess a solubility
in solvents that
will also dissolve the polymer. Where the HIP complex does not dissolve in the
solvent used
it will suspend easily as a result of its hydrophobic surface.
30 The invention wherein the drugs being delivered are included in the polymer
matrix
in an HIP complex represents three advantages over the biodegradable polymer
systems: ( 1 )
the hydrophobic polymers can be better mixed with the drug in its lipophiIic
ion-pair state;
(2) the drug fortes hydrophobic particles within the polymer, and avoids the
problem of the
formation of a concentration of polar particles at the interface of the
polymer leading to the
14
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCT/US99/06198
"burst" effect; (;) the hydrophobic particles dispersed within the
biodegradable polymer are
not leached out by biological fluids which result in a predictable release
rate. The inventor
have discovered the use of the HIP complex to control (retard or extend) the
release of a drug
at a predictable rate, resulting in part from a more uniform formulation.
One embodiment of this invention includes a method for achieving a true
homogeneous solution of biologically active proteins and polypeptides in a
organic solvent.
None of the methods by which enzymatic activity is achieved in a nonaqueous
environment
employs a true protein solution. The inventors have discovered that the HIP
complex can be
redissolved in an organic solvent such that a true homogeneous solution is
formed. This
discovery has important ramifications for controlling the ertzytnatic activity
of prot~~ ~ ~e
body. Through the formation of HIP complexes, enzymes and other proteins can
be
solubilized in a variety of organic solvents, including ethanol, propylene
glycol and glycols
in general, N-methyl pyrrolidone (NMp) and others. These materials should have
altered
errzytaatic activity and specificity. It is important to note that use of HIP
complexes to form
true solutions of biologically active proteins and polypeptides is a
fundamentally different
approach from any previously described for achieving enrymatic activity in non-
aqueous
media.
Also included in this invention is the discovery that the HIP complex
dissolved in
organic solvent can be extracted back into aqueous medium with retention of
the native
protein stivcnrre. This discovery has potential use in the purification of
proteins. A protein
having a pH different from others in a mixture may be extracted or
preferentially precipitated
from the mixture by HIP comple:c formation.
The invention further includes a method of obtaining a stabilized protein
comprising
precipitating a protein in the HIP complex. Much research effort has been
directed into
developing stabilized lyophilized formulations of proteins, including by the
addition of
cryoprotectants. The HIP complex may, in many cases,
provide a simple alternative to obtaining a stabilized protein. A protein in
the solid HIP
complex has enhanced stability and resistance to degradation through storage,
shipping, and
handling. Chemical stability is conferred because the amount of water present
is relatively
low, as in lyophilized powders. To reconstitute the protein, the HIP complex
is suspended
in a diluent containing a significant chloride concentration (e.g., phosphate
buffered saline
(PHS) or normal saline). Most HIP complexes r~edissolve rapidly and
completely, leaving a
solution whose only additive is a small amount of surfactant. T'he protein can
also be stored
as a stable entity by dissolving or suspending the HIP complex in an organic
solvent or
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCT/US99/06198
solvent mixture. To form an aqueous solution of the protein, the solution or
suspension can
be shaken with water containing chloride. In cases where the organic solvent
is immiscible
with water, the protein will partition into the water.
An additional embo.::ment of this :.-tvention is ~ :.~.-_':ed of :ncotpors;izg
prod ~d
other drugs into lipid vesicles, liposomes. or dete:ge :: ~i:-I:es. Shaking of
an oil-v~'ater
mixture with an HIP complex of a protein leads to emulsification, indicating
that a HIP
complex can more easily be introduced into emulsion delivery systems than the
dmg done.
Systems for such use can be designed using either the insoluble material in
suspension
formulations or in oil formulation. such as oil in water emulsions. Other
e:camples include
nasal and pulmonary aerosols, ophthalmic suspensions, transdermai patches,
lozenges,
chewing gum, buccal and sublingual systems. and suppositories.
Another aspect of this invention is the reduction of the bitter taste of drugs
incorporated into HIP complexes. since only compounds in solution are tasted.
Therefore,
this invention includes a method for improving the taste of orally
administered drugs by
1 ~ formation of insoluble HIP complexes with such drugs. The taste of a
substance is detected
by receptors in the tongue. A maior zrnrnach to modiy~'.r.g. . ~he tas;e of a
dn:g is ;o alter i~
solubility in saliva If the solubility is sufficiently low the taste will not
be noted. The low
solubility of the HIP complex in biological fluids, including saliva, can be
used to mask the
flavor of a drug, optionally, the HIP complexes may be incorporated into a
polymer to further
mask the taste of the drug. Another way to mask taste is to partition the drug
into an oil. such
as olive oil. This can then be given as an oil in water emulsion with
flavoring agents added
to the outer water phase. HIP cortple~c fer_mstion would provide the drug with
the necessary
high oil to water partition coeflacient.
The term "hydrophobic ion-pairing (HIP)" as used in this disclosure refers to
the
interaction between an amphiphilic material and a pharmaceutical substance.
Preferred
amphiphilic materials include detergents which interact with proteins. other
polypeptides and
nucleic acids. HIP complex ~P,;~a.;~..~~~ ~.e ~,.~..»_____ __~.~ . . _
hydrophobic ion-pair. The detergent interacts with an oppositely charged
compound, such
as a polypeptide or nucleic acid. This interaction has been termed HIP because
it appears to
be primarily electrostatic in nature.
As used in the present invention, the term "anionic detergents" encompasses
any
hydrophobic material that is a salt of an acid which can be employed to modify
solubility
Properties in the described way, including sulfates, sulfonates, phosphates,
and carboxylates.
Sulfates are the salts of the stronger acids in this series and, therefore,
the most efficient at
16
SUBSTITUTE SHEET (RULE 28)
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WO 99/47543 PCT/US99/06198
forming ion pairs. Provided that the alkyl chains or aryl rings are of 8-18
carbons in length,
they are potential candidates for HIP methodology.
As used in the presem invention, the term "cationic surfactants" encompasses
any
material having a hydrophobic moiety and, a cationic moiety which can be
employed to
modify solubility properties in the dtsc~:ibed way. Preferred are the
biocompatible cationic
surfactants o: tt:~ invention (see below).
Although the solution having the HIP complex dissolved in the organic solvem
is
itself a valuable product, the solution may also be used in the preparation of
additional
pharmaceutical products. In particular the solution may be used to prepare a
powder of solid
10 particles comprising the pharmaceutical substance and the amphiphilic
material. In a
preferred embodiment, the solution is subjected to. antisolvent precipitation
processing to
prepare a powder of solid particles. Powders may be prepared having particles
of an ultrafine
size and a relatively narrow size distribution. Also, hollow elongated. fiber-
like particles of
a small size may be prepared. These particles have unique properties which may
be desirable
15 for various pharmaceutical applications.
With reference to Fig. 12, one embodiment of as antisolvent precipitation
method of
the present invention is shown. A liquid feed solution 102 is provided having
a
pharmaceutical substance and an amphiphilic material dissolved together in an
organic
solvent, which is used as a carrier liquid for processing of the
pharmaceutical substance. The
20 liqtud feed solution 102 is subjected to antisolvent precipitation 104 in
which the liquid feed
solution 102 is contacted with an antisoivent fluid 106. During the
antisolvent precipitation
104, the antisolvent fluid 106 invades the organic solvent of the liquid feed
solution 102,
resulting in precipitation of solid particles comprising the pharmaceutical
substance and the
amphiphilic material. The resulting mixnue 108, having the precipitated
particles, is
25 subjected to separation 110 in which solid particles 112 are separated from
the exiting fluid
114. A portion 116 of the exiting fluid 114 is recycled to form a part of the
antisolvent fluid
106 and a portion 118 of the exiting fluid 114 is bled from the system to
prevent an
undesirable build-up of the organic solvent in the system. Continuous or batch
processes
other than the process shown in Fig. 12 may also be used according to the
present invention.
The antisolvent fluid is a fluid in which the pharmaceutical substance and the
amphiphilic material, in association, arc substantially not soluble. It should
be understood
that it is possible that the antisolvent fluid may be capable of dissolving
some amount of the
pharmaceutical substance and the amphiphilic material without departing from
the scope of
the present invention. The antisolvent fluid. however, is substantially
incapable of dissolving
17
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PGTNS99/06198
a significant portion of the pharmaceutical substance and the amphiphilic
material from the
liquid feed solution such that at least a signi$cant portion of pharmaceutical
substance and
the amphiphilic material are, in effect, not soluble in the antisolvent fluid.
e~lso, the
antisolvent fluid is at least partially miscible with the orgaaic solvent such
that the antisolvent
fluid is capable of penetrating into the organic solvent sufficiently to cause
the desired
precipitation of the pharmaceutical substance and the amphiphilic material.
Preferably, the antisolvent precipitation 104 is conducted under thermodynamic
conditions which are near critical or supercritical relative to the
antisolvent fluid. Preferably,
the antisolvent precipitation is such that the antisolvent fluid is at a
reduced pressure of
I O greater than about 0.5, with the reduced pressure being the ratio of the
total pressure during
the antisolvent precipitation 104 to the critical pressure of the antisolvent
fluid 106. More
preferably, the contacting occurs at a reduced pressure of from about 0.8 to
about 3.0 relative
to the antisolvent fluid and even more preferably at a reduced pressure of
from about 0.8 to
about 1 ?. Preferably, the antisolvent precipitation 104 is at a reduced
temperature of greater
15 than about 0.75, with the reduced temperature being the ratio of the
temperature (in K) doting
the antisolvent precipitation 104 to the cortical temperature (in IC) of the
antisolvent fluid 106.
More preferably, the contacting cxcurs at a reduced temperature of grater than
about 0.85,
even more preferably greater than about 0.9 and most preferably greater thaw
about 0.95.
Typically, the reduced temperature is smaller than about 1.2.
20 The antisolvent fluid may comprise any suitable fluid for near critical or
supercritical
processing. These fluids include carbon dioxide. ammonia, nitrous oxide,
methane, ethane,
ethylene, propane, butane, pentane, benzene, methanol, ethaaol, isopropanol,
isobutanol,
fluorocarbons (including chlorotrifluoromethane. monof?uoromethane,
hexafluoraethane and
1,1-difluoroethylene), toluene, pyridine, cyclohexane, tn-cresol, decalin,
cyclohexanol, o-
25 xylene, tetraIin, anilin, acetylene, chlorotrifluorosiIane, xenon, sulfur
hexafluoride, propane,
and others. Carbon dioxide, ethane, propane, butane and ammonia are preferred
antisolvent
fluids.
For many pharmaceutical substances, it is desirable to use an antisolvent
fluid which
permits processing at relatively mild temperatures. This is particularly
important for
30 processing proteins and other polypeptides which are susceptible to a loss
of biological
activity when subjected either to very low temperatures or to very high
temperatures. For
applications involving proteins and other large polvpeptides, the antisolvent
fluid should
preferably have a critical temperature of from about 0°C to about
50°C. Included in this
category of antisolvent fluids are carbon dioxide, nitrous oxide, ethane,
ethylene,
18
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCT/US99/06198
chlorotrifluoromethane. monofluoromethane, acetylene. 1,1-difluoroethylene,
hexsfluoroethane. chlorotrifluorosilane, and xenon. A particularly preferred
antisohent fluid
is carbon dioxide because it is readily available, non-toxic, and has a
critical temperature of
31 °C and a critical pressure of T_'.9 atm, which permits processing
under relatively mild
conditions.
The contacting of the liquid feed solution 102 with the antisolvent fluid 106
during
the antisolvent precipitation 104 may be accomplished using any suitable
contacting
technique and contacting apparatus. Preferably, the liquid feed solution 102
is sprayed as
small droplets into the antisolvent fluid 106. A sonicated spray nozzle, which
is vibrated
ultrasonically, has been found to work well because it is capable of producing
very small
droplets of a relatively uniform size and is, therefore, conducive to
preparation of ultrafine
Powders having particles of a narrow size distribution. The contacting may be
performed in
a batch operation or continuously. Also. continuous operation could involve
contacting by
concurrent flow or countercuaent flow.
The separation 110 may be accomplished using any suitable separation technique
and
aFP. Fur example, the separation may involve simple density separation,
filtration or
use of a centrifuge.
The antisolvent precipitation process of the present invention may be used to
produce
ultrafine particles of a narrow size distribution and which are often of
spheroidal shape.
Thesecar.:.~.-._,:~....__...:.~,:~_. _'..,_;..,..::'.t,'--~-
...:_.;,rs c: xay ye 1 a°.ic:cn or staallc-.
The size of the particles produced will depend upon the particular
pharmaceutical substance
and the processing conditions used.
In general, particle size becomes larger as the viscosity and surface tension
of the
organic solvent increases. For example, the use of ethanol as an organic
solvent would
generally produce smaller particles than the use of isopropanol as an organic
solvent. Also,
particles generally tend to become larger in the vicinity of the critical
temperature as the
process temperature approaches the critical temperature from above. If the
process
temperature is too high, however, then particle sizes generally tend to become
larger again.
For example, using carbon dioxide, the smallest particles seem to be produced
around a
temperature of about 35 °C, with larger particles generally being
produced at substantially
higher and Power temperatures. When using carbon dioxide, the pressure is
preferably within
the range of from about 70 bars to about 90 bars.
It has been found that the method of the present invention may be used to
produce
particles of a narrow size distribution. Preferably, panicles produced in the
gas antisolvent
19
SUBSTITUTE ShlEET (RULE 26~
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WO 99/47543 PCTNS99/06198
precipitation method of the present invention are such that greater than about
90 weight
percent of the particles are within about 50 percent larger or smaller than a
weight average
particle size.
In addition to varying the size of the particles, it is also possible to vary
the shape of
the particles produced. For example, it is possible to produce spheroidal
shaped pa~~l~s
which have good flowabiliry properties. Also, it has been found that hollow
fiber-like
particles may be made according to the present invention, the length of which
may vary
depending upon processing conditions. These fiber-like particles have a
tubular quality in
that they comprise an elongated body, of a substantially rounded cross-
section, which has a
IO hollow interior. which typically is open at least one end of the elongated
body, and is
preferably open at both ends of the elongated body.
It has been found that these fiber-like particles tend to form when the
pharmaceutical
substance is subjected to gas antisolvent precipitation at a very high
concentration in the
organic solvent, such that the molecules of the pharmaceutical substance tend
to be entangled
15 when dissolved in the organic solvent. Macromolecules are particularly
susceptible to such
entanglement in solution and are, therefore, preferred for making these fiber-
like particles.
Such macromolecules include polymers and polypeptides, including proteins. The
concentrations required for any particular pharmaceutical substance will
depend upon the
specific pharmaceutical substance being processed, but concentrations of 3 to
10 weight
20 percent or higher, relative to the organic solvent, may be required for
many polypeptide
macromolecules.
The fiber-like particles typically have a diameter of smaller than about l00
microns,
preferably smaller than about SO microns. In some cases. the diameter may be
as small as 10
microns or less. Length may vary from about 0.3 mm or less to as long as I cm
or more, and
25 is preferably longer than about 0.5 mm and more preferably longer than
about 1 mm.
Generally, a tower flow rate of the liquid feed solution during gas
antisolvent precipitation
tends to produce longer fiber-like particles and a higher flow rate tends to
produce shorter
fiber-like particles.
These hollow, fiber-like particles offer a number of advantages for use in the
30 pharmaceutical industry. one advantage is that these fiber-Iike particles
have a shape that will
not, upon ingestion, pass as easily as a spheroidal particle through the
stomach. The fiber-
like particles should, therefore, tend to have a longer retention time in the
stomach region and
would, accordingly, be available in a stomach region for a longer period of
time for the
desired pharmaceutical treatment. Another advantage of the fiber-like
particles is that,
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99147543 PCTIUS99/06198
because they are hollow, it is possible to place smaller particles of another
phacsnaceuticai
substance inside the hollow interiors. For example. small particles of
morphine or
pentatnidine could be loaded into the hollow inte.~'.ors of a protein-based
fiber-Like particle.
In addition to the pharmaceutical substance and the amphiphilic material, a
biodegradable polymer may also be incorporated into the solid particles of the
present
invention, as noted previously, for conaolled release of the pharmaceutical
substance. A
biodegradable polymer may be incorporated in the antisolvent precipitation
method of the
present invention by co-dissolving the biodegradable polymer in the organic
solvent along
with the pharmaceutical sucstance and the amphiphilic material. The particles
produced
during antisolvent precipitation w911 then contain the biodegradable polymer
as well as the
amphiphilic material"and the pharmaceutical substance. The biodegradable
polymer may be
used in any convenient amount relative to the pharmaceutical substance. The
weight ratio
of the biodegradable polymer to the phatrnaceutical substance could vary from
about 0.1 to
I to about 100,000 to I depending upon the application. Most controlled
release
IS applications, however, will involve a ratio of from about 10 to 1 to about
100 to 1.
Incorporation of L'te binder-adcble Foly:re: into the solid particles may be
used to
delay release of the pharmaceutical substance and to permit sustained release
of the
pharmaceutical substance over some extended period of time. It has been found
that the
release profile from a particle of the present invention in an aqueous buffer
solution for the
pharmaceutical substance is relatively constant and that a sudden initial
release, or "burst
effect." is avoided. T'nis indicates that the pharmaceutical substance is not
concentrating near
the surface of the particle and that the particle comprises an intimate and
homogeneous
mixture of the phatinaceutical substance. the amphiphilic material and the
biodegradable
polymer.
Any biodegradable polymer may be used which may be co-dissolved in the organic
solvent along with the Ff.,~.t~nace~ c :I sabstmca and _':c amphiphilic
material. Examples of
such biodegradable polymers include those having at least some repeating units
representative of polymeriattg at least one of the following: an
alphahydroxycarboxylic acid,
a cyclic diester of an alphahydroxvcarboxvlic acid, a dioxanone, a lactone, a
cyclic carbonate,
a cyclic oxalate, an epoxide, a glycol, and anhydrides. Preferred is a
biodegradable polymer
comprising at least some repeating units representative of polymerizing at
least one of lactic
acid, glycolic acid, lactide, glycolide, ethylene oxide and ethylene glycol.
The biodegradable
polymers may be a homopolymer or a copolymer of two or more different
monomers.
21
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCTNS99/06198
Preferred homopolvmers include poly(lactic acid), polylactide, poly(glycolic
acid),
p<::yv~:ycoliae ar.d po;~~(t;;ly:e:.a glycol).
~ ~;rt,e..,..~~,.. _ e..t ~~..~
= -~: -rte.. . . n~ie~ :r_~w~~,~~~ ..~" .,~_,,~,d .,._-.: a ; of the pr-..
c.e rM-
invention in pilarn:ac~sticaI delivery applications. To deliver a
phar:naceuacal substance,
solid particles having the pharmaceutical substance and the amphiphilic
material according
to the present invention are introduced into a human or animal host.
In one embodiment, the solid particles are inhaled for pulmonary delivery. For
pulmonary delivery, it is prefernd that greater than about 90 weight percent
of all of the solid
particles in an administered pharmaceutical formulation are of a size smaller
than about 10
microns and more preferably at least about 90 weight percent of said particles
are smaller
thaw about 6 microns. and even more preferably at least about 90 weight
percent of all of said
solid particles are from about 1 micron to about 6 microns. Particularly
pre°e:red for
pulmonary delivery applications are particles of from about 2 microns to about
~ microns in
size. These particles may also comprise a biodegradable polymer for delayed
and/or
sustained release of the pharmaceutical substance. The ultrafme size and
narrow size
distribution of the solid particles of the present invention permit a much
higher utilization of
the pharmaceutical substance for pulmonary delivery than the low utilization
experienced
with present methods for pulmonary delivery of pharmaceutical substances.
Whereas current
aerosol and nebuIiTation techniques may use only 10 percent of a
pharmaceutical substance
which is administered, with the particles of the present invention, 80 percent
or more of a
pharmaceutical substance which is administered may be utilized.
The solid particles of the present invention may also be placed in a
suspension and
the suspension injected into the host. For intramuscular or subcutaneous
injection, the
particles will often comprise a biodegradable polyme: for sustained release of
tht
pharataceutical substance. For intramuscular or subcutaneous injection, the
particles should
be less than about 100 microns in size, most preferably less than about 50
microns in size,
although smaller or larger particles may be used in some applications.
For intravenous injection, substantially all particles should be of a size
smaller than
about 1 micron so that the particles will not be susceptible to creating a
blockage within the
circulatory system. T-te particles may comprise a biodegradable polymer, if
desired.
For any treatment requiring injection of a suspension over a prolonged period,
such
as for a micropump which continuously injects a suspension at a slow rate,
greater than about
90 weight percent of the particles are preferably smaller than about 1 micron
to reduce
22
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCTNS99/06198
problems associated with settling of the solid particles. More preferably,
substantially all
panicles are sma'.ler _hart about 1 ri:cron.
The fiber-like particles should ix useful in a number of pharmaceutical
applications
to deliver a pharmaceutical substance to a location where it is needed. For
example, due to
their hollow, fibrous shape, these particles should tend to absorb water due
to capillary
action. The fiber-like particles, may, therefore accelerate biodegradation of
a biodegradable
polymer relative to a particle which is not hollow. Also, the fiber-like
particles could be
woven or spun. alone or with other f brous materials, to incorporate a
pharmaceutical
substance into a medical product using the woven or spun materials. For
example, the fiber
like particles could be made to include a growth factor. Some of the fiber-
like particles then
may be used in making wound coverings, from which the growth factor could be
delivered
to the wound. In addition, the fiber-like particles could be used as a support
for the growth
of cells. Also, the fiber-like particles could be incorporates into
2ra.°~s. suc : as arte:;al Qra;,s,
by spinning with other fibers such as DacmnTM or another material. The fiber-
like particles
could include a pharmaceutical substance to rnhance healing in the vicinity of
the graft or the
acceptance of the graft. Moreover, the fiber-like particles could be used in
the manufacture
of patches for delivery of a pharmaceutical substance, including patches for
sublingual or
buccal delivery of a pharmaceutical substance.
Particles of the present invention, having the ion-paired pharmaceutical
substance,
may also be used to enhance properties of immune sys;e:r. boostea to elicit an
imrau:.e
system response. Rather than injecting a solution of an antigenic protein or
other peptide
with an adjuvant, such as aluminum hydroxide, to cause precipitation after
injection, a
suspension of the ion-paired particles of the present invention could be used.
In another
embodiment, the particles of the present invention could be used in cements,
to deliver a
2j growth factor to help heal broken bones or teeth.
The invention further provides novel cationic stu~factants having the formula:
P-L-C
wherein:
P is a biocompatible hydrophobic moiety;
'0 C is a biocompatible cationic moiety; and
L is a biodegradable linkage linking P and C,
"Biocompatible" is used herein to mean that the hydrophobic or cationic moiety
is
naturally-occurring in, or is well-tolerated by, cells (including prokaryotic
and eukaryotic
cells) or an organism (including animals (e.g., humans) and plants). .4
"biodegradable
23
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCTNS99/06I98
linkage" is one which is degraded by normal conditions or processes found in a
cell or
organism. Thus, the biodegradable linkage of a cationic surfactant of the
invention is
degraded into two biocompatible components in a cell or organism to which the
cationic
surfactant is delivered. As a result, the cationic surfactants of the
invention are much less
toxic than currently existing cationic surfactants.
P is preferably a saturated or ~~~d. linear, branched or cyclic hydrocarEon
(e.g.,
alkyl, cyclic alkyl, aryl, or combinations thereon containing at least 8
carbon atoms, more
preferably 8-40 carbon atoms, most preferably 10-30 carbon atoms. Presently
preferred is
P which is an alkyl containing 10-20 carbon atoms. Also presently preferred is
P which
comprises the steroid backbone, the steroid backbone preferably being
substituted with C-L-
at C3 and/or containing at least one double bond, P most preferably being the
cholesterol
nucleus. By steroid backbone is meant the fused tetracyclic structure common
to all steroids
(shown Figure 2~. By cholesterol nucleus is meant cholesterol without the
hydroxyl group
at C3 and being substituted at C3 with C-L-.
P may be substituted or unsubstitttted. The substituent may be any moiety that
has at
least some degree of hydrophobicity and is of low toxicity to cells or in
vivo. Suitable
substituents include alkyl, cyclic alkyl, aryl, alkyl esters, alkyl amines,
alkyl ethers, etc.
L is preferably an ester, carbonate, carbamate or ketal linkage.
C must be positively charged at pH 7.4 or less. C preferably comprises a
guanidinium
gmup or one or more primary, secondary, tertiary or quaternary amines. Thus, C
may be an
arginine, lysine, choline. ethanolamine, or ethylene diamine residue. C is
most preferably an
arginine residue.
Particularly preferred cationic surfactants are arginine esters having the
following
formula:
O W
R,-O-C-CH-CHI-CHI-CH=-NH-CH-NHz
R" which may be substituted or unsubstituted, is a saturated or unsaturated,
linear,
branched or cyclic hydrocarbon (e.g., alkyl, cyclic alkyl. aryl, or
combinations thereof)
24
suBSr~n~ sHe~ ~u~ zs~
CA 02324254 2002-12-20
74667-173
containing at least 8 carbon atoms. l~~tore preferably R, contains 8-~l0
carbon amms, most
preferably 10-30 carbon atoms. Presently prefemd is a P which is an alkyl
containing 10-20
carbon atoms or rs the chofesterof nucleus. Suuabie substituents are those
listed above for
P. R, may comprise one or more neutral amino acids.
R, is H, one or more neutral or basic amino acids, including additional
arinines. or
a linear. branched or cyclic hydrocarbon ~e.g.. alkyl, cyclic alkyl, aryl, or
combinations
thereof) containing at least l, preferably 1-l~, most preferably Z-10, carbon
atoms and also.
optionally, containing at least one amine croup within the hydrocarbon,
attached to ra5e
hydrocarbon (including at either end), or both. Yrefetz-ed amine groups are
quaternary amines
and gtrartidinitrm groups.
When intended for repeated use irr vivo, R, and R~' are preferably chosen so
that they
are not immunogenic. Thus, when R, or R, is a peptide. it will preferably
comprise fewer
than 6 amino acids. Methods of making peptides are, at course, well known
(also see below).
Suitable peptides can also be purchased ;,ommercially
1 S R, may also be linked to t1e arginine residue tluough other biodegradable
linkages.
Other preferred linkages include ketal, carbonate and c:arbamate linkages.
the arginine esters of the invention may be synthesized by known methods of
synthesizing argittine esters. See, e.g., Guglielmi et al.. Z- Pftvsivl.
Chem., 351, 1617-1630
(1971) and U.S. Patents Nos. 5,364,884 and 4,308,280
These prior syntheses have been linuted to short-chain
alkyl and benzyl esters (six carbons or less). but the methods can be employed
for synthesis
of the arginine esters of the invention. F or instance. the arginine esters
may be prepared by
the reaction of R=-arginine with an alcohol, R1 OII, in the presence of dry
gaseous hydrogen
chloride or using thionyl chloride (see Figures 20A-E). It has been found
necessary to modify
these syntheses by using sulfuric acid to catalyze the titer formation when
more hydrophobic
R, groups are used- In Figures 20D-F., arFinine is first protected as in
pepad:: synthetic
methods and then deblocked after the formation of the ester. For a description
of peptide
synthetic methods, see Merrifield, J Am Chem. Soc.. 85, 2149 ( 1963);
Merrifield, in Chem.
Polypeprides, pp. 335-361 (Katsoyannis and Panayous eds. 1973); Davis et al.,
piochem.
Inr'l, 10, 394-414 ( 1985); Stewart and Young, Solid Phase Peptide Synthesis (
1969); U.S.
Patent No. 3,941,763; Finn et al., in The Proteins. 3rd ed" vol. 2, pp. 105-
253 (1976); and
Erickson et al., in The Proteins, 3rd ed.. vol. 2, pp. 257-~?7 (1976).
Arginine esters of the
invention can also be synthesized using the conditiotL, described in
Mitsunobu, Synthesis
1981, 1-<8, with R,-arginine first being protected a_s rn peptide synthetic
methods and then
'? 5
CA 02324254 2000-09-15
WO 99/47543 PCTNS99/06198
deblocked after the formation of the ester (see Figures 20F-G). Other possible
methods
include the use of protected arginine derivatives and dicyclohexylcarbodiimide
as the
coupling agent and the use of Lewis acids. such as BFI ethecate.
Also preferred are cationic cholesterol surfactants having the following
formula:
R,-L-CHOL
CHOL is the cholesterol nucleus. L is an ester, carbamate, carbonate or ketat
linkage.
R~ is a linear, branched or cyclic hydrocarbon (e.g., alkyl, cyclic alkyl,
aryl, or combinations
thereof] containing at least 1, preferably 1-15, most preferably 2-10, carbon
atoms and also
containing at least one amine group within the hydrocarbon, attached to the
hydrocarbon
(including at either end), or both. Preferred amine groups are quaternary
amines and
guanidinium groups. Vlost pre:ercd is an arginine residue (-CH(NH:)-CH,-CH,-
CHI-NH-
C(NH~NH,'. R, may be substituted with neutral or other basic groups, including
alkyls,
aryls, amides, ester groups, and ether groups containing no more than 10
carbon atoms.
The synthesis of arginine esters of cholesterol was described above (see
Figtues 20C-
F and the description of these figures). These methods may be used to
synthesize other esters
of cholesterol. Additional methods of synthesizing esters of cholesterol and
methods of
synthesizing carbamates of cholesterol are schematically shown in Figures 21 A-
E. A method
of synthesizing a ketal is illustrated in Figure 27. Choiesterol carbonates
can be synthesized
by reacting cholesterol chloroformate with an amino alcohol (see Example 37).
The cationic surfactanu of the invention can be used for the same purposes as
prior
art cationic surfactants. However, due to their much lower toxicity compared
to the prior art
cationic surfactants, the cationic surfactants of the invention are especially
useful in
pharmaceutical preparations and in other situations where cell survival is
important. In
Particular, they can be used as the amphiphilic material in the methods and
compositions
described above.
In addition, the cationic surfactants of the invention can be used to deliver
negatively
charged compounds, such as acidic proteins and nucleic acids, into cells. This
is
accomplished by simply contacting the cells with a cationic surfactant of the
invention and
a compound desired to be delivered into the cell. The cells may be any type of
eukaryotic or
prokaryotic cell, but is preferably a mammalian cell, including human cells.
The contacting
may take place in vitro or in vivo.
26
SUBSTITUTE SHEET (RUL.E 26)
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WO 99/47543 PCT/US99/06198
The cationic surfactanu are particularly suitable for transforniirtg ceps, '~
mils may
be transformed with any type of nucleic acid. including recombinant DNA
molecules coding
for a desired protein or polypeptide, recombinant DNA molecules coding for a
desired
aatisense RNA or ribozyme, cloning vectors, expression vectors, viral vectors,
plasmids, a
transgene for producing transgenic animals or for gene therapy, antisen~ ~A,
~d
ribozymes. The cells may be any type of cell, but are preferably
microorganisms (e.g..
bacteria and yeast and other fungi) and animal (including human) cells (e.g.,
cell lines,
piuripotent stem cells and fertilized embryos). The contacting may take place
in vitro or in
vivo.
To transform a cell, the cell is contacted with a nucleic acid and a
surfactant accu~g
to the invention. Preferably, the nucleic acid and surfactant are combined and
incubated
together before contacting them with the cell. The time of incubation is that
time sufficient
to allow the nucleic acid and surfactant to complex. This time can be
determined empirically.
A time of about 4~ minutes has been found to be sufficient for incubation of
arginine dodecyl
ester and a plasmid (see Example 39). The cell is contacted with the nucleic
acid and
surfactant for a time suffcient to allow the nucleic acid to be delivered into
at Least some of
the cells. This time can also be determined empirically. A time of about 30
hours has been
found to be sufficient when using the combination of arginine dodecyl ester
and plasmid (see
Example 39). Other conditions for contacting the cell with the nucleic acid
and surfactant
are known in the art or may be determined empirically.
The cationic surfactanu of the invention may be used alone to transform cells.
Preferably, however, they are used in combination with helper lipids for
transforming cells.
The lipids may be any of those lipids known in the art to be useful in
transforming cells,
including dioleoyl phosphatidyl ethanolamine (DOPE) and cholesterol. The lipid
should
preferably promote fusion of the nucleic acid/surfactaat/lipid complex with
the membrane
of the cell so that the nucleic acid may be transported into the interior of
the cell.
To transform a cell, the cell is contacted with a nucleic acid, a surfactant
according
to the invention and a lipid. Preferably, the nucleic acid. surfactant and
lipid are combined
and incubated together before contacting them with the cell. The three may be
combined
simultaneously or sequentially (in any possible order of the three). The time
of incubation
is that time sufficient to allow the nucleic acid, surfactant and lipid to
complex. This time
can be determined empirically. The cell is contacted with the nucleic
acid/surfactant/lipid
for a time sufficient to allow the nucleic acid to be delivered into at least
some of the cells.
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SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCT/US99/06198
This time can also be determined empirically. Other conditions for contacting
the cell with
the nucleic acid, surfactant and lipid are known in the art or may be
determined empirically.
The cationic surfactants of the invention may also be used. with or without
helper
lipids, in combination with other methods of transformation, such as
electroporation. 'This
may be particularly advantageous in transformation of plant cells.
After transformation in virro, the cells may be cultured to produce a desired
protein,
polypeptide or RNA. Alternatively, the cells may be injected into as animal
for gene therapy.
In yet another alternative. the cells may be allowed to grow and differentiate
into a transgenic
animal or plant.
When the cells are to be transformed in vivo, the cationic surfactant or the
lipid are
preferably selected or modified so that they are targeted to selected cells to
be transformed.
For instance, the nucleic acid/surfactant combination could be incorporated
into liposomes
composed of the lipids. The liposomes could be targeted to particular cells by
having an
antibody specific for a molecule on the surface of the cells attached to the
exterior of the
liposomes.
The invention also provides a kit for delivering nucleic acids or other
negatively
charged compounds into cells. I;us ka comprises a container of a cationic
surfactant of the
invention. The kit may further comprise a container containing a nucleic acid,
such as a
cloning vector, expression vector or gene. The kit may further comprise other
reagents and
materials normally used for transforming cells, such as restriction enrymes,
lipids,
polymerase chain reaction reagents, and buffers.
In yet another important aspect of the present invention. it has,
surprisingly, been
found that the antisolvert precipitation method of the present invention may
be operated to
produce compositions having particularly desirable characteristics for
sustained release of a
pharmaceutical substance. The composition is characterized as including a
pharmaceutical
material, in the form of an HIP complex with an amphiphilic material, and a
biocompatible
polymer, with the biocompatible polymer being highly amorphous. The highly
amorphous
character of the polymer is particularly desirable for reducing immune system
responses and,
therefore, reducing the likelihood of causing significant inflammation during
use by a human
or other mammalian patient. Furthermore, the highly amorphous sustained-
release
composition also provides a desirably stable sustained release profle for
release of the
pharmaceutical substance. and typically with little or negligible burst
effect. The highly
amorphous sustained-release composition is particularly well suited for
delivery of a
pharmaceutical substance for sustained release by pulmonary delivery,
subcutaneous
28
SUBSTITUTE SHEET (RULE 26)
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WO 99I4~543 PC'f/US99/06198
placement, intraperitoneal ptacement and inaaocular placement. The antisolvent
process for
making particles of :he i:~=lily amorphous sustained-release composition, the
particles and
composition so made, product forms incorporating the composition, and uses of
the
composition for administration to a tnattunalian patient for sustained-release
purposes are all
within the scope of the present invention.
The sustained-release composition includes the biocompatible polymer in a
highly
amorphous form. By highly amorphous, it is meant that the polymer is typically
no more
than about 25% crystalline. Most often, however, it will be desirable to keep
the crystalline
content of the biocompatible polymer as low as possible. Through careful
control of
operating parameters of the antisolvent precipitation process, the sustained-
release
composition may be prepared typically with the biocompatible polymer being no
more than
about 20% crystalline, preferably no more than about 15% crystalline, more
preferably no
more than about 10% crystalline, and even more preferably no more than about
5%
crystalline. Particularly preferred is for the biocompatible polymer to be
substantially entirely
amorphous. As used herein, the crystalline content of the biocompatible
polymer is as
calculated based or. x-ray defraction results, a technique well known in the
art.
Careful control of the antisolvent precipitation process is important for
making the
highly amorphous sustained-release composition of the present invention with
the desirable
features noted above. In that regard, it is particularly important that the
relative flows of the
antisolvent fluid and the liquid feed, which includes a solvent having
codissolved therein the
biocompatible polymer and the HIP complex, be carefully controlled within
certain ranges.
In that regard, the volumetric ratio of the antisolvent fluid flow rate to the
liquid feed flow
rate should typically be Larger than about ~, and more typically be in a range
of from about
5 to about 100. Preferred amorphous characteristics. however, are more
advantageously
obtained with a volumetric ratio of antisolvent fluid flow rate to liquid feed
flow rate of
larger than about 15, more preferably larger than about 20, and even more
preferably larger
than about 25. A particularly prefewed range for the volumetric ratio of
antisolvent fluid
flow rate to liquid feed flow rate for preparing the highly amorphous
sustained-release
composition is from about 10 to about 50, more particularly from about 15 to
about 40, even
more particularly from about 15 to about 30, and most particularly from about
20 to about
30.
In addition to controlling the relative flows of antisolvent fluid and liquid
feed, the
manner in which the antisolvent fluid and the liquid feed are contacted is
also important.
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Although contacting may be in counterflow, it has been found that concurrent
flow. or co-
I3ow, df the liquid feed and the antisolvent fluid generally produces a
superior product
Furthermore. control of the relative concentrations of the biocompatible
polymer, the
pharmaceutical substance and the amphiphilic material is an important
consideration when
making the highly amorphous sustained release composition. The relative
amounts is the
liquid feed of the HIP complex, comprised of the pharmaceutical substance and
the
amphiphilic material, and the biocompatibie polymer will depend upon the
specific
application. The HIP complex may comprise about 0.5 weight percent or more of
the total
weight of the HIP complex and the biocompatible polymer, although amounts of
grtater than
1 weight percent a:e more common and greater than 5 weight percent are even
more
common. For the highly amorphous sustained-release composition of the present
invention,
however, it is generally desirable to include large relative amount of the HIP
complex. Of
the total weight of the HIP complex and the biocompatible polymer in the
liquid feed, the
HIP complex should typically comprise at feast about 15 weight percent,
preferably at least
about 20 weight percent, more preferably at least about 25 weight percent, and
even more
preferably at least about 30 weight percent. Typically, however, the HIP
complex content
will be no larger than about 70 weight percent, preferably no larger than
about 60 weight
percem and more preferably no larger than about ~0 weight percent.
Because the antisolvent precipitation process is extremely efficient at
incorporating
biocompatible polymer and HIP complex material into the manufactured product,
~e ~g~y
amorphous sustained-release composition will typically include a very high
loading of the
HIP complex material. Typically, the highly amorphous sustained-release
composition will
comprise the pharmaceutical substance and the amphiphilic material, in the
form of a HIP
complex, in an amount of at least about 15 weight percent, preferably at least
about 20 weight
percent, more preferably at least about 25 weight percent and even more
preferably at lest
about 30 weight percent. At extremely high levels of loading with the HIP
complex, the
structural integrity of the composition may be compromised. Therefore, the HIP
content will
typically comprise no greater than about 70 weight percent of the composition,
preferably no
greater than about 60 weight percent of the composition and even more
preferably no greater
than about 50 weight percent of the composition. The biocompatible polymer
typically
makes up the balance of the composition.
The antisolvent precipitation process of manufacture, with carefully
controlled
operation, permits manufacture of the composition with the biocompatible
polymer in a
highly amorphous state and with a heavy loading of the HIP complex material,
Furthermore,
SUBSTITUTE SHEET (RULE 26j
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WO 99/47543 PCTNS99/06198
even though the composition is heavily Ioaded with the HIP complex material.
the HIP
complex material is, nevertheless, typically substantially homogeneously
dispersed
throughout a matrix of the polymer. such that the HIP complex and the
biocompatible
p°13'm~ ~ m an intimate mixture on a microscopic level. This intimate
and homogeneous
mixture is very important to achieving a stable release profile for release of
the
pharmaceutical substance from the sustained-~:lease composition, as is the
high Ievet of
loading of HIP complex material in the composition, as is discussed farther
below.
Also important is that the mass content of the amphiphilic material in the HIP
complex material is typically as large as or larger than the mass content of
the pharmaceutical
substance. In that regard, the mass ratio of the amphiphilic material to the
pharmaceutical
substance in the composition will typically be larger titan about 1, and is
often in a range of
about 1 to about 5 with a range of from about 3 to about 6 being preferred.
Even though the HIP complex includes a large proportion of amphiphilic
material,
the highly amorphous sustained-release composition is still heavily loaded
with the
pharmaceutical substance. The pharmaceutical substance typically comprises
greater than
about 5 weight percent of the composition, preferably greater than about 10
weight percent
of the composition, more preferably greater than about 15 weight percent of
the composition,
and even more preferably greater than about 20 weight percent of the
composition.
The antisolvent fluid for making the lug~y ~orphous sustained-release
composition
will typically be carbon dioxide. It is preferred, however, that contacting of
the antisolvent
fluid and the liquid feed occur at subcritical conditions. Preferably, the
temperature is
suberitical with the reduced temperature. reiztive to the antisolvent fluid,
preferably being in
a range having a lower limit of about 0.75, more preferably about 0.85, event
more preferably
about 0.90, and most preferably about 0.95; and having an upper limit of about
0.95 and more
preferably about 0.99 and even more preferably about 0.995. The reduced
pressure, relative
to the antisolvent fluid, is typically from about 0.5 to about 2. When carbon
dioxide is the
antisolvent fluid, the temperature during the antisolvent precipitation step
is preferably in a
range of froth about 20°C to about 30°C, and more preferably
from about 20°C to about
30°C.
For the highly amorphous sustained-release composition, the pharmaceutical
substance may be any ionic phatirtaceutical material and the amphiphilic
material may be any
compatible surfactant. Any of the pharmaceutical substances or amphiphilic
materials
described previously may be used. Preferred pharmaceutical substances include
antibiotics,
chemotherapeutic agents, and biologic agents. Prcfeaed surfactants are AOT and
SDS, as
31
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCTNS99/06198
well as the cationic materials discussed previously. The solvent may be any
suitable solvent,
but often includes at least one of methylene chloride or trichloromethane.
The biocompatible polymer may be any polymer that may be processed in the
antisolvent precipitation process to form the composition such that the
polymer is in a highly
amorphous state, as discussed previously. Most often, the biocompatible
polymer is a
biodegradable polymer, as discussed previously. When biodegradable, the
polymer is most
often hydrolvtically degradable.
The biocompatible polymer. as it is commercially available, is frequently
available
only in a highly crystalline state. The polymer is, however, converted to a
highly amorphous
state during the antisolvent precipitation process. Preferred polymers for the
biocompatible
Polymer are poly(lactic acid) homopolymers, including poly(1-lactic acid) and
poly(d-lactic
acid), poly(glycolic acid) homopolymer, polyanhydrides, such as poly(sebacic
acid),
poly(carboxyphenoxyhexane), polybutyrates and cellulosic polymers such as
polyhydroxypropyl ethylcellulose. Any suitable molecular weight polymer may be
used that
is soluble in the solvent used for the liquid feed. Typical molecular weights
are from about
2 kDa to about 500 kDa. In some instances, the biocompatible polymer could be
a mixttue
of two or more different polymers or a copolymer of nvo or more different
monomers. Also,
it should be noted that, at least in the case of poly(lactic acid) and
poly(glycolic acid)
polymers, the polymers are typically not prepared from the acids, but from the
cyclic diesters,
lactide or glycolide, as the case may be. It should be recognized that, as
used herein,
poly(lactic acid) and poly(glycolic acid) polymers are inclusive of polymers
prepared directly
by condensation polymerization of the acids or by ring-opening polymerization
of the cyclic
diesters. The polymers made from ring.opening polymerization of lactide and
glycolide are
often referred to as polylactide and polglycolide.
As produced in the antisolvent precipitation process, the highly amorphous
sustained-
release composition will be in particulate form. The particulate product may
include particles
of a variety of sizes aad shapes. In that regard, any of the particulate
products having ~rcicle
characteristics as previously described may be made in the form of the highly
amorphous
sustained-release composition. For example, for pulmonary delivery
applications, the
particulate product preferably includes ultrafine particles of a spheroidal
shape and with
greater than about 90 weight percent of the particles being smaller than about
10 microns,
more preferably smaller than about 6 microns, and even more preferably of a
size of from
about 1 micron to about 6 microns. Furthermore, the highly amorphous sustained-
release
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composition may be made in the form of the fiber-like particles, or in the
form of extremely
fme particles of a siu smaller than about 1 micron.
The highly amorphous. sustained-release composition may be incorporated into a
variety of product forms for use. One product form is a macrostructure formed
by
agglomeration of particles of the particulate product prepared by the
antisolvent precipitation
method. The agglomeration is typically accomplished by compression. For
example,
cylinders, pellets, beads (e.g., spheroidal, ellipsoidal or other shape),
discs and other
macrostructures could be prepared from smaller particles. Preferred uses for
such
macrostructtues arc for subcutaneous and intraperitoneal surgical
implantation. Depending
upon the specific application, the macrostructure will typically have a mass
in a range with
a lower limit of about 0.01 gram, about 0.05 gram, about 0.1 gram, about 0.5
gram or about
1 gram; and an upper limit of about 100 grams, about 50 grams, about 10 grams
or about 1
gram. Any mass range having any one of the stated lower limits and any one of
the stated
upper limits is within the scope of the present invention, so long as the
upper limit is larger
I S than the lower limit. For example, for many applications, the
macrostructure mass will be
in a range of from about 0.05 to about 0.5 gram. To the extent that more of
the
pharmaceutical substance is desired than contained in a single macrostructure,
then multiple
macmstructures may be implanted together. For some applications, however, it
will be
desirable to have a very long sustained-release period, such as over a month
or more. In these
cases, the macrostructure will typically have a mass in a range of from about
1 to about 10
grams, although a larger mass may be desirable at times. Also, depending upon
the specific
application, the macrostructure will typically occupy a volume within a range
having a lower
limit of about 0.01 cubic centimeter. 0.0~ cubic centimeter, 0.1 cubic
centimeter, 0.5 cubic
centimeter or 1 cubic centimeter; and an upper limit of about 100 cubic
centimeters, about
50 cubic centimeters, about 10 cubic centimeters or about 1 cubic centimeter.
Arty volume
range having any one of the stated lower limits and any one of the stated
upper limits is
within the scope of the present invention. so Iong as the upper limit is
larger than the tower
limit.
Another product form is a suspension of panicles of the highly amorphous
sustained-
release composition in a liquid vehicle. Preferred uses for such suspensions
are for
placement by injection subcutaneously, inuaperitoneally and inttaocularly.
Another preferred
use for the liquid vehicle is for oral administration, for example, when
uptake by
gastrointestinal tissue is desired. For injection applications, the particles
of the highly
amorphous sustained-release composition should typically be smaller than about
50 microns,
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more preferably smaller than about 20 microns and most preferably smaller than
aim 10
microns. Particles of a size of from about 0.~ micron to about I O microns are
preferred for
most injection applications. the liquid vehicle for the suspension may be an
aqueous liquid
or an organic liquid. When an aqueous liquid is used as the liquid vehicle,
however, the
particles of the highly amorphous sustained-release composition and the liquid
vehicle are
preferably mixed immediately before administration to a patient. Otherwise,
significant
release of the pharmaceutical substance into the aqueous liquid vehicle could
occur prior to
administration. The particles and the liquid vehicle could be provided in a
kit including the
particles in one container and the aqueous liquid in a second container for
easy mixing prior
to use. For most applications, however, it will be desirable to use an organic
liquid vehicle
that is premixed with the particles and stored for later use, without
significant release of the
pharmaceutical substance during storage. Examples of preferred organic liquid
vehicles are
ethanol and propylene glycol.
In another product form, a powder of the highly amorphous sustained-release
composition could be packaged in a nebuIizer or other aerosol-producing device
for
pulmonary delivery applications. Particle sizes for pulmonary delivery are
preferably as
discussed previously.
It should be noted that, in addition to being preferred for the highly
amorphous
sustained-release composition, the product forms of macrostructures, liquid
suspensions and
for aerosol generation are preferred even for compositions in which the
biocompatible
polymer may not have the desired highly amorphous character.
A major advantage of the highly amorphous sustained-release composition of the
present invention is that, because of the highly amorphous character of the
biocompatible
polymer, the composition is less likely to cause an immune response when
administered to
a patient. More highly crystalline materials are more likely to cause as
immune response
and, therefore, accompanying inflammation.
Another significant advantage of the highly amorphous sustained-release
composition
of the present invention is that the composition exhibits very favorable
release characteristics
for sustained release of the pharmaceutical substance. This desirable result
is believed to be
related to the highly amorphous character of the composition and to the heavy
loading of the
composition with the HIP complex material. In that regard, when immersed in a
phosphate
buffer solution at a temperature of about 37°C, the highly amorphous
sustained-release
composition of the present invention typically exhibits a release profile for
release of the
pharmaceutical substance that plots as a single substantially straight line
when plotted as
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cumulative pharmaceutical substance released versus the square root of time.
'The highly
amorphous sustained-release composition, therefore, exhibits only a single
diffusion-
controlled stage of release, and does not exhibit the occutzence of multiple
diffusion-
contmlled stages of release, as has been reported for othez amorphous
compositions due to
S crystalli~tion of polymer during use. Even though the release profile of the
composition of
the present invention exhibits substantially only a single stage of diffusion-
conttnlled release,
it should be recognized that anomalies in the release profile may be expected
to occur at the
very beginning of release. Also, during later stages of release, degradation
of the polymer,
in the case of a biodegradable polymer, will affect the release profile. For
the highly
amorphous sustained-release composition of the present invention, however,
typically greater
than about 70 percent, preferably greater than about 80 percent and more
preferably greater
than about 90 percent, of the pharmaceutical substance is released during the
aforementioned
single diffusion-controlled stage of release when immersed in the phosphate
buffer solution
at the noted temperature. The phosphate buffo: solution, for comparison
purposes, should
typically be an aqueous solution including about 0.9 gram per liter of sodium
chloride, about
0.144 gram per liter of monobasic potassium phosphate and about 0.795 gram per
liter of
dibasic sodium phosphate heptahydrate.
Yet another significant advantage of the highly amorphous sustained-release
composition to the present invention is that it typically exhibits little, if
any, significant burst
e::ect during the initial stages of release of the pharmaceutical substance.
In that regard,
when immersed in the phosphate buffer solution, described previously, at a
temperature of
about 37°C, typically no more than about 15% of the pharmaceutical
substance is released
during the first 24 hours following immersion, preferably no more than about ~
10% and even
more preferably no more than about 5%. Furthermore, the highly amorphous
sustained-
release composition will typically exhibit this low burst effect as
manufactured by the
antisolvent precipitation process. In some instances, however, it may be
desirable to subject
the composition to a post-manufacture wash with a buffer solution to rrrrtove
pharmaceutical
substance that may be adhering to the outside surface of particles. Such a
wash may be
accomplished, for example, by sonication for a short duration in a bath of
buffer solution.
Other methods for washing the particles are also possible.
The noted advantages of the highly amorphous sustained-release composition
concerning pharmaceutical release characteristics are believed to be
attributable, at least in
part, to the highly amorphous character of the composition and to the heave
loading of the
composition with the HIP complex. The amorphous character of the composition
is desirable
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because sustained-release composition made with highly crystalline polymers
often have
pharmaceutical release rates that are too slow. Furthermore, the amorphous
character of the
sustained-release composition of the present invention appears to be retained
for at least a
significant time during release. The initial formation of the highly amorphous
biocompatible
polymer in the composition and the apparent maintenance of a highly amorphous
character
during use are believed to be due. at least in par to heave loading with the.
HIP complex
material. Because both the HIP complex material and tht biocompatible polymer
both
typically have a high hydrophobiciry, they typically form a homogeneous and
intimate
mixture which may retard crystallization of the polymer during use.
Still a further significant advantage of the highly amorphous sustained-
release
composition of the present invention is that it typically includes only very
small quantities
of residual organic solvent. In that regard, residual organic solvent levels.
as manufactured.
are typicaIfy less than about 50 parts per million by weight, preferably less
than about 25
P~ per' million by weight, and even more preferably less than about 10 parts
per million by
weight. Frequently, residual organic solvent levels are less than about 3
parts per million by
weight. To maintain rsidual orgwn:c solvent levels at an extremely low level,
the antisolvent
precipitation process is preferably conducted such that after the particles
have been
precipitated in a reactor vessel, they arc retained in the reactor vessel and
flushed with a
volume of substantially pure antisolvent fluid for a sufficient time to reduce
the residual
solvent content to the desired level. A post-precipitation flush with a volume
of substantially
pure antisolvent fluid should preferably include at lease about one reactor
volume of
antisolvent fluid, with a flush of at least about two times the reactor volume
being more
preferred.
One particularly preferred embodiment for the highly amorphous sustained-
release
composition of the present invention includes poly(1-lactic acid) as the
biocompatible
polymer. When making a composition with poly(I-lactic acid), the preferred
solvent is either
methylene chloride or ttichloromethane, although ethyl acetate may also be
used if the poly
(1-lactic acid) has been suitably end-capped or otherwise modified for
enhanced solubility.
The pharmaceutical substance may be any of the pharmaceutical substances
previously listed. In one preferred embodiment of the present invention,
however, the
pharmaceutical substance in the highl; wr.:erY'nous sustained-release
composition is isoniaad,
which is preferably in the form of an ion pair with AOT. This composition is
particularly
preferred for treating tuberculosis. The preferred biocompatible polymer is
poly(1-lactic
acid). The preferred method for administering the highly amorphous sustained-
release
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composition with isoniaud is via subcutaneous placement, typically by
implantation of a
macrostructure, as previously described, comprised of agglomerated particles
made by the
antisolvent precipitation process.
In another aspect of the present invention, the antisolvent precipitation
process may
be used to make particles including a highly amorphous biocompatible polymer,
even in the
absence of a HIP complex material. For example, a pharmaceutical substance
that is not in
an HIP complex form could be suspended in or, for a few phannaoeuticals, co-
dissolved in
a solvent containing the polymer. Alternatively, particles made by the
antisolvent
precipitation process could be of substantially pure biocompatible polymer.
The preferred
processing conditions with respect to flow rates. conditions for contacting
the solvent and
antisolvent, pressure, temperature, antisolvent fluids, solvents and
biocompatible polymers
are as previously described for the highly amorphous sustained-release
composition including
an HIP material. Compositions without the HIP complex material, however, are
no; pre.~':ar~
because the presence of the HIP complex material. as previously discussed,
significantly
enhances the properties of the composition for use in sustained-release
applications. In these
embodiments, the biocompatible polymer in the particles typically preferably
is not more than
about 25% crystalline, preferably not more than about 30 weight percent, more
preferably not
mor t::an about 15°.o crystalline, even more prefe:ubiy not more than
about Z O% crystalline,
and most preferably not more than about 5% crystalline.
The invention will now be further described with reference to the following
non-
limiting examples.
EXA.IvIpLES
The methods used for measuring apparent partitioning coeff~.cients are
described in
Example 1. The measurement of the behavior of the Gly-Phe-:~IH::SDS complex is
described
in Example 2. The behavior of the 8-Argvasopressin:SDS complex, leuprolide:SDS
complex, neurotensin:SDS complex, and bradykinin:SDS complex are described in
E.~cample
3. The behavior of the insulin:SDS complex is described in Example 4. The
dissolution of
the insulin:SDS complex as a function of the organic solvent is described in
Example 5.
Further behavior o° :.~:: :~~Y:~;;~::SDS complex is described in
Example 6. Example 7
describes the CD ~; ._.-:..:: ~ ~ ;!-.~ irsulin:SDS complex. Example 8
describes the thermal
stability of the insulin:SDS complex. Example 9 describes the behavior of
other large
Proteins with SDS, specifically, human growth hormone. The behavior of bovine
pancreatic
trypsin inhibitor with SDS is described in Example 10, and Example 11
describes the
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behavior of human serum albumin with SDS. The melting point of the SDS:insulin
Hip
complex was studied (Example 12).
Example 13 describes a method for forming a fine HIP complex suspension
suitable
for pulmonary delivery. Example 14 describes a method for achieving uniform
distribution
of a protein throughout a hydrophobic polymer suitable for use as an
injectable implant.
Example 15 describes the use of the HIP complex for improved storage of
proteins. The use
of pmtein precipitation in the HIP complex for protein purification is
described in Example
16. A method of administering a protein dissolved as an HIP complex in organic
solvent is
described in Example 17. Example 18 describes the preparation of a drug with
reduced bitter
taste.
Examples 19-29 demonstrate batch preparation of particles using gas
antisolvent
precipitate. Examples 30-32 demonstrate continuous preparation of particles
using gas
antisolvent precipitation.
Examples 33-.~0 describe the preparation. characterization and use of cationic
surfactants of the invention.
Example 1. ~y~ ' a oefficie
The relative solubilities in two phases is given in terms of an apparent
partition
coefficient. The apparent partition coet$cient is defined as the ratio of the
equilibrium
concentration in an organic phase to that in an aqueous phase. The actual
value of the
apparent partition coefficient, P, is dependent on the two solvent systems
employed. In all
cases herein described, the organic phase is 1-octanol and the aqueous phase
is water alone
or with a minimal amount of HCI added.
Apparent partition coefficients were measured by dissolving a peptide in 1:?5
ml of
=5 an aqueous solution. Before SDS addition. it:e YH was measured on a
Beck.:zan pH meter.
Upon addition of an SDS solution, the solutions turned cloudy and a
precipitate formed
immediately. An equal volume of I-octanol was added and the mixtures agitated,
and then
left undisturbed for several hours. Prior to analysis, the tubes were spun for
10 minutes at
4000 g. Each layer was removed and the absorbance measured on a Beckman DU-64
UV
visible spectrophotometer using 1 cm quartz cells. All apparent partition
coeffcients were
corrected for changes in pH with differing SDS concentrations.
Results are described as logarithms of the apparent partition coefficient. A
log P
value of 0 means that the compound is equally soluble in water and the organic
phase, A
positive log P value means the peptide is more soluble in the organic phase
than in water and
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a negative log P values indicate a greater aqueous solubility than in the
organic solvent. All
of the log P values reported herein have been corrected for slight changes in
solubility with
pH.
Example 3. Annarent P rritioning Coefl'tcient for lvPhe ty'Hi,
The logarithm of the appat~ent wattr/1-octanol partition coefficients for GIy-
Phe-NH_
Gly-Phe amid. 0.6 mg/ml, pH about 5) and Gly-Phe (0.6 mg/ml at pH 7 and pH 3)
as a
function of SDS to peptide ratio are shown in Fig. 1. Apparent partition
coefficients were
measured as described in Example 1.
In order for HIP to occur, the polypeptide must contain at least one basic
group (either
a lysine or arginine side chain or a free N-terminal amino group). Gly-Phe~NH:
contains a
single basic group, and at pH 7 forms a 1:1 complex with SDS. The complex
precipitates
from aqueous solution, but readily partitions into 1-octanol. as shown in Fig.
1. For Gly-Phe
itself which exists in a zwitterionic form at neutral pH, a complex with SDS
is formed with
difficulty, and little enhancement of the partition coefficient is observed.
However, by
lowering the pH to less than 4, the carboxylate group of Gly-Phe becomes
protonated, leaving
the molecule with an overall positive charge and again. a hydrophobic ioa pair
can be formed.
Partitioning of ulyPhe at pH 3 mirrors the marked increase seen for Gly-
PheNH=. Thertfore,
even for acidic peptides. lowering the pH may permit hydrophobic ion pair
complexes to be
formed.
Example 3. Behavior of Prote'n~~D omple~_
The logarithms of the apparent water/1-octanol partition coefficient for AVP
(0.49
mg/m1, pH 5), leuprolide (LPA)(0.5 mg/ml, pH 6), neurotensin (N'I~ (O.y mg/ml,
pH x), and
bradykinin (BK) (O.y mg/ml, pH x) are shown in Fig. 2. Apparent partition
coefficients were
measured as described in Example 1.
Peptides larger than Gly-Phe-NH: can interact with SDS to form HIP-complexes
with
enhanced solubiiiry in organic solvents. AVP is a nonapeptide hormone which
controls water
and salt elimination in the body. it contains two basic groups, the N-terminal
amino group
and the guanidinium side chain of Arg', and no acidic groups. Stoichiometric
addition of
SDS produces a precipitate from as aqueous solution (pH 7) which readily
partitions into a
1-octanol (Fig. 2). At a mole ratio of 2:1 (SDS:peptide). the solubility in 1-
octanol actually
exceeds the solubility in water by more than tenfold (i.e., log P > I).
Overall, the apparent
partition coefficient for AVP was increased by nearly four orders of
magnitude.
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Example 4. Behavior of Inc ~liw D~ ('~..,1~,
The logarithm of the apparent partition coefficient of insulin as a function
of SDS
ratio is shown in Fig. 3.
Polypeptides which contain both acidic and basic groups can also farm
hydrophobic
ion pairs. Insulin contains six basic groups (one Arg, one Lys, two His, and
two F-terminal
amino groupsl and four acid:: groeps. By lowering the pH to 2.5, all of the
acidic groups
(which are carboxylic acids) become protonated and the only remaining ch~ges
are due to
the basic functional groups, producing an overall charge of+(.
The solubility of insulin is altered dramatically upon addition of
stoichiometcic
amounts of SDS (Fig. 3). The solubility of an insulin-SDS complex approaches 1
mg/ml
(0.17 mlvl] in I-octanol, and its apparent partition coefficient inct~rases by
nearly four orders
of magnitude. At higher SDS concentrations, the apparent partition coefficient
decreases,
because the solubility of insulin in water increases again, presumably due to
micelle
formation.
Example 5. ~ of Insulin- DS'omnle~c as a Fu~r~;on of O
~E.antc foment.
Dissolution of insulin-SDS complexes in other solvents was investigated as
well
( :.rile i ). Precipitates of SDS-insulin complexes were isolated and added to
various organic
solvents. Some degrr.. of polarity appears to be necessary to obtain
measurable solubility in
the organic phase, as partitioning into chlorocarbons (CH_CI= 1-chlorooctane,
and CCI,} and
alkanes (mineral oil, hexane) could not be detected using UY-visible
absorption
spectroscopy. Besides alcohols. SDS-insulin complexes are soluble in N-
methylpyrrolidone
(NMP). trimethylphosphate (T1ZP), polyethylene glycol, ethanol, and t-butanol.
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TABLE 1. PARTITIONING OF INSULIN INTO NON-AQUEOUS SOLVENTS
Organic Solvent Lo P
8 Apparent Sot.
!m ml)
I -octanol 2 1.2 2 1.0 _
CCI, not de.ec.a~:r ~ insoluble
Mineral Oil not detectable insoluble
CH,CI= not detectable insoluble
Dimethoxyethane not detectable not determined
Hexane not detectable insoluble
1-Chlorooctane not detectable insoluble
THF miscible not determined
Acetone miscible not determined
Ether not detectable insoluble
DMF not determined 2 1.0
NMP miscible
2 1.0
Ethyl acetata miscible ' insoluble
PEG 400 miscible z 0.2
Trimethyl phosphatemiscible ~ 0.15
Ethanol miscible Z 1.0
i-Propanol miscible
2 1.0
Methanol miscible
2 1.0
Propylene Glycol miscible 2 0.5
TMI' miscible
Z 0.2
Trifluoroethanol miscible 2 0.5
Example 6. Behavior of Leurzolide~SD om, iex.
Leuprolide acetate is a luteinizing hormone releasing hormone (LHRH) agonist
used
in the treatment of endometriosis. It contains 9 amino acid residues and two
basic
functionalities (a histidine and an arginine group). Both termini are blocked.
Stoichiometric
amounts of SDS were added to an aqueous solution of leuprolide (0 and 0.5
mg'ml, pH 6.0),
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resulting in formation of a precipitate. The apparent partition coefficient of
the SDS-leuprolid
complex (Fig. 2) exhibited a log P into 1-octanoi greater than 1Ø
Example 7. CD Snectrometrv of the DC-Incutin omeiex
Two important considerations for proteins dissolved in non-aqueous solvents
are
whether native structures are retained and whether the material can be
extracted ~k into an
aqueous phase. The secondary composition of a 6:1 SDS-insulin complex
dissolved in neat
1-octanol at 5 °C is shown in Fig. 3. The insulin concentration was 61
ug/ml.
CD spectra were recorded on an Aviv 62DS spectrophotometer equipped with a
thermoelectric temperature unit. All temperatures were measured t0.2°C.
Samples were
placed in strain-free quartz cells (pathlength of 1 mm) and spectra obtained
taking data every
0.25 nm using a three second averaging time, and having a spectral bandwidth
of 1 nm.
Analysis of the CD spectrum. using an algorithm based on the methods of
Johnson
(I990) Genetics 7:205-214 and van Stokkum gt ~j. (1990) Anal. Biochem.
j,Q~:110-118,
indicates that the alpha-helix content of insulin in octanol is 57%, similar
to that found for
insulin in aqueous solution (57%) (Vlelberg and Johnson (1990} Genetics
$:280~286) and in
the solid state by x-ray crystallography (53%) (Baker ~ ~j. (1988} Phil.
Tracts. R Soc.
London 8319, 369-456). The spectra arr slightly more intense than those
reported for insulin
in water (Docker and Biswas ( 1980) Biochemistry IQ:5043-5049; Melberg and
Johnson
(1990) ; Brew gl ~, (1990) Biochemisuy,',~,Q:9289-9393). The relative
intensity of the
222 ntn band to the 208 nm band is similar to that observed for insulin at
high concentrations
(Docker and Biswas (I980) ~). This represent the first example of native-tike
structure
in a protein dissolved in a neat organic solvent.
Fig. 4 shows the far ultraviolet CD spectrum of insulin extracted from 1-
octartol into
an aqueous solution of 0.10 M HCI. The pathlength was 1 mm, the sample
concentration 53
ug/ml. and the sample temperature 5 °C. Upon shaking an octanol
solution of insulin with an
aqueous solution containing 0.10 M HCI, insulin can be extracted back into the
aqueous
phase, presumably due to rtplacement of the SDS counierion with chloride.
Lower HCI
concentrations did not at~ect extraction of insulin from 1-octanol.
Examination of the CD
specmcm of the redissolved material (Fig. 4) indicates an overall structure
similar to that of
native insulin.
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Example 8. Increased Thermal Stabilit<~ of the ~T)S f"enlin r"".,i.~
The stability of insulin to thermal denaturation is difficult to assess as
chemical
degradation rates are rapid at elevated temperatures (Ettinger and Timasheff
(1971)
Biochetnistry IQ:824-831 ). In aqueous solution, the thermal denaturation of
insulin occurs
at a T°, of about 65'C [define Tm. The Tm of insulin in 1-octanol has
been measured,
following molar ellipticiry at 322 nm, to occur at 98°C (Fig. 6), which
is more than 30
degrees above that observed in water. This observation supports the conclusion
that proteins
dissolved in organic solvents demonstrate exceptional thermal stability.
Although prior
reports have observed that proteins suspended in organic solvents exhibit
increased chemical
stability due to lack of water (Ahem and Klibanov (1987) references), the
present disclosure
is the first report to find increased protein stability of the SDS:protein
complex in organic
solvent with respect to denaturation. Furthermore, as shown in Fig. 9, the SDS-
insulin
complex appears to maintain its native structure in 1-octanol. even after
prolonged heating
at 70°C for more than 1 hour.
Example 9. Pehav~n~ of T ~.~~~ v-,.r~:~~ r"
I
Larger proteins car: aim Form cumpiexes with SDS. At pH 7.8, the aqueous
solubility
of human growth hormone (hGH) was not affected by addition of SDS, even at
ratios of
100:1. However, at pH 2. hGH precipitates from aqueous solution at the ratios
ranging from
10:1 to 40:1. At higher SDS concentrations, hGH redissolves, presumably via
micellar
solubilization. The hGH precipitate was not found to be soluble in 1-octanol,
as determined
by spectrophotometric assay. however, it was easily suspended in water and
various oils,
such as olive oil.
Example 10. Bshavior of Bovin P ncreatic Trn2cin Inhibitor omnisy~~.; ~~,~rh
cnc
Other proteins can also form complexes with SDS. Bovine pancreatic trypsin
inhibitor (BPTn is a small basic protein (hiW 5900) with a well defined and
stable structure
(Wlodawer~~j. (1984) J. Mol. Biol. 180:301-339, and (1987) J. Mot. Biol.
],Q~:145-156).
At pH 4, it partitions into 1-octanol upon addition of SDS (Fig. 7). As with
insulin, the
structure is maintained (data and shown) and the SDS-BPTI complex is soluble
in other
solvents as well, such as NMP and trimethyl phosphate (TMP). In TMP, the
globular
si:ucture is compromised, as determined by CD spectroscopy. Apparently, TMP is
a strong
enough solvent to displace water from the hydration sphere and destabilize the
structure of
43
SUBSTITUTE SHEET (RULE 26)
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WO 99/47543 PCT/US99/06198
BPTI. This mechanism of protein denaturation has been described in detail by
Arakawa and
Timasheff ( 1982) Biochemistry x:6536-644, and ( 1982) Biochemistry 21:6545-
6552.
Example 11.
Stoichiometric addition of SDS to human serum albumin (HSA) (MW.68 kD)
produces precipitates as a hydrophobic ion pair complex is formed. While
partitioning into
1-octanol could not ba detected by W-visible absorption spectroscopy, the SDS-
HSA
complex was found to be soluble in NNIP (Fig. 8), yielding solutions of
concentrations
greater than i mg/ml (pathIength = 1 cm. sample temperance = 27°C),
Wit(iout SDS, the
solubility of HSA in Ni~IP is less than 0.03 mQ/ml.
Example 12. Melting Point of DS~Tncui;r, C'nmTIPw
T'ne melting point (SIP) of SDS:insulin ion pairs in 1-octanol was studied at
SDS:insulin ratio ranging from 1:1 to 1:24.
Insulin at 1 mg/ml in 0.005 N HCl was prepared containing SDS at 1, 2, 3. 4.
5, 6, 7,
8, 9,12,15, 18, 21 and 24 moles of SDS per mole of insulin. Equal volumes of
octanol were
added to each SDS:insulin solution to partition the insulin into the octanol
phase. The
concentration of the SDS:insulin complex extracted into the octanol was
estimated by its
absorbance at 278 nm and the solution diluted to 200 ug/ml. The melting point
of the various
insulin in octanol solutions was then determined with an AVIV 62DS circular
dichroism
spectrometer. Hoth circular dichroism (CD) signal and light scattering (as
measured by
changes in absorbance) were measured at ~22 nm and the melting point
determined by an
inflection point in the measured scan.
Fig. 9 shows the graph of melting point as a function of SDS:insulin molar
ratios,
with an apparent maximum at 6: I molar ratio and a melting point of about I I6
°C. The molar
ratio of 6:1 is also the stoichiometric ratio and show the highest thermal
stability for insulin
in octanol.
Fig. 10 shows a typical CD scan at '_''_'= nm as a function of temperature. A
melting
point of 106°C was determined by the maxima of the first derivative of
the pictured data.
Fig. lI shows a typical absorbance scan at 222 nm as a function of temperature
and
effectively mimics the CD scan. showing a melting point of 106°C.
Example 13. F~ation of a Fine ~cDen_cion HIP Comnler for PLlmon n i riW
44
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99~6198
For the formation of particles for pulmonary delivery, a protein solution is
stirred
vigorously using a homogenizer. SDS is added dropwise to the agitated
solution. Particles
in the 2-10 micron range are obtained. These particles are separated from the
mixture by
centrifugation or filtration. The particles are then suspended in a mixttue of
Freon'a 11 and
12, such than when placed in a meter dose inhaler, a therapeutic amount of
protein is
delivered on each actuation.
Example 14. Uniform Distribution of Protein Lgho ~t ~ Hy,~mPhobic Poh~rner f r
1cr
The biodegradable polymer consisting of a 50:50 mixnire of poly-lactic acid
and poly-
glycolic acid is dissotved :r: a vol~~.:.~ oreanic solvent. such as V-methyl-
p.ntolidone GIMQ).
An appropriate amount of an HIP-protein complex such as insulin-SDS (0.5%-5.0%
by
weight relative to the polymer) is dissolved in the same solvent. The two
solutions are mixed
and stirred for one hour. After the mixing is complete, the solvent is removed
by
evaporation. This is done in a mold to form an implant, or by a spray drying
procedure to
form small uniform particles for injection. The resulting solid material can
also be ground
to a powder and formulated as an injectable suspension. The protein is
released from these
systems as the polymer biodegrades and the HIP complex hydrolyses.
Example 15. Use of HIP Comelex Formation for Prot ~r Crnr~o'
~,..
The HIP complex is formed by dissolving the protein or polvpeptide in water at
minimal ionic strength. The pH is adjusted to as low a pH value as is
practical to ensure
'-5 stabilir~ and acti city. ? st;,ck ~olucion of SDS is added so ~hat the
number of eauivaltnts of
SDS matches the number of basic groups. For insulin, the pH is adjusted to
2.~, and 6 molar
equivalents of SDS are used per mole of insulin. The resulting complex
precipitates from
soltnion, is collected, and dried at room temperattue. The solid HIP complex
may be stored
at higher humidifies and temperatures than the native proteins without
noticeable loss of
activity.
Dissolution in a non-reactive organic solvent, such as I-octanol, produces a
true
solution of a protein. The HIP complex of insulin stored in 1-octanol is much
more stable
than insulin in water, as shown by its enhanced thermal stability.
Example 16. Use of HIP Comnle~c Formation for Protein P ~rification.
SUBSTITUTE SHEET (RULE 2B)
CA 02324254 2000-09-15
WO 99/47543 PCTIUS99/06198
The hydrophobicities of HIP complexes of proteins will differ according to the
fraction of the proteins surface covered by the alkyl sulfate molecules. In
turn. the HIP
protein complexes are separated using a variety of methods, including
hydrophobic
interaction columns.
Further. proteins may be purified by selective precipitation out of solution.
For
example, a protein is separated from additives such as human serum albumin
(HSA), which
may be present in amounts 20-50 times greater than the protein. Since HSA does
not
precipitate out of solution at pH 5.0 with SDS, a basic protein may be
selectively precipitated
and purified from HSA under those conditions.
Example 17. v
aProtein to a Pati ~~
The administration of HIP complexes to a patient may be accomplished in a
number
of ways. A biodegradable polymer/HIP complex system may be dissolved in an
organic
solvent, for example N-methyl pyrrolidone, and injected subcutaneously to form
an implant,
processed to form microspheres which can be injected subcutaneously or
intramuscularly,
processed to form an implant which is placed sttrgic~~ly under tt:e skin. or
given orally as part
of an oral delivery system for peptides and proteins. T:ze solid HIP complex
may also be
Prepared as a suspension or a non-aqueous solution, which may be injected or
placed on the
skin where the complex may partition into the skin. The HIP complex may also
be nebuiized
and administered to a patient via inhalation, for pulmonary drug delivery. The
HIP complex
may also be formulated to be given orally, such that it is protected from
degradation in the
stomach via an enterically coated capsule, and released in either the upper or
lower intestinal
tract. The HIP complex may be loaded alone or in conjunction with oils, bile
salts, or other
enhancers to increase absorption. The HIP complex may also be suspended or
dissolved in
oil and introduced to the patient as a rectal or vaginal suppository.
Example 18. P~aration of a Dr y With Reduced Bitt r T cre
The low solubility of the HIP complex results in diminished taste of bitter
tasting
drugs taken orally. The HIP complex may also be dissolved in oil so as to
further reduce
bitter taste. The slow rate of hydrolysis, especially in an oil-type vehicle,
prevents the bitter
tasting drug from dissolving in the mouth and being tasted.
~t6
SU8ST1TUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99106198
Examples 19-29. Batch Preparation of Pan;~W~ r rw~
EIS~1I21L~I1~.
Examples 19-29 demonstrate batch manufacture of particles having a
pltartnace»~
substance and an amphiphilic material using supercritical carbon dioxide as a
gas antisolvetu.
Fig. 13 shows a process flow diagram for the batch processing of Examples 19-
29.
Referring to Fig. 13, supercritical carbon dioxide. from the anisolvent tank
122 is fed into the
antisolvent chamber 124 and is pressurized using a hand syringe pump 126, with
valve 128
and valve 130 closed and valve 132 and valve 134 open. After the antisolvent
chamber is
pressurized, then valve 134 is closed and the test solution 136 is fed into an
injection port
138. Nitrogen from a propellant tank 140 is pressurized behind the injection
port 138 and
is used to force the solution through a sonicated orifice 142 to spray the
test solution 136 into
the antisolvent chamber 1=4. The test solution 136 for each example has a
pharmaceutical
substance and an amphiphilic material dissolved together as a hydrophobic ion
pair complex
I S in an organic solvent. Some examples have a biodegradable polymer also
dissolved in the
organic solvent. Solid particles which precipitate are allowed to settle, with
all valves closed,
onto a scanning electron microscope (SEM) stub in the anisolvent chamber 124.
The
anisolvent chamber 124 is then slowly depressurized through the valve I30 aad
the SEM
stub is removed for analysis. Any remaining solid particles from the
antisolvent chamber 124
are collected on the filter 144.
The makeup of each test solution for Examples 19-29 is shown in Table 2. Test
conditions aad results, including a description of particles which are
precipitated, are shown
in : a'cia 3. c igs. I4 and 13 are SEVI photomicrographs of imipratnine
panicles of E.~catnple
'?, showing the elongated f ber-like shape of the particles. In Fig. 15 it may
be seen that the
fiber-Iike particle has a hollow interior in which small particles of another
pharmaceutical
substance could be loaded for some pharmaceutical applications. Fig 16. is a
SEM
photomicrograph of a particle of ribonuclease and poly (ethylene glycol) of
Example 27,
swing an opening in the end of the particle into a hollow interior space. Fig.
17 is a SEM
photomicrograph of particles of a-chymotrypsin of Example 19, showing
ultrafine sphernidal
particles of a size smaller thaw about 10 microns, with many of a size of
around 1 micron.
Fig. 18 is a SEM photomicrograph of pentamidine particles of Example 29 of a
size smaller
than about 1 micron.
47
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
a
a
a
v v~ ~ c r
'
> ~ ~ ~ a ;v r'-~ ~ e a a
a, - a
o ~ ~ s o ~ s t s e~a
_
o o a c a , a a -
~ r.. a
a
V .N G E G G v
a
i i i i i ~
i i
a
0
i i i i i v
~ i
::,
a
n
O O O N
N N Q
.
v
a
N
W ~ C
Ca : '
'r~~ m a o o .o ~ HOC
.%
0 o c o
a a a a ~ ~ ~ ~
3
N
>
v _
~ O
H
~
O r ~ C
~ t=
o '~'~ '_. ~; ~' M M o o N _ ~ p o
', 8
0
a C , ~ o vc
H
i a o x
O
>
C C C O
C
~
>
a
~ c ~c ~ ~ H V a ~
~
E
c
. .
.
o
y a a a = = E ~ ~
' . . ~
?
W
~
o
C O C - _ = a a o a.
'~ ~ ~ ~ E ~..
~
~
o
c. E-E E E ~ H ~, ~ _~
c
>, a, a, ~ c a ~ ~ o ~ c,
C ~
_~
~
'
a
~
O
.~
V U C ~ ~
~
>>
H
i
~ 'i w i~~ V
t~
v
X
~
~
CD
~
_
iS i3 i! a Q
~
-c
c3
~
~
..
C
C
cC
.~
~S
NV
~.~
g~
~
'~~~
mo
N N N N N N N N N m
E
E
N E
.G
X
W
,~~
~~~~~.~.-.
N
M
et
~1
~p
N
pp
~
W
r
wr
~/
r
yr
1n O V1 O
N
SUBSTITUTE SHEET (RULE 28)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/U6198
TABLE 3
Test C onditions
"
Example Particles
Temp ( Press. (bar)
C)
19 34 76 spheroidal. approx.
10~s and smaller
20 28 76 irregular shape,
approx. I a dia.
21
spheroidal. approx.
2-3u dia.
22 36 85 fiber-like, approx.
1 Ou dia. and 1 cm
lone
23 34.5 85 spheroidal
I0 24 34.6 85 irregular, approx.
I-5 u,
25 35.2 g5
26 35.5 85.5 spheroidal, approx
50 a
27 35.3 85 fiber-Like, approx.
l0u dia. and tmm long,
spheroidal, approx
0.5-lu
28 35.3 77 collapsed spheres,
approx 5 a dia.
15 29 35 82 spheroidal, approx. .
0.1-lu dia.
Examples 30-32. Contintouc ManLfac ~ of solid P rrirlPC by Cas nticnh~r»
Examples 30-32 show continuous manufacture of solid particles comprising a
pharmaceutical substance and an amphiphilic material.
Fig. I9 shows a process flow diagram for the continuous manufacture lost for
Examples 30-32. The antisolvent chamber I24 is first pressurized with an
automatic syringe,
pump 126 with a back pressure regulator 146 adjusted maintain the desired
antisolvent
pressure in the antisolvent chamber 124 at a given antisolvent flow rate
through the system.
This initial pressurization is performed with the valve 148, the valve 134 and
the valve 130
closed and with the valve 150 and the valve 132 open. One of two methods for
metering the
49
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCTNS99/06198
solution 136 into the antisolvent chamber 124 is used for each example. One
method is to
load the pump 152 with pure solvent and to spray the pure solvent into the
antisolvent
chamber 124 until a steady state is achieved. The solution 11.6 is then loaded
into the
injection port 138 and spiked into the solvent delivery line 1~4 to the
antisolvent
chamber I=4. The second method is to load the pump 152 with the solution and,
bypassing
the injection port, to deliver the solution to the antisolvent chamber 124.
Both delivery
techniques are operated at a flow rate of 1 milliliter per minute with a
carbon dioxide flow
rate of 20 milliliters per minute. In both cases, the solution enters the
antisolvent
chamber I24 through the sonicated orifice 142. During operation, carbon
dioxide is vented
from the top of the antisolvent chamber to allow particles to settle and not
be entrained in the
exiting carbon dioxide. Any particles that are washed out of the antisolvent
chamber 124 are
collected on the filter 144.
After spraying the solution 136 into the antisolvent chamber, then valves 150
and 130
are closed and valves 134 and 148 are opened and carbon dioxide is metered
into the
I S antisolvent chamber 124 liom bottom to top to flush any residual solvent
from the antisolvent
chamber 124. The system is then slowly depressurized and particles which have
precipitated
are collected from either the antisolvent chamber 124 or the filter 144.
The makeup of the solution for each of Examples 30-32 is shown in Table 4.
Table 5
shows the test conditions for each of Examples 30-32 and results of the
examples, including
a description of particles which are produced.
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
w a o
.'.' .'
~_o_ o
y G U J
> aJ4l N
v L
_v _
T T
t
J CJ d
O
n
C i ~
N N
O
a a
E., r
Gr
r
d n 6l
;p a
M M M
a
~ a
ea .
H
.., R
d ~ ~ C7
.
~'~'a a m
>
~
3
O s ei
'v
a
o
~
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V ~ H
..
~
.~ E >
~
E
~
a
o
c c c
J U U N t O ~.
~ ~ C C E ~ O
R >. c o 0 ~ ;=
ue ~ .
.. E- u ~ ;v
C U ~
y .
VlN H
o 's v
R .~
,
~
~ r~
V. ~
O~C O~
p, CO O 00
H_
~ ~ E
.O
R
M M
k
N M ~ V1
'..i ~ rr
~ ~r
V1 O V1
51
SUBSTITUTE SHEET (RULE 26~
CA 02324254 2000-09-15
WO 99147543 PCT/US99/06198
TABLE 5
Test Conditions
Ex ~-
l
amp Particles
e Temp (C) Press. (bar)
30 35 88 spheroidal, approx.
l,u
31 36.8 89 spheroidal, approx.
0.4~s
32 36.2 88? spheroidal, approx.
0.4~c
Example 33. Svnthec,_'c of _ r~im'ne Ocy~] Ester.
This example describes the synthesis of arginine octyl ester. This ester w~
synthesized by the in situ generation of the acid chloride of arginine,
followed ~y ;:irect
I S esterification with the appropriate alcohol (see Figure 20A).
One millimole of L-arginine free base (Sierra) was suspended in 50 mL of neat
1-
octanol (Sigma). A rubber septum was used to keep excess water in the
atmosphere from
reacting with the thionyl chloride (SOCI=; Aldrich). One equivalent of thionyl
chloride was
added, and the reactants were slowly heated to 90°C. The mixture was
allowed to cool to
60°C, one more equivalent of thionyl chloride was added, and the
mixture was heated again
to 90°C; all solid (presumably arginine free base) disappeared. The
reaction mixture was
allowed to sit at 90°C for 2 hours exposed to the atmosphere to remove
excess thionyl
chloride. A five-volume excess of diethyl other was added to the mixture, and
a gummy
precipitate formed and coagulated. This precipitate was washed with saturated
sodium
bicarbonate solution, whereupon a powder precipitate formed from the gummy
precipitate.
This was removed by gravity filtration and washed 2x with sodi~ bicarbonate
aad
2x with diethyl ether.
The powder was found to be insoluble in a variety of organics, including
alcohols,
hydrocarbons, aromatics, DMF and pyridine. The powder was also insoluble in
water, and
would only dissolve in 0.1 N or stronger HCI.
TLC Assay n"~ (Sigma) showed distinct differences in mobility for substrate
and
product (the product traveled with the solvent finnt). To perform this assay,
product and
substrate wen dissolved in 0.1 N HCl at 1 mg/ml, and the product and substrate
solutions
were then spotted onto a Selecto silica gel TLC plate which was placed in a
vapor-saturated
vessel containing 60% isopropanol. I S% methyl ethyl ketone, and 25% 1 N HCI.
The
chromatograms were developed with ninhydrin.
53
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
The molecular structure of the product was verified by NMR and fast atom
bombardment (FAB) mass spectrometry to be arginine octyl ester
dihydrochloride. The
melting point was 155°C. The yield was approximately 100%.
Example 34. Svnthesie Of . r~ininsy ctrr,
One millimole thionyl chloride was added to a stirred suspension of one
millimole
L-arginiae fee base in 50 mL of octanol under nitrogen. The mixture was heated
to 90°C,
and the temperature was maintained with stirring for 2 hours. The mixture was
cooled to
60°C, one more equivalent of thionyl chloride was added, and the
mixture was stinrd at 60°C
for an additional 2 hours, at which time the reaction was seen to be complete
by TLC
(performed as described in Example 33). Excess thionyl chloride was allowed to
evaporate.
Then, the solution was cooled to room temperature, and 250 ml diethyl ether
was added.
Washing of the resultant soft white precipitate with saturated sodium
bicarbonate solution
gave a white solid. Filtration of this suspension and washing of the filtrate
with saturated
sodium bicarbonate solution (3x with 20 ml), water (3x with 20 ml), acetone
(3x with 20 ml)
and diethyl ether (3x with 20 ml) gave arginine octyl ester. The yield was
85%. FAH mass
spectrometry gave the expected parameters for arginine octyl ester.
Example 35. Svnthesi .cvl Ester.
20 This ester was synthesized using approximately the same procedure as
described in
Example 33 for the octyl ester. 1-Dodecanol (Aldrich) was used in place of the
1-octanol.
After several rounds of thionyl chloride addition, the substrate did not
disappear as
in the octyl synthesis. As the macture was heated to approximately
80°C, the substrate began
to clump together. Additional rounds of thionyl chloride addition did not
change the
appearance of the clumped substrate. TLC of the supernatant showed some
product. Five
volumes of diethyl ether caused some opaque precipitate to form, but it did
not coagulate as
in the octyl synthesis. Attempts using Whatman filter paper to filter out the
precipitate by
both gravity and Buchner filtration were unsuccessful, so the precipitate was
collected by
centrifugation. The resulting pellet had a gummy appearance like the octyl
product. This
pellet was washed with saturated sodium bicarbonate, and a product with a more
powdery
appearance formed. Centrifugation could not separate the prodact from the
aqueous
bicarbonate solution, so the precipitate was collected in a Buchner funnel
with Whatman
filter paper. Washing with either saturated sodium bicarbonate or diethyl
ether seemed to
reduce the amount of product.
53
SUBSTTTUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
TLC. NMR and FAB mass spectrometry gave the expected resulu for arginine
dodecyl ester dihydrochloride. The melting point was 1~5-130°C. The
yield was 110 mg
(about 1 %).
Clearly, this synthetic approach did not work well. In view of the tow yield,
other
synthetic approaches utilizing the Vilsmeier route (Figure 209) were tried,
but none gave
greater yields (the highest yield obtained was 0.~%).
Example 36: Svnthec_i Of. rQinine Dode~vl ~et--
A suspension of L-arginine free base (0.6 g, 3.5 mmol), sulfuric acid (0.31
mI,
7 mmol). and dodecanol (~5 ml) were stirred together at 140°C under
nitrogen. After 6
hours, a clear light yellow solution resulted, and TLC indicated the reaction
to be complete.
The reaction miacture was diluted with diethyl ether (50 ml), and washed with
water (3 x 25
ml). The combined aqueous extracts were washed with diethyl ether (2 x 25 ml),
and
basified with IV KOH solution, upon which a white solid precipitated.
Filtration of the
suspension and washing of the filtrate with water (3x with 25 ml), acetone (3x
with 25 ml)
and diethyl ether (3x with 35 ml) gave arginirte dodecyl ester. The yield was
86%. Melting
point was 125-130°C. NMR gave the expected resulu for arginine dodecyl
ester.
Example 37: ~vnthesic Of 4 hole terol r onatg
N,N-dimethyl ethanolamine (Aldrich; 0.24 ml. 2.44 mmol) was added dropwise
over
the course of 30 minutes at room temperature to a stirred solution of
cholesterol
chloroformate (Aldrich; 1.0 g, 2.2 mmol) in dichloromethane (Fisher; 30 ml).
The resulting
white suspension was stirred at room temperature for 10 minutes, at which time
TLC (20: I
hexanes:ethyl acetate) showed the reaction to be complete. Saturated sodium
bicarbonate
solution (10 ml) was added to the suspension, at which point a clear solution
resulted. The
organic layer was extracted. washed with water and saturated brine, and dried
over
magnesium sulfate. Filtration and evaporation gave the product (CC-CHOL) as a
syrup,
which crystallized on standing at room temperature. The yield was 85%. CC-CHOL
has the
following formula:
(CH3}=-N-CH,-CH_,-O-C-O-CHOL.
II
O
s~
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
~XamplC 3$: ~ra_Cteri atiOn Of 4r inin' Fctr,c
Stock solutions of the argin:ne esters were made by first dissolving the
powder in 0.1
N HCl to give a 10 mM solution and then raising the pH to a value between ~
and 6. The pH
should not be raised above 8.
S
A. p
Anionic compounds were dissolved in pH 5.~ bu8br ( 10 mM bis-tris propaae,10
mM
CaCI=, 10 mM KCI). Appropriate amounts of the stock solution of arginine ester
(see eve),
the anionic compound and buffer were mixed so that the final concentration of
the anionic
material was 1 mg/mL. An equal volume of organic solvent was added, and the
samples
wen vortexed for 15 seconds on high speed. Layers were separated by
centrifugation at
4000 rpm for 5 minutes. Concentrations of the anionic material in the aqueous
and organic
layers were determined by L'V spectroscopy on a Beckman DU-64 series
spectrophotometer.
The results are given in Table 6 below.
Table 6
~lYl~t 1
p-toluenesulfonic acid,
sodium salt none octanol -1.62
" Cg~ octattol -0.353
" C12" octanol -0.336
"
none i~~s -2.7
C8 isooctane -2.2
sodium benzoate none octaaol -1.2
C8 octanol 0.05
" C 12 octanol -0.072
DNA ("degraded free acid")
none octanol -1.52
" Cg octanol -1.24
adenosine triphosphate
none octanol -3.23
" C12(1:1)' octanol -1.48
" C 12(3:1 )' octanol 0.022
SUBSTITUTE SHEET (RULE 26)
CA 02324254 2000-09-15
WO 99/47543 PCT/US99/06198
sp-Toluenesulfonic acid, sodium salt was purchased from Kodak. Sodium benzoate
and adenosine triphosphate were purchased from Sigma.
'Log p is log (concentration in organic phaseiconcentration in aqueous phase).
"C8 is arginine octyl ester, and C12 arginine dodecyl ester.
'Ratio of detergent to anionic compound.
For DNA and bovine serum albumin (data not shown), the solutions tuned cloudy
when arginine dodecyl ester was added, but none would partition into octanol
layer, although
some was trapped at the interface. Cloudiness could not be spun out in
centrifuge.
B. Surface Tension
Surface tension was measured using a Fisher surface tensiometer. Briefly, a
platinum
iridium ring with a diameter of 6 cm was lowered into the appropriate dilution
of detergent
in 0.1 N HCI. Surface tension was read at the point where the force on the
ring upwards
caused the nine to break contact with the liquid surface. The results are
shown in
Figures 22A-B.
The results show that arginine octyl ester is a relatively poor detergent with
a critical
micelle concentration (cmc) of about 6 mM (2.3 mg/ml) (see Figure 22A).
However, the
dodecyl ester is a much better surfactant, with a cmc of approximately 0.3 mM
(0.10 mg/ml)
(see Figure 2''B). Considering the better detergent properties of the dodecyl
ester, all
subsequent studies focused on the dodecyl ester.
C. Cvtotoxiciri
The cytotoxicity of arginine dodecyl ester was investigated in cell culture
with two
types of cells (see Cory et al., Cancer Commun., .1, 107-?l: (1991)): CCRF-CEM
cells, a
human T-cell leukemia cell line that grows in suspension (obtained from the
American Type
Culture Collection, ATCC); and a green monkey kidney cell line (COS-7) that
grows in
monoiayers (also obtained from ATCC). For comparison, the cells were also
exposed to
tetradecyltrimethylammonium bromide (DTAB) (Sigma).
Cells were plated into 96-well plates (Corning) in a total of 200 ~sL
Dulbecco's
modified minianal essential medium for COS-7 cells, RPMI 1640 for CEM cells,
supplemented with penicillin G (50 U/ml), streptomycin sulfate (50 ~g/ml), and
10% fetal
calf senun, at 10,000 cells/well for COS-7 amd 50,000 ceils/well for CEM
cells. The plates
were incubated at 37°C for 34 hours after plating. The cells were then
exposed to various
concentrations of the detergents. Each detergent concentration was used in 8
replicate wells.
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After 2-6 hours, media/detergent solutions were aspirated. and the wells were
washed twice
with PBS. For CEM suspension cells, centrifugation of the suspension at 1000 x
g for 5 min
between each wash was required. After washing, 200 ~cL of fresh medium were
added, and
the cells were incubated for 72 hours. After 72 hours, cell proliferation was
determined using
the Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay. To
do so,
cells were exposed to MTS substrate (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxvmethoxyphenyl)-2-(4sulfophenyl)-3H-tetrazolium for 3 hours. Cellular
respiration
was assessed by monitoring the appearance of a soluble fotmazan reduction
product by
spectrophotometry at 490 nm. Absorbance was read using a Molecular Devices
spectrophotometric plate reader. Absorbance was directly proportional to the
number of
living cells in each well. Survival was plotted versus detergent
concentration, with the
untreated control group representing 100% survival. Detergent concentrations
producing
half maximal growth inhibition (IC.° values) were extrapolated from the
resulting curves.
The results are shown in Figures 23A-B. In CCRF-CEM cells, the ICS for DTAB
was 20 ~sM, whereas the arginine dodecyl ester had an ICi° of 150 ~eM
(Figure 23A). This
is seven-fold less toxicity for arginine dodecyl ester. Similar results wen
obtained in the
COS-7 cells, where the ICs° for DTAB was 80 ~cM, whereas the arginine
dodecyl ester had
an ICS of 175 ~cM (Figure 23B). This is approximately two-fold less toxicity
for arginine
dodecyl ester.
Example 39: Transfection ~%ith ArEi~~,Dodecy ~r'r
The plasmid used was pR~V4001uc. It was obtained from Dr. David Gordon, Div.
Endocrinology, Universiy of Colorado School of Medicine, Denver, C0. It was
propagated
in Escherichia toll strain DH~a (A T CC), isolated by a standard alkaline-SDS
lysis procedure,
and purified twice by isopycnic centrifugation on CsCI gradients (Sambrook et
al., Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory) (1989). COS-7
cells at
approximately 50,000 cells per 60 mm diameter plate (Falcon) were used for
transfection.
Control experiments were done with Lipofectamine (GIBCO/Life Technologies,
Gaithersburg, MD).
In 200 total uL of serum-free medium, plasmid (20 ~cg) and Lipofectamirte or
arginine
dodecyl ester wen mixed and allowed to interact for 45 minutes. The volume was
then
brought to 1 mL with serum-free medium. Plates with cells were washed with
serum-free
medium. Then, 1 mL serum-free medium was added to plates already containing 2
mL
serum-free medium and the plates were incubated at 37°C for 4 hours.
After 4 hours, serum
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was added so the final serum concentration was 10%. In another experiment, the
time of
incubation was varied.
After allowing cells to grow and express gene product for 36-50 hours, the
cells were
harvested. Harvested cells wen lysed and processed for measurement of
luciferase activity
using potassium luciferin substrate as described in Fraser et al., Mol.
Pharmacol., 47, 696-
706 (1995). Intensity of luminescence should be proportional to the amount of
expt~essed
luciferase and, therefore. the efficiency of transfection. "Background"
is the reading from just the substrate mixture on the luntittometer before
addition of cell
lysate. Average background is approximately 50 units. Any reading over 100
units is
considered significant.
The results are shown in Figures 24A-B. The results demonstz~ate that arginine
dodecyl ester promoted transection of the plasmid in a concentration and time
dependent
manner. Note that the transfection studies were performed without formation of
liposomes
or the addition of helper lipids, which should provide a much larger increase
in transfection
effciency. The intent of these experiments was to demonstrate that, even in
serum-
containing medium, there is sufficient interaction between the arginine esters
and DNA to
effect transfection of cells. The e~ciency of transfection was about 100 x
higher for
Lipofectamine than for arginine dodecyl ester.
Example 40: Characteriztion of CC-CHOL
CC-CHOL was tested for cytotoxiciry as described in Example 38 using COS-7 and
JEG-3 cells. 1EG-3 cells are a human choriocarcinoma cell line available from
ATCC. The
culture medium was Eagle's minimum essential medium containing 10% serum.
The results are shown in Figures ?SA-B. The results show that CC-CHOL was not
toxic to COS-7 and JEG-3 cells.
While various embodiments of the present invention have been described in
detail,
it should be understood that any feature of any embodiment may be combined
with any other
feature of any other embodiment. Any compatible combination of pharmaceutical
substance,
amphiphilic material, polymer and/or solvent may be used. Also, any feature of
any
processing method may be used with any solvent. Furthermore, the hollow, fiber-
like
particles may be prepared for any suitable combination of pharmaceutical
substance and
amphiphilic material. Moreover, the fiber-like particles may be made of a
biocompatible
polymer, alone or in combination with other materials, or a pharmaceutical
substance, alone
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or in combination with other materials, which are directly soluble in the
organic solvent.
Such features are expressly included within the scope of the present
invention.
Also, while various embodiments of the present invention have been described
in
detail, it is apparent that modifications and adaptations of those embodiments
will occur to
those skilled in the art. It is to be expressly understood. however, that such
moditic3tions at:d
adaptations are within the scope of the present invention, as set fort.'t in
the following claims.
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