U.S. patent application number 11/117732 was filed with the patent office on 2005-12-08 for particle delivery techniques.
This patent application is currently assigned to PowderJect Research Limited. Invention is credited to Burkoth, Terry Lee, Muddle, Andrew Gordon, Porter, Linda Maree, Sarphie, David Francis.
Application Number | 20050271733 11/117732 |
Document ID | / |
Family ID | 34577381 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050271733 |
Kind Code |
A1 |
Burkoth, Terry Lee ; et
al. |
December 8, 2005 |
Particle delivery techniques
Abstract
A method is provided for in vivo or ex vivo delivery of a
preparation of powdered nucleic acid molecules into vertebrate
tissue for transformation of cells in the tissue using needleless
injection techniques. The method can be used to deliver
therapeutically relevant nucleotide sequences to cells in mammalian
tissue to provide gene therapy, elicit immunity or to provide
antisense or ribozyme functions. A method for providing densified
processed pharmaceutical compositions is also described. The method
is used to convert non-dense pharmaceutical powders or particulate
formulations into densified particles optimally suited for
transdermal delivery using a needleless syringe. The method is also
used to optimize the density and particle size of powders and
particulate formulations for subsequent transdermal delivery
thereof. Densified pharmaceutical compositions formed by the
present methods are also provided.
Inventors: |
Burkoth, Terry Lee; (Palo
Alto, CA) ; Sarphie, David Francis; (Madison, WI)
; Muddle, Andrew Gordon; (Cambs, GB) ; Porter,
Linda Maree; (The Gap, AU) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
PowderJect Research Limited
|
Family ID: |
34577381 |
Appl. No.: |
11/117732 |
Filed: |
April 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11117732 |
Apr 29, 2005 |
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09216641 |
Dec 17, 1998 |
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6893664 |
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09216641 |
Dec 17, 1998 |
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PCT/GB97/01636 |
Jun 17, 1997 |
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09216641 |
Dec 17, 1998 |
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PCT/GB97/02478 |
Sep 11, 1997 |
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Current U.S.
Class: |
424/489 ;
435/459; 514/44R |
Current CPC
Class: |
A61K 9/0021 20130101;
C12N 15/89 20130101; A61K 48/00 20130101; A61K 9/145 20130101; A61K
9/14 20130101 |
Class at
Publication: |
424/489 ;
514/044; 435/459 |
International
Class: |
A61K 048/00; A61K
009/14; C12N 015/87 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 1996 |
GB |
9612629.7 |
Sep 11, 1996 |
GB |
9619002.0 |
Claims
What is claimed is:
1. A method for delivering densified particles to a target tissue
or cell, the method comprising the steps of: (i) forming densified
particles from a particulate pharmaceutical preparation, the method
for forming the densified particles comprising compacting the
preparation to provide a compacted pharmaceutical preparation and
size-reducing the compacted preparation into densified particles of
suitable size and density for transdermal delivery thereof by
needleless injection; and (ii) administering said densified
particles to the target tissue or cell by needleless injection.
2. The method of claim 1, wherein the particles have an average
size predominantly in the range of about 10 to 250 .mu.m.
3. The method of claim 1, wherein the particles are delivered to a
cell in epidermal tissue.
4. The method of claim 1, wherein the particles are delivered to a
cell in the stratum basal layer of skin tissue.
5. The method of claim 1, wherein the particles are comprised of a
nucleic acid molecule and a pharmaceutically acceptable
excipient.
6. The method of clam 1, wherein the particles are delivered to the
target tissue or cell in vivo or ex vivo.
7. The method of claim 1, wherein the nucleic acid molecule
comprises a nucleotide sequence encoding an immunogen.
8. A method for forming densified particles from a particulate
pharmaceutical preparation, comprising compacting the preparation
to provide a compacted pharmaceutical preparation and size-reducing
the compacted preparation into densified particles of suitable size
and density for transdermal delivery thereof by needleless
injection.
9. A method according to claim 8, wherein the suitable size is in
the range of about 0.1 to 150 .mu.m mean diameter.
10. A method according to claim 8, wherein the densified particles
have a particle density in the range of about 0.5 to 3.0
g/cm.sup.3.
11. A method according to claim 8, wherein size reducing of the
compacted material is carried out by milling and/or sieving.
12. A method according to claim 8, wherein the method further
comprises selecting densified particles using size
classification.
13. A method according to claim 8, wherein the size classification
of the densified particles is carried out using sieving or cyclone
separation.
14. A method according to claim 8, wherein the particulate
pharmaceutical preparation is a preparation of a gene
construct.
15. A densified particulate pharmaceutical composition formed from
a lyophilised or spray-dried pharmaceutical preparation, said
densified composition having an average particle size in the range
of about 0.1 to 250 .mu.m mean diameter and a particle density in
the range of 0.1 to 25 g/cm.sup.3.
16. A composition according to claim 15, wherein the lyophilised or
spray-dried pharmaceutical preparation is a heat-sensitive
biopharmaceutical preparation.
17. A composition according to claim 15, wherein the lyophilised or
spray-dried pharmaceutical preparation is a preparation of a
peptide or protein.
18. A composition according to claim 15, wherein the particulate
pharmaceutical preparation is a preparation of a gene
construct.
19. A composition according to claim 15, wherein the particle size
is in the range of about 0.1 to 150 .mu.m mean diameter.
20. A composition according to claim 15, wherein the particle
density is in the range of about 0.5 to 3.0 g/cm.sup.3.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/216,641, filed Dec. 17, 1998, now allowed, which is a
continuation-in-part of International Patent Application Numbers
PCT/GB97/01636, filed Jun. 17, 1997, and PCT/GB97/02478, filed Sep.
11, 1997, both designating the United States, from which
applications priority is claimed pursuant to 35 U.S.C.
.sctn.365(c), and which applications are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to particle delivery
methods. More particularly, the invention pertains to in vivo and
ex vivo delivery of powdered nucleic acid molecules into mammalian
tissue using needleless injection techniques. The invention also
relates to methods for forming dense, substantially solid particles
from non-dense particulate pharmaceutical compositions such as
those prepared using freeze-drying or spray drying techniques. The
densified compositions obtained using the method are particularly
suitable for transdermal particle delivery from a needleless
syringe system.
BACKGROUND OF THE INVENTION
[0003] The ability to deliver agents into and through skin surfaces
(transdermal delivery) provides many advantages over oral or other
parenteral delivery techniques. In particular, transdermal delivery
provides a safe, convenient and noninvasive alternative to
traditional drug administration systems, conveniently avoiding the
major problems associated with oral delivery, e.g., variable rates
of absorption and metabolism, gastrointestinal irritation and/or
bitter or unpleasant drug tastes. Transdermal delivery also avoids
problems associated with traditional needle and syringe delivery,
e.g., needle pain, the risk of introducing infection to treated
individuals, the risk of contamination or infection of health care
workers caused by accidental needle-sticks and the disposal of used
needles. In addition, such delivery affords a high degree of
control over blood concentrations of administered drugs.
[0004] However, despite its clear advantages, transdermal drug
delivery presents a number of its own inherent logistical problems.
The passive delivery of drugs through intact skin necessarily
entails the transport of molecules through a number of structurally
different tissues, including the stratum corneum, the viable
epidermis, the papillary dermis, and the capillary walls in order
for the drug to gain entry into the blood or lymph system.
Transdermal delivery systems must therefore be able to overcome the
various resistances presented by each type of tissue. In light of
the above, a number of alternatives to passive transdermal delivery
have been developed. These alternatives include the use of skin
penetration enhancing agents, or "permeation enhancers," to
increase skin permeability, as well as non-chemical modes such as
the use of iontophoresis, electroporation or ultrasound. However,
such techniques often give rise to unwanted side effects, such as
skin irritation or sensitization. Thus, the number of drugs that
can be safely and effectively administered using traditional
transdermal delivery methods has remained limited.
[0005] More recently, a novel transdermal delivery system that
entails the use of a needleless syringe to fire solid
drug-containing particles in controlled doses into and through
intact skin has been described. In particular, commonly owned U.S.
Pat. No. 5,630,796 to Bellhouse et al. describes a needleless
syringe that delivers pharmaceutical particles entrained in a
supersonic gas flow. The needleless syringe is used for transdermal
delivery of powdered drug compounds and compositions, for delivery
of genetic material into living cells (e.g., gene therapy) or
nucleic acid immunization, and for the delivery of
biopharmaceuticals to skin, muscle, blood or lymph. The needleless
syringe can also be used in conjunction with surgery to deliver
drugs and biologics to organ surfaces, solid tumors and/or to
surgical cavities (e.g., tumor beds or cavities after tumor
resection). In theory, practically any pharmaceutical agent that
can be prepared in a substantially solid, particulate form can be
safely and easily delivered using such devices.
[0006] One particular needleless syringe generally comprises an
elongate tubular nozzle having a rupturable membrane initially
closing the passage through the nozzle and arranged substantially
adjacent to the upstream end of the nozzle. Particles of a
therapeutic agent to be delivered are disposed adjacent to the
rupturable membrane and are delivered using an energizing means
which applies a gaseous pressure to the upstream side of the
membrane sufficient to burst the membrane and produce a supersonic
gas flow (entraining the pharmaceutical particles) through the
nozzle for delivery from the downstream end thereof. The particles
can thus be delivered from the needleless syringe at delivery
velocities as high as Mach 1 to Mach 8, which velocities are
readily obtainable upon the bursting of the rupturable
membrane.
[0007] Another needleless syringe configuration generally includes
the same elements as described above, except that instead of having
the pharmaceutical particles entrained within a gas flow, the
downstream end of the nozzle is provided with a diaphragm which is
moveable between a resting "inverted" position (in which the
diaphragm presents a concavity on the downstream face to contain
the pharmaceutical particles) and an "everted" position (in which
the diaphragm is outwardly convex on the downstream face as a
result of a supersonic shockwave having been applied to the
upstream face of the diaphragm). In this manner, the pharmaceutical
particles contained within the concavity of the diaphragm are
expelled at a high initial velocity from the device for transdermal
delivery thereof to a targeted tissue surface.
[0008] Transdermal delivery using the above-described needleless
syringe configurations is carried out with particles having an
approximate size that generally ranges between 0.1 and 250 .mu.m.
For drug delivery, a typical particle size is usually at least
about 10 to 15 .mu.m (the size of a typical cell). For gene
delivery, a typical particle size is generally substantially
smaller than 10 .mu.m. Particles larger than about 250 .mu.m can
also be delivered from the device, with the upper limitation being
the point at which the size of the particles would cause untoward
damage to the skin cells. The actual distance which the delivered
particles will penetrate depends upon particle size (e.g., the
nominal particle diameter assuming a roughly spherical particle
geometry), particle density, the initial velocity at which the
particle impacts the skin surface, and the density and kinematic
viscosity of the skin. In this regard, optimal particle densities
for use in needleless injection generally range between about 0.1
and 25 g/cm.sup.3, preferably between about 0.8 and 1.5 g/cm.sup.3,
and injection velocities generally range between about 150 and
3,000 m/sec.
[0009] A particularly unique feature of the needleless syringe is
the ability to optimize the depth of penetration of delivered
particles, thereby allowing for targeted administration of
pharmaceuticals to various sites. For example, particle
characteristics and/or device operating parameters can be selected
to provide for penetration depths for, inter alia, epidermal or
dermal delivery. One approach entails the selection of particle
size, particle density and initial velocity to provide a momentum
density (e.g., particle momentum divided by particle frontal area)
of between about 2 and 10 kg/sec/m, and more preferably between
about 4 and 7 kg/sec/m. Such control over momentum density allows
for tissue-selective delivery of the pharmaceutical particles.
[0010] Accordingly, there is a need to provide a reliable method
for preparing sufficiently dense particles (having a density of
about 0.8 to 1.5 g/cm.sup.3) which have an average size of about
0.1 to 150 .mu.m from a wide variety of pharmaceutical
compositions. These pharmaceutical particles can thus be
transdermally delivered to a subject using a needleless syringe
system.
[0011] Needleless syringes, such as those described above, also
provide a unique means for gene therapy and nucleic acid
immunization. These techniques provide for the transfer of a
desired gene into a subject with the subsequent in vivo expression
thereof. Gene transfer can be accomplished by transfecting the
subject's cells or tissues ex vivo and reintroducing the
transformed material into the host. Alternatively, genes can be
administered directly to the recipient.
[0012] A number of methods have been developed for gene delivery in
these contexts. For example, viral-based systems using, e.g.,
retrovirus, adenovirus, and adeno-associated viral vectors, have
been developed for gene delivery. However, these systems pose the
risk of delivery of replication-competent viruses. Hence, nonviral
methods for direct transfer of genes into recipient cells and
tissues are desirable.
[0013] Nonviral methods of gene transfer often rely on mechanisms
employed by mammalian cells for the uptake and intracellular
transport of macromolecules. For example, receptor-mediated methods
of gene transfer have been developed. The technique utilizes
complexes between plasmid DNA and polypeptide ligands that can be
recognized by cell surface receptors. However, data suggests that
this method may permit only transient expression of genes and thus
has only limited application.
[0014] Additionally, microinjection techniques have been developed
for the direct injection of genetic material into cells. The
technique, however, is laborious and requires single cell
manipulations. Thus, the method is inappropriate for use on a large
scale.
[0015] Direct injection of DNA-containing solutions into the
interstitial space for subsequent uptake by cells has also been
described. For example, International Publication No. WO 90/11092,
published 4 Oct. 1990, describes the delivery of isolated
polynucleotides to the interior of cells wherein the isolated
polynucleotides are delivered into the interstitial space of the
tissue and then taken up by individual cells to provide a
therapeutic effect. Such methods entail the injection of the
DNA-containing solutions into tissue using conventional needles or
cannulas, and are therefore not well suited for long term therapies
or for field or home applications.
[0016] Biolistic particle delivery systems (particle bombardment
systems) have also been developed for gene delivery into plant
cells. Such techniques use a "gene gun" to introduce DNA-coated
microparticles, such as DNA-coated metals, into cells at high
velocities. The coated metals (biolistic core carriers) are
generally propelled into cells using an explosive burst of an inert
gas such as helium. See, e.g., U.S. Pat. No. 5,100,792 to Sanford
et al. The technique allows for the direct, intracellular delivery
of small amounts of DNA.
[0017] Biolistic core carriers upon which the DNA is coated, such
as tungsten, gold, platinum, ferrite, polystyrene or latex, have to
date been needed to achieve adequate gene transfer frequency by
such direct injection techniques. See, e.g., International
Publication No. WO 94/23738, published Oct. 27, 1994. In
particular, these materials have been selected based on their
availability in defined particle sizes around 1 .mu.m in diameter,
as well as providing a sufficiently high density to achieve the
momentum required for cell wall or cell membrane penetration.
Additionally, common biolistic core carriers are chemically inert
to reduce the likelihood of explosive oxidation of fine
microprojectile powders, are non-reactive with DNA and other
components of the precipitating mixes, and display low toxicity to
target cells. See e.g., Particle Bombardment Technology for Gene
Transfer, (1994) Yang, N. ed., Oxford University Press, New York,
N.Y. pages 10-11.
[0018] However, such biolistic techniques are not appropriate for
use with large DNA molecules since precipitation of such molecules
onto core carriers can lead to unstable configurations which will
not withstand the shear forces of gene gun delivery.
[0019] Accordingly, there remains a need to provide a highly
efficient method for introducing therapeutically relevant DNA or
other nucleic acid molecules into mammalian tissue cells wherein
the method avoids the problems commonly encountered with prior gene
delivery techniques.
DISCLOSURE OF THE INVENTION
[0020] The present invention is based on the surprising discovery
that substantially solid particles of nucleic acid molecules having
a nominal average diameter of at least about 0.1 .mu.m, preferably
at least about 10 .mu.m (which are therefore larger than the
average mammalian cell), can be delivered into cells of mammalian
tissue without the need for biolistic core carriers. The result is
unexpected because it was heretofore believed that only small
DNA-coated core carrier particles, having an extremely high
particle density and a much smaller size than a typical mammalian
cell, could adequately be used as microprojectiles in biolistic
gene delivery techniques. See e.g., Particle Bombardment Technology
for Gene Transfer, (1994) Yang, N. ed., Oxford University Press,
New York, N.Y. pages 10-11.
[0021] In the practice of the invention, powdered nucleic acid
molecules are delivered using needleless injection techniques. In
particular, a novel delivery system that uses a needleless syringe
to fire solid particles of therapeutic agents in controlled doses
into and through intact skin has recently been described in
commonly owned U.S. Pat. No. 5,630,796. The patent describes a
needleless syringe that delivers pharmaceutical particles entrained
in a high velocity gas flow. The needleless syringe can be used for
transdermal delivery of powdered drug compounds and compositions,
for delivery of genetic material into living cells (e.g., gene
therapy) and for the delivery of biopharmaceuticals to skin,
muscle, blood or lymph. The needleless syringe can also be used in
conjunction with surgery to deliver drugs and biologics to organ
surfaces, solid tumors and/or to surgical cavities (e.g., tumor
beds or cavities after tumor resection)
[0022] Furthermore, the nucleic acids to be delivered can be
converted from non-dense pharmaceutical powders or particulate
formulations (e.g., those having particle densities below that
required for transdermal delivery from a needleless syringe) into
densified (compacted) particles that are optimally suited for
transdermal delivery using a needleless syringe. The method is
equally applicable to densification of pharmaceutical agents other
than nucleic acids.
[0023] Accordingly, in one embodiment, the invention is directed to
a method for delivering solid particles comprised of nucleic acid
molecules to mammalian tissue for the genetic transformation of
cells in the tissue with the delivered nucleic acids. In a
substantial departure from conventional particle bombardment
techniques, the nucleic acid particles transferred using the method
of the present invention are not delivered using biolistic core
carriers. Furthermore, the molecules can have a particle size that
is equal to or larger than the average mammalian cell size.
[0024] More particularly, densified particles comprised of selected
nucleic acid molecules and, optionally, suitable vehicles or
excipients, are prepared for delivery to mammalian tissue via a
needleless syringe which is capable of expelling the particles at
delivery velocities approaching Mach 1 to Mach 8 speeds. The
particles have an average size that is at least about 0.1 .mu.m,
wherein an optimal particle size is usually at least about 10 to 15
.mu.m (equal to or larger than the size of a typical mammalian
cell). However, nucleic acid particles having average particle
sizes of 250 .mu.m or greater can also be delivered using the
present method. The depth that the delivered particles will
penetrate the targeted tissue depends upon particle size (e.g., the
nominal particle diameter assuming a roughly spherical particle
geometry), particle density, the initial velocity at which the
particle impacts the tissue surface, and the density and kinematic
viscosity of the tissue. In this regard, optimal individual
particle densities (e.g., in contrast to bulk powder density) for
use in needleless injection generally range between about 0.1 and
25 g/cm.sup.3, and injection velocities generally range between
about 150 and 3,000 m/sec.
[0025] In various aspects of the invention, the above method can be
practiced in vivo to provide targeted delivery of the nucleic acid
particles to a target tissue, such as delivery to the epidermis
(for gene therapy applications) or to the stratum basal layer of
skin (for nucleic acid immunization applications). In these aspects
of the invention, particle characteristics and/or device operating
parameters are selected to provide optimal tissue-specific
delivery. One particular approach entails the selection of particle
size, particle density and initial velocity to provide a momentum
density (e.g., particle momentum divided by particle frontal area)
of between about 2 and 10 kg/sec/m, and more preferably between
about 4 and 7 kg/sec/m. Such control over momentum density allows
for precisely controlled, tissue-selective delivery of the nucleic
acid particles.
[0026] In other aspects of the invention, the needleless syringe is
used to transfect cells or tissues ex vivo with the particulate
nucleic acid molecules, wherein the transformed cells are
subsequently reintroduced into the host.
[0027] In another embodiment of the invention, a method is provided
for converting non-dense pharmaceutical powders or particulate
formulations (e.g., having density characteristics below that
required for transdermal delivery from a needleless syringe) into
densified (compacted) particles that are optimally suited for
transdermal delivery using a needleless syringe. Such particles
have an optimal particle density ranging from about 0.1 to about 25
g/cm.sup.3, preferably ranging from about 0.5 to about 3.0
g/cm.sup.3, and most preferably ranging from about 0.8 to about 1.5
g/cm.sup.3. The densified particles are processed to obtain optimal
particle sizes ranging from about 0.1 to about 250 .mu.m,
preferably ranging from about 0.1 to about 150 .mu.m, and most
preferably ranging from about 20 to about 60 .mu.m. The method
entails the compaction of a pharmaceutical composition using high
pressure and, optionally vacuum, to densify the composition. The
resulting compacted material is then size-reduced using
conventional methods to provide densified particles of optimized
size.
[0028] In a related embodiment of the invention, a method is
provided for optimizing the density and particle size of a
particulate pharmaceutical composition that has particle size and
density characteristics that fall within the above ranges. These
particles are rendered more suitable for needleless syringe
delivery using the above-described compaction and size-reduction
techniques. In this manner, the penetration depths that are
obtained when the optimized particles are delivered using a
needleless syringe can be adjusted to provide targeted dermal or
intra-dermal delivery.
[0029] In a further related embodiment, the invention pertains to a
method for optimizing the particle size and density of a
lyophilized or spray-dried biopharmaceutical composition. The
method entails the compaction of a lyophilized or spray-dried
pharmaceutical powder to obtain a densified material. The densified
material can then be reground to produce compositions in which the
individual particles approach the theoretical maximum density and
are thus optimal for delivery by impact with and penetration into
the target tissue at high velocities when delivered from a
needleless syringe. In a particular embodiment, lyophilized
recombinant human growth hormone (rhGH) powder is densified to
obtain particles in the range of about 20 to 50 .mu.m and having a
bulk density of about 0.8 to 1.5 g/cc.sup.3. The densified rhGH
particles are ideally suited for delivery from a needleless syringe
device.
[0030] In another embodiment, the invention is directed to a
compacted particulate pharmaceutical composition formed from a
porous pharmaceutical preparation. The compacted composition has an
average particle size in the range of 0.1 to 250 .mu.m mean
diameter, a particle density in the range of 0.1 to 25 g/cm.sup.3,
and a bulk density of at least about 0.5 g/cc.sup.3. Needleless
syringes comprising the compacted particulate pharmaceutical
preparation, as well as single-dose containers for a needleless
syringe comprising the same, are also provided.
[0031] In another embodiment, the invention is directed to a method
of delivering a selected pharmaceutical agent to a vertebrate
subject. The method comprises providing a densified (compacted)
particulate pharmaceutical preparation as described above and
delivering the preparation to a target tissue of the vertebrate
subject by needleless syringe.
[0032] In yet a further embodiment, the invention is directed to
particles of a suitable size and density for transdermal delivery
by needleless injection, consisting of a gene construct and an
excipient selected from the group consisting of pharmaceutical
grades of dextrose, sucrose, lactose, trehalose, mannitol,
sorbitol, inositol, erythritol, dextrans, cyclodextrans, starch,
cellulose, sodium or calcium phosphates, calcium sulfates, citric
acid, tartaric acid, glycine, albumin, gelatin, polyacrylates, high
molecular weight polyethylene glycols, and combinations
thereof.
[0033] These and other embodiments of the subject invention will
readily occur to those of skill in the art in light of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 is a pictorial representation of an ex vivo delivery
apparatus having a needleless syringe arranged over a tissue
culture plate containing cells to be transformed with the
particulate nucleic acid preparations described herein.
[0035] FIG. 2 is a histogram depicting transformation efficiencies
obtained using the apparatus of FIG. 1 to deliver DNA particles at
30 bar pressure over a 60 mm target distance as described in
Example 1. In the Figure, "B/P" refers to transformation efficiency
(expressed as the number of blue cells/per dish), "F#2" and "F#3"
refer to preparation 2 and preparation 3, respectively, "TCC"
refers to the contemporaneous delivery of DNA-coated tungsten
particles, and "THC" refers to a historical delivery of DNA-coated
tungsten particles.
[0036] FIG. 3 is a graph depicting the transformation efficiencies
obtained using the apparatus of FIG. 1 to deliver DNA particles at
30 bar pressure over a range of target distances, also as described
in Example 1. In the Figure, "B/P" refers to transformation
efficiency (expressed as the number of blue cells/per culture
dish), and "d(mm)" refers to target distance expressed in mm.
[0037] FIG. 4 depicts a comparison of the mean in vivo serum levels
of recombinant human growth hormone (rhGH) in animals that were
administered lyophilized rhGH powder by needleless injection
(.tangle-solidup.), densified rhGH particles (prepared by the
method of the invention) by needleless injection (.box-solid.), or
lyophilized rhGH powder by sub-cutaneous injection
(.circle-solid.)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of molecular biology and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory
Manual (1982); DNA Cloning: A Practical Approach, vol. I & II
(D. Glover, ed.); Perbal, A Practical Guide to Molecular
Cloning.
[0039] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
pharmaceutical formulations or process parameters as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments of
the invention only, and is not intended to be limiting.
[0040] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a nucleic acid molecule" includes
a mixture of two or more nucleic acid molecules, reference to "an
excipient" includes mixtures of two or more excipients, and the
like.
[0041] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
A. DEFINITIONS
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
following terms are intended to be defined as indicated below.
[0043] The term "transdermal" delivery captures both transdermal
(or "percutaneous") and transmucosal administration, i.e., delivery
by passage of a drug or pharmaceutical agent through the skin or
mucosal tissue. See, e.g., Transdermal Drug Delivery: Developmental
Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel
Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and
Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987);
and Transdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner
(eds.), CRC Press, (1987). Aspects of the invention which are
described herein in the context of "transdermal" delivery, unless
otherwise specified, are meant to apply to both transdermal and
transmucosal delivery. That is, the compositions, systems, and
methods of the invention, unless explicitly stated otherwise,
should be presumed to be equally applicable to transdermal and
transmucosal modes of delivery.
[0044] By "needleless syringe" is meant an instrument which
delivers a particulate composition transdermally, without a
conventional needle that pierces the skin. Needleless syringes for
use with the present invention are discussed throughout this
document.
[0045] As used herein, the term "drug" or "pharmaceutical agent"
intends any compound or composition of matter which, when
administered to an organism (human or animal) induces a desired
pharmacologic and/or physiologic effect by local and/or systemic
action. The term therefore encompasses those compounds or chemicals
traditionally regarded as drugs, vaccines, as well as
biopharmaceuticals including molecules such as peptides, hormones,
nucleic acids, gene constructs and the like. More particularly, the
term "drug" or "pharmaceutical agent" includes compounds or
compositions for use in all of the major therapeutic areas
including, but not limited to, anti-infectives such as antibiotics
and antiviral agents; analgesics and analgesic combinations; local
and general anesthetics; anorexics; antiarthritics; antiasthmatic
agents; anticonvulsants; antidepressants; antihistamines;
anti-inflammatory agents; antinauseants; antineoplastics;
antipruritics; antipsychotics; antipyretics; antispasmodics;
cardiovascular preparations (including calcium channel blockers,
beta-blockers, beta-agonists and antiarrythmics);
antihypertensives; diuretics; vasodilators; central nervous system
stimulants; cough and cold preparations; decongestants;
diagnostics; hormones; bone growth stimulants and bone resorption
inhibitors; immunosuppressives; muscle relaxants; psychostimulants;
sedatives; tranquilizers; proteins peptides and fragments thereof
(whether naturally occurring, chemically synthesized or
recombinantly produced); and nucleic acid molecules (polymeric
forms of two or more nucleotides, either ribonucleotides (RNA) or
deoxyribonucleotides (DNA) including both double- and
single-stranded molecules, gene constructs, expression vectors,
antisense molecules and the like).
[0046] The above drugs or pharmaceutical agents, alone or in
combination with other drugs or agents, are typically prepared as
pharmaceutical compositions which can contain one or more added
materials such as carriers, vehicles and/or excipients. The terms
"carriers," "vehicles" and "excipients" are used interchangeably
herein and generally refer to substantially inert materials which
are nontoxic and do not interact with other components of the
composition in a deleterious manner. However, these terms do not
encompass biolistic core carriers. The terms capture materials that
can be used to increase the amount of solids in particulate
pharmaceutical compositions, such as those prepared using
spray-drying or lyophilization techniques. Examples of suitable
carriers include water, silicone, gelatin, waxes, and like
materials. Examples of normally employed "excipients," include
pharmaceutical grades of dextrose, sucrose, lactose, trehalose,
erythritol, dextrans, cyclodextrans mannitol, sorbitol, inositol,
starch, cellulose, sodium or calcium phosphates, calcium sulfate,
citric acid, tartaric acid, glycine, albumin, gelatin,
polyacrylates high molecular weight polyethylene glycols (PEG), and
combinations thereof.
[0047] "Gene delivery" refers to methods or systems for reliably
inserting foreign DNA into host cells. Such methods can result in
expression of non-integrated transferred DNA, extrachromosomal
replication and expression of transferred replicons (e.g.,
episomes), or integration of transferred genetic material into the
genomic DNA of host cells.
[0048] The nucleotide sequences are generally present in a suitable
nucleic acid molecule and delivered in the form of vectors. By
"vector" is meant any genetic element, such as a plasmid, phage,
transposon, cosmid, chromosome, virus, virion, etc., which is
capable of replication when associated with the proper control
elements and which can transfer gene sequences between cells.
[0049] A "nucleotide sequence" or a "nucleic acid molecule" refers
to DNA and RNA sequences. The term captures molecules that include
any of the known base analogues of DNA and RNA such as, but not
limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycaronylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0050] A "coding sequence" or a sequence which "encodes" a
particular polypeptide, is a nucleic acid sequence which is
transcribed (in the case of DNA) and translated (in the case of
mRNA) into a polypeptide in vitro or in vivo when placed under the
control of appropriate regulatory sequences. The boundaries of the
coding sequence are conventionally determined by a start codon at
the 5' (amino) terminus and a translation stop codon at the 3'
(carboxy) terminus. A coding sequence can include, but is not
limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA
sequences from procaryotic or eukaryotic DNA, and even synthetic
DNA sequences. A transcription termination sequence will usually be
located 3' to the coding sequence.
[0051] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites ("IRES"), enhancers, and
the like, which collectively provide for the replication,
transcription and translation of a coding sequence in a recipient
cell. Not all of these control sequences need always be present so
long as the selected gene is capable of being replicated,
transcribed and translated in an appropriate recipient cell.
[0052] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0053] By "isolated" when referring to a nucleotide sequence, or a
nucleic acid molecule containing the nucleotide sequence, is meant
that the indicated molecule is present in the substantial absence
of other biological macromolecules of the same type. Thus, an
"isolated nucleic acid molecule which encodes a particular
polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that no not
encode the subject polypeptide; however, the molecule may include
some additional bases or moieties which do not deleteriously affect
the basic characteristics of the composition.
[0054] The term "transfection" is used to refer to the uptake of
foreign DNA by a host cell, and a host cell has been "transformed"
as a result of having been transfected. The foreign DNA may or may
not be integrated (covalently linked) to chromosomal DNA making up
the genome of the cell. By "host cell," or "host mammalian cell" is
meant a cell which has been transfected, or is capable of being
transfected, by a nucleic acid molecule containing a nucleotide
sequence of interest. The term includes the progeny of the parent
cell, whether or not the progeny is identical in morphology or in
genetic make-up to the original parent, so long as the nucleotide
sequence of interest is present within the cell.
[0055] By "biolistic core carrier" is meant a carrier on which a
nucleic acid (e.g., DNA) is coated in order to impart a defined
particle size as well as a sufficiently high density to achieve the
momentum required for cell wall penetration, such that the DNA can
be delivered using biolistic techniques, such as by use of a gene
gun (see, e.g., U.S. Pat. No. 5,100,792). Biolistic core carriers
typically include dense solids such as tungsten, gold, platinum,
ferrite, polystyrene and latex. See e.g., Particle Bombardment
Technology for Gene Transfer, (1994) Yang, N. ed., Oxford
University Press, New York, N.Y. pages 10-11.
[0056] By "vertebrate subject" is meant any member of the subphylum
cordata, particularly mammals, including, without limitation,
humans and other primates. The term does not denote a particular
age. Thus, both adult and newborn individuals are intended to be
covered.
B. MODES OF CARRYING OUT THE INVENTION
[0057] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0058] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0059] As explained above, the present invention allows for the
highly efficient delivery of solid particles of nucleic acid
molecules having a nominal average diameter of at least about 0.1
.mu.m, preferably at least about 10 .mu.m, to mammalian tissues.
The method utilizes biolistic gene transfer techniques yet
surprisingly allows for the delivery of nucleic acid molecules
without the need for biolistic core carriers.
[0060] A wide variety of nucleic acid molecules can be delivered
using the methods of the invention. Generally, the molecules
contain coding regions with suitable control sequences or other
therapeutically relevant nucleotide sequences. The nucleic acid
molecules are prepared in the form of vectors which include the
necessary elements to direct transcription and translation in a
host cell. If expression is desired using the host's enzymes (such
as by the use of endogenous RNA polymerase) , the gene or genes
will be present in the vectors operatively linked to control
sequences recognized by the particular host, or even particular
cells within the host. Thus, eucaryotic and phage control elements
will be present for expression in mammalian hosts. Such sequences
are known in the art and are discussed more fully below.
[0061] Suitable nucleotide sequences for use in the delivery
methods of the present invention include any therapeutically
relevant nucleotide sequence. Thus, the present invention can be
used to deliver one or more genes encoding a protein defective or
missing from a target cell genome or one or more genes that encode
a non-native protein having a desired biological or therapeutic
effect (e.g., an antiviral function). The invention can also be
used to deliver a nucleotide sequence capable of providing
immunity, for example an immunogenic sequence that serves to elicit
a humoral and/or cellular response in a subject, or a sequence that
corresponds to a molecule having an antisense or ribozyme
function.
[0062] Suitable genes which can be delivered include those used for
the treatment of inflammatory diseases, autoimmune, chronic and
infectious diseases, including such disorders as AIDS, cancer,
neurological diseases, cardiovascular disease, hypercholestemia;
various blood disorders including various anemias, thalassemia and
hemophilia; genetic defects such as cystic fibrosis, Gaucher's
Disease, adenosine deaminase (ADA) deficiency, emphysema, etc. A
number of antisense oligonucleotides (e.g., short oligonucleotides
complementary to sequences around the translational initiation site
(AUG codon) of an mRNA) that are useful in antisense therapy for
cancer and for viral diseases have been described in the art. See,
e.g., Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:4313; Uhlmann
et al. (1990) Chem. Rev. 90:543; Helene et al. (1990) Biochim.
Biophys. Acta. 1049:99 Agarwal et al. (1988) Proc. Natl. Acad. Sci.
USA 85:7079; and Heikkila et al. (1987) Nature 328:445. A number of
ribozymes suitable for use herein have also been described. See,
e.g., Cech et al. (1992) J. Biol. Chem. 267:17479 and U.S. Pat. No.
5,225,347 to Goldberg et al.
[0063] For example, in methods for the treatment of solid tumors,
genes encoding toxic peptides (e.g., chemotherapeutic agents such
as ricin, diphtheria toxin and cobra venom factor), tumor
suppressor genes such as p53, genes coding for mRNA sequences which
are antisense to transforming oncogenes, antineoplastic peptides
such as tumor necrosis factor (TNF) and other cytokines, or
transdominant negative mutants of transforming oncogenes, can be
delivered for expression at or near the tumor site.
[0064] Similarly, genes coding for peptides known to display
antiviral and/or antibacterial activity, or stimulate the host's
immune system, can also be administered. Thus, genes encoding many
of the various cytokines (or functional fragments thereof), such as
the interleukins, interferons, and colony stimulating factors, will
find use with the instant invention. The gene sequences for a
number of these substances are known.
[0065] For the treatment of genetic disorders, functional genes
corresponding to genes known to be deficient in the particular
disorder can be administered to the subject. The instant methods
will also find use in antisense therapy, e.g., for the delivery of
oligonucleotides able to hybridize to specific complementary
sequences thereby inhibiting the transcription and/or translation
of these sequences. Thus, DNA or RNA coding for proteins necessary
for the progress of a particular disease can be targeted, thereby
disrupting the disease process. Antisense therapy, and numerous
oligonucleotides which are capable of binding specifically and
predictably to certain nucleic acid target sequences in order to
inhibit or modulate the expression of disease-causing genes are
known and readily available to the skilled practitioner. Uhlmann et
al. (1990) Chem. Rev. 90:343, Neckers et al (1992) Crit. Rev.
Oncogenesis 3:175; Simons et al. (1992) Nature 359:67; Bayever et
al. (1992) Antisense Res. Dev. 2:109; Whitesell et al. (1991)
Antisense Res. Dev. 1:343; Cook et al. (1991) Anti-Cancer Drug
Design 6:58S; Eguchi et al. (1991) Annu. Rev. Biochem. 60:631.
Accordingly, antisense oligonucleotides capable of selectively
binding to target sequences in host cells are provided herein for
use in antisense therapeutics.
[0066] For nucleic acid immunizations, antigen-encoding expression
vectors can be delivered to a subject for the purpose of eliciting
humoral and/or cellular immune responses to antigens encoded by the
vector. In particular, humoral, cytotoxic cellular and protective
immune responses elicited by direct intramuscular injection of
antigen-encoding DNAs have been described. Tang et al. (1992)
Nature 358:152; Davis et al. (1993) Hum. Molec. Genet. 2:1847;
Ulmer et al. (1993) Science 258:1745; Wang et al. (1993) Proc.
Natl. Acad. Sci. USA 90:4156; Eisenbraun et al. (1993) DNA Cell
Biol. 12:791; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA
90:12476; Fuller et al. (1994) AIDS Res. Human Retrovir. 10:1433;
and Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519. In
addition, these immune responses have also been elicited using
biolistic techniques. See, e.g., EP 0500799.
[0067] Isolation of Genes and Construction of Vectors:
[0068] Nucleotide sequences selected for use in the present
invention can be derived from known sources, for example, by
isolating the same from cells containing a desired gene or
nucleotide sequence using standard techniques. Similarly, the
nucleotide sequences can be generated synthetically using standard
modes of polynucleotide synthesis that are well known in the art.
See, e.g., Edge et al. (1981) Nature 292:756; Nambair et al. (1984)
Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.
Generally, synthetic oligonucleotides can be prepared by either the
phosphotriester method as described by Edge et al. (supra) and
Duckworth et al. (1981) Nucleic Acids Res. 9:1691, or the
phosphoramidite method as described by Beaucage et al. (1981) Tet.
Letts. 22:1859, and Matteucci et al. (1981) J. Am. Chem. Soc.
103:3185. Synthetic oligonucleotides can also be prepared using
commercially available automated oligonucleotide synthesizers. The
nucleotide sequences can thus be designed with appropriate codons
for a particular amino acid sequence. In general, one will select
preferred codons for expression in the intended host. The complete
sequence is assembled from overlapping oligonucleotides prepared by
standard methods and assembled into a complete coding sequence.
See, e.g., Edge et al. (supra); Nambair et al. (supra) and Jay et
al. (supra).
[0069] A particularly convenient method for obtaining nucleic acid
sequences for use herein is by recombinant means. Thus, a desired
nucleotide sequence can be excised from a plasmid carrying the same
using standard restriction enzymes and procedures. Site specific
DNA cleavage is performed by treating with the suitable restriction
enzyme (or enzymes) under conditions which are generally understood
in the art, and the particulars of which are specified by
manufacturers of commercially available restriction enzymes. If
desired, size separation of the cleaved fragments may be performed
by polyacrylamide gel or agarose gel electrophoresis using standard
techniques.
[0070] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) using
standard techniques. The Klenow fragment fills in at 5'
single-stranded overhangs but digests protruding 3' single strands,
even though the four dNTPs are present. If desired, selective
repair can be performed by supplying only one, or several, selected
dNTPs within the limitations dictated by the nature of the
overhang. After Klenow treatment, the mixture can be extracted with
e.g. phenol/chloroform, and ethanol precipitated. Treatment under
appropriate conditions with 51 nuclease or BAL-31 results in
hydrolysis of any single-stranded portion.
[0071] PCR techniques can also be used in order to obtain a nucleic
acid molecule of interest. Generally, the technique involves
amplification of sequences from a human genomic or cDNA library.
Degenerate or nondegenerate oligonucleotide primers for PCR may be
prepared based on known amino acid sequences or on sequences of
homologous genes. The products of such PCR reactions may be
selected according to size by gel electrophoresis. Such PCR methods
are described in e.g., U.S. Pat. Nos. 4,965,188; 4,800,159;
4,683,202; 4,683,195.
[0072] Once coding sequences for desired peptides or proteins have
been prepared or isolated, such sequences can be cloned into any
suitable vector or replicon. Numerous cloning vectors are known to
those of skill in the art, and the selection of an appropriate
cloning vector is a matter of choice. Ligations to other sequences
are performed using standard procedures, known in the art.
[0073] Selected nucleotide sequences can be placed under the
control of regulatory sequences such as a promoter, ribosome
binding site and, optionally, an operator (collectively referred to
herein as "control" elements), so that the sequence encoding the
desired protein is transcribed into RNA in the host tissue
transformed by a vector containing this expression construct. The
coding sequence may or may not contain a signal peptide or leader
sequence.
[0074] The choice of control elements will depend on the host being
transformed and the type of preparation used. Thus, if the host's
endogenous transcription and translation machinery will be used to
express the proteins, control elements compatible with the
particular host will be utilized. In this regard, several promoters
for use in mammalian systems are known in the art and include, but
are not limited to, promoters derived from SV40, CMV, HSV, RSV,
MMTV, T7, T3, among others. Similarly, promoters useful with
procaryotic enzymes are known and include the tac, spa, trp,
trp-lac .lambda.-p.sub.L, T7, phoA promoters, as well as
others.
[0075] In addition to control sequences, it may be desirable to add
regulatory sequences which allow for regulation of the expression
of protein sequences encoded by the delivered nucleotide sequences.
Regulatory sequences are known to those of skill in the art, and
examples include those which cause the expression of a coding
sequence to be turned on or off in response to a chemical or
physical stimulus, including the presence of a regulatory compound.
Other types of regulatory elements may also be present in the
vector, for example, enhancer sequences.
[0076] An expression vector is constructed so that the particular
coding sequence is located in the vector with the appropriate
control and, optionally, regulatory sequences such that the
positioning and orientation of the coding sequence with respect to
the control sequences allows the coding sequence to be transcribed
under the "control" of the control sequences (i.e., RNA polymerase
which binds to the DNA molecule at the control sequences
transcribes the coding sequence). Modification of the sequences
encoding the particular protein of interest may be desirable to
achieve this end. For example, in some cases it may be necessary to
modify the sequence so that it is attached to the control sequences
with the appropriate orientation; i.e., to maintain the reading
frame. The control sequences and other regulatory sequences may be
ligated to the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site.
[0077] Preparation of Particulate Molecules:
[0078] Once obtained and/or constructed, the nucleic acid molecules
are prepared for delivery in particulate form. For example,
particulate molecules can be produced using particle formation
techniques well known in the art, such as but not limited to
spray-drying, spray-coating, freeze-drying (lyophilization) and
super critical fluid precipitation.
[0079] In one embodiment, the invention entails a procedure for
forming dense particles from low density particulate pharmaceutical
preparations. In particular, manufacturing processes for preparing
pharmaceutical particles from delicate molecules such as DNA,
proteins or peptides generally result in low density particles
having either a hollow spherical or open lattice monolithic
structure. Such particles are poorly suited for use in needleless
syringe delivery systems, wherein the particles must have
sufficient physical strength to withstand sudden acceleration to
velocities approaching the speed of sound and the impact with, and
passage through, the skin and tissue.
[0080] One common method of preparing particulate
biopharmaceuticals, such as nucleic acids, is lyophilization
(freeze-drying). Lyophilization relates to a technique for removing
moisture from a material and involves rapid freezing at a very low
temperature, followed by rapid dehydration by sublimation in a high
vacuum. This technique typically yields low-density porous
particles having an open matrix structure. Such particles are
chemically stable, but are rapidly reconstituted (disintegrated
and/or brought into solution) when introduced into an aqueous
environment.
[0081] Another method of providing particulate preparations that
can be used with these and other delicate or heat-sensitive
biomolecules is spray-drying. Spray-drying relates to the
atomization of a solution of one or more solids using a nozzle,
spinning disk or other device, followed by evaporation of the
solvent from the droplets. More particularly, spray-drying involves
combining a highly dispersed liquid preparation (e.g., a solution,
slurry, emulsion or the like) with a suitable volume of hot air to
produce evaporation and drying of the liquid droplets. Spray-dried
pharmaceuticals are generally characterized as homogenous spherical
particles that are frequently hollow. Such particles have low
density and exhibit a rapid rate of solution.
[0082] The low-density particulate solids produced by
lyophilization and spray-drying techniques are ideal for
redissolution for parenteral administration in solution via syringe
or catheters. However, such particles are not useful for delivery
from a needleless syringe in a solid form. Accordingly, for
purposes of the present method, the preparations are densified to
provide particles including nucleic acid molecules that are much
better suited for delivery using a needleless syringe (e.g.,
substantially solid particles having a size of about 50 .mu.m and a
density of at least about 0.9 to 1.5 g/cc.sup.3). In particular,
the open lattice or hollow shell particles provided by spray-drying
or lyophilization can be condensed without heating or shear to
provide dense materials that can be milled or otherwise
size-reduced to yield pharmaceutical particles having both size and
density characteristics suitable for delivery by needleless
injection.
[0083] The nucleic acids for delivery by the method of the
invention, may be initially prepared in a formulation suitable for
spray-drying or lyophilization. Such formulations generally require
only a solution in which the nucleic acids will be stable for
freezing and lyophilization and, optionally, an excipient for the
drying procedure which is acceptable for parenteral delivery. In
this regard, suitable excipients may be added to the formulations
to provide sufficient mass for an individual dose, enabling
measurement of doses by practical processes, e.g., by weight or
volume. Typical dosages can be about 0.5 to about 5 mg, preferably
about 1 to about 2 mg. Suitable excipients include, but are not
limited to, carbohydrates (such as trehalose, glucose, dextrose and
sucrose) or polyols (such as mannitol). Amino acids such as glycine
and its hydrochloride salt can be used as buffers as well as
phosphate, lactate or citrate buffers, among others. Additionally,
any known composition for DNA stabilization will find use in the
present formulations. The compositions may optionally include
additive agents such as cryoprotectants, antioxidants, or the like.
Adjusting compositions to enhance physical and chemical stability
of the various particulate nucleic acid formulations provided
herein is within the ordinary skill in the art.
[0084] One particular approach to stabilization during reprocessing
of the nucleic acid formulations entails the use of additives which
are combined with the solution prior to freezing for lyophilization
to cause the nucleic acids to coil or ball and thus provide the
genetic material as a discontinuous phase in the otherwise
microscopically homogeneous particles. In such formulations, the
bulking agent would be the continuous phase in the dried solid so
that any grinding prior to compression, compression densification
and regrinding (as described in detail below), and any particle
attrition during sizing via sieve or air classification,
acceleration and injection, would be less likely to disrupt the
long chain nucleic acids. Homogeneity of the particles with respect
to nucleic acid content is critical because of the potential for
segregation by size during storage or injection.
[0085] Condensing the nucleic acid powders can be conducted by
compaction in a suitable press (e.g., a hydraulic press, tableting
press or rotary press), wherein the powders are compressed at about
1,000 to 24,000 pounds/square inch (e.g., 0.5 to 12 tons/square
inch or 7 to 170 MPa) for a suitable time. Compaction can be
carried out under vacuum if desired. The resulting compacted
material is then coarsely reground until visually broken up. The
particle size is then reduced to about a 20 to 50 .mu.m average
size with an optimal bulk particle density of around 0.9 to 1.5
g/cm.sup.3, or as close to absolute or theoretical density as
possible. Particle size reduction can be conducted using methods
well known in the art including, but not limited to, roller
milling, ball milling, hammer, air or impact milling, attrition
milling, sieving, sonicating, or combinations thereof. The
compression parameters and particle sizing will, of course, vary
depending upon the starting material used, the desired target
particle size and density, and like considerations.
[0086] Particle density can be ascertained using helium pycnometry
to treasure absolute density and various techniques to establish
porosity such as mercury intrusion BET, flotation in a density
gradient, and the like. These techniques are all well known in the
art.
[0087] Thus, the method can be used to obtain nucleic acid
particles having a size ranging from about 10 to about 250 .mu.m,
preferably about 10 to about 150 .mu.m, and most preferably about
20 to about 60 .mu.m; and a particle density ranging from about 0.1
to about 25 g/cm.sup.3, and a bulk density of about 0.5 to about
3.0 g/cm.sup.3, or greater.
[0088] A particularly preferred method for providing nucleic acid
molecules suitable for biolistic delivery is the novel
densification/compaction technique described herein. The technique
is useful not only for preparing nucleic acid biopharmaceuticals,
but can be used to prepare almost any desired physiologically
active composition for needleless delivery.
[0089] Accordingly, in another embodiment of the invention, a
method is provided for densifying (compacting) a non-nucleic acid
pharmaceutical preparation.
[0090] As explained above, current manufacturing processes for
preparing pharmaceutical particles from delicate molecules such as
proteins or peptides are poorly suited for use in needleless
syringe delivery systems. For example, as discussed above,
lyophilization typically yields low-density porous particles having
an open matrix structure. Exemplary biopharmaceuticals available as
lyophilized particles include recombinant human growth factor
(e.g., Genotropin.RTM., Pharmacia, Piscataway, N.J.); somatrem
(e.g., Protropin.RTM., Genentech, S. San Francisco, Calif.);
somatropin (e.g., Humatrope.RTM., Eli Lilly, Indianapolis, Ind.);
recombinant interferon .alpha.-2a (e.g., Roferon.RTM., Hoffman-La
Roche, Nutley, N.J.) recombinant interferon .alpha.-2b (e.g.,
Intron A.RTM., Schering-Plough, Madison, N.J.); and recombinant
alteplase (e.g., Activase.RTM., Genentech, S. San Francisco,
Calif.).
[0091] In addition, spray-dried pharmaceuticals are generally
characterized as homogenous spherical particles that are frequently
hollow. Such particles have low density and exhibit a rapid rate of
solution. Exemplary heat-sensitive pharmaceuticals that are
prepared using spray-drying techniques include the amino acids;
antibiotics such as aureomycin, bacitracin, penicillin and
streptomycin; ascorbic acid; cascara extracts; pepsin and similar
enzymes; protein hydrolysates; and thiamine.
[0092] When spray-dried and lyophilized pharmaceutical particles
are ground or milled, they yield very small, light and non-dense
particles that are poorly suited for delivery through skin or
mucosal tissues. In particular, such particles, when delivered from
a needleless syringe, are often too light to have the momentum
necessary to penetrate intact skin (e.g., pass through the stratum
corneum) and would thus fail to enter the systemic circulation. In
this regard, the stratum corneum is a thin layer of dense packed,
highly keratinized cells, generally about 10-15 .mu.m thick and
which covers most of the human body. The stratum corneum thus
provides the primary skin barrier which a transdermally-delivered
particle must cross.
[0093] Accordingly, the present method entails densifying
(compacting) such preparations to provide particles that are much
better suited for delivery from a needleless syringe (e.g.,
substantially solid particles having a size of about 50 .mu.m and a
bulk density of at least about 0.5 to 1.5 g/cc.sup.3). In
particular, open lattice or hollow shell particles provided by
spray-drying or lyophilization can be condensed without heating or
shear to provide dense, compacted materials that can be milled or
otherwise size-reduced to yield pharmaceutical particles having
both size and density characteristics suitable for delivery by
needleless injection.
[0094] Condensing of the spray-dried or lyophilized powders is
typically conducted by compaction in a suitable press (e.g., a
hydraulic press, tableting press or rotary press), wherein the
powders are compressed at about 1,000 to 24,000 pounds/square inch
(0.5 to 12 tons/square inch) for a suitable time. This compaction
can be carried out under vacuum if desired. The resulting compacted
material is then coarsely reground until visually broken up. The
particle size is then reduced to about a 20 to 50 .mu.m average
size to yield a bulk density of around 0.5 to 1.5 g/cc.sup.3 (with
a particle density of about 0.1 to 25 g/cm.sup.3). Particle size
reduction can be conducted using methods well known in the art
including, but not limited to, roller milling, ball milling, hammer
or impact milling, attrition milling, sieving, sonicating, or
combinations thereof. The compression parameters and particle
sizing will, of course, vary depending upon the starting material
used, the desired target particle size and density, and like
considerations. The starting material can be any pharmaceutical
preparation having a particle size and density which one is
desirous of changing to obtain more optimal size and density
characteristics for use in needleless syringes.
[0095] Following densification, particles of suitable size can be
selected and classified using standard techniques, known in the
art, such as by vibratory, sonic or jet sieving, cyclone
separation, or like techniques, well known in the art.
[0096] Actual particle density, or "absolute density," can be
readily ascertained using known quantification techniques such as
helium pycnometry and the like.
[0097] Alternatively, envelope density measurements can be used to
assess suitable densification of the particulate pharmaceutical
compositions. Envelope density information is useful in
characterizing the density of porous objects of irregular size and
shape. Envelope density, or "bulk density," is the mass of an
object divided by its volume, where the volume includes that of its
pores and small cavities. A number of methods of determining
envelope density are known in the art, including wax immersion,
mercury displacement, water absorption and apparent specific
gravity techniques. A number of suitable devices are also available
for determining envelope density, for example, the Geopyc.TM. Model
1360, available from the Micromeritics Instrument Corp. The
difference between the absolute density and envelope density of a
sample pharmaceutical composition provides information about the
sample's percentage total porosity and specific pore volume. In the
practice of the invention, compaction of porous particulate
pharmaceutical compositions will generally result in a reduction of
porosity, and a concomitant increase in bulk (envelope)
density.
[0098] Thus, the method can be used to obtain particles having a
size ranging from about 0.1 to about 250 .mu.m, preferably about
0.1 to about 150 .mu.m, and most preferably about 20 to about 60
.mu.m; a particle density ranging from about 0.1 to about 25
g/cm.sup.3, and a bulk density of preferably about 0.5 to about 3.0
g/cm.sup.3, and most preferably about 0.8 to about 1.5
g/cm.sup.3.
[0099] The above-described method can also be used to optimize the
density and particle size of a particulate pharmaceutical
composition that has particle size and density characteristics that
fall within the above ranges. In this manner, the penetration
depths that are obtained when the optimized particles are delivered
at high velocities using a needleless syringe can be adjusted to
optimize targeted dermal or intra-dermal delivery.
[0100] However, as noted hereinabove, the invention is particularly
suited for preparing densified particles having optimized density
from heat-sensitive biopharmaceutical preparations of peptides,
polypeptides, proteins, nucleic acids and other such biological
molecules. Exemplary peptide and protein formulations which can be
densified using the instant method include, without limitation,
insulin; calcitonin; octreotide; endorphin; liprecin; pituitary
hormones (e.g., human growth hormone and recombinant human growth
hormone (hGH and rhGH), HMG, desmopressin acetate, etc); follicle
luteoids; growth factors (such as growth factor releasing factor
(GFRF), somatostatin, somatotropin and platelet-derived growth
factor); asparaginase; chorionic gonadotropin; corticotropin
(ACTH); erythropoietin (EPO); epoprostenol (platelet aggregation
inhibitor); glucagon; interferons; interleukins; menotropins
(urofollitropin follicle-stimulating hormone (FSH) and luteinizing
hormone (LH)); oxytocin; streptokinase; tissue plasminogen
activator (TPA); urokinase; vasopressin; desmopressin; ACTH
analogues; angiotensin II antagonists; antidiuretic hormone
agonists; bradykinin antagonists; CD4 molecules; antibody molecules
and antibody fragments (e.g., Fab, Fab.sub.2, Fv and sFv
molecules); IGF-1; neurotrophic factors; colony stimulating
factors; parathyroid hormone and agonists; parathyroid hormone
antagonists; prostaglandin antagonists; protein C; protein S; renin
inhibitors; thrombolytics; tumor necrosis factor (TNF); vaccines
(particularly peptide vaccines including subunit and synthetic
peptide preparations); vasopressin antagonists analogues; and
.alpha.-1 antitrypsin. Exemplary nucleic acid molecules are
detailed above.
[0101] Administration of the Particles:
[0102] Following formation, the particulate preparations are
delivered to mammalian tissue using a needleless syringe. The
compacted particles of the present invention can be packaged in
individual unit dosages. As used herein, a "unit dosage" intends a
dosage receptacle containing a therapeutically effective amount of
a pharmaceutical powder produced according to the methods of the
present invention. The dosage receptacle is generally one which
fits within a needleless syringe device to allow for transdermal
delivery from the device. Such receptacles can take the form of
capsules, foil pouches, sachets, cassettes, and the like.
Appropriate needleless syringes are described herein above.
[0103] Transdermal delivery from these various needleless syringe
configurations is carried out with particles having an approximate
size that generally ranges between 0.1 and 250 .mu.m. However, the
optimal particle size is usually at least about 10 to 15 .mu.m (the
size of a typical cell). Particles larger than about 250 .mu.m can
also be delivered from the device, with the upper limitation being
the point at which the size of the particles would cause untoward
damage to the skin. Other particulate biopharmaceuticals, such as
peptide and protein preparations, will generally have an
approximate size of 0.1 to about 250 .mu.m, preferably about 0.1 to
about 150 .mu.m, and most preferably about 20 to about 60
.mu.m.
[0104] The actual distance which the delivered particles will
penetrate depends upon particle size (e.g., the nominal particle
diameter assuming a roughly spherical particle geometry), particle
density, the initial velocity at which the particle impacts the
skin surface, and the density and kinematic viscosity of the skin.
In this regard, optimal particle densities for use in needleless
injection generally range between about 0.1 and 25 g/cm.sup.3, and
optimal bulk densities range from about 0.5 and 3.0 g/cm.sup.3.
Injection velocities generally range between about 100 and 3,000
m/sec.
[0105] Compositions containing a therapeutically effective amount
of the powdered molecules described herein can be delivered to any
suitable target tissue via the above-described needleless syringes.
For example, the compositions can be delivered to muscle, skin,
brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,
blood, bone cartilage, pancreas, kidney, gall bladder, stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye,
gland and connective tissues. For nucleic acid molecules, delivery
is preferably to, and the molecules expressed in, terminally
differentiated cells; however, the molecules can also be delivered
to non-differentiated, or partially differentiated cells such as
stem cells of blood and skin fibroblasts.
[0106] The powdered compositions are administered to the subject to
be treated in a manner compatible with the dosage formulation, and
in an amount that will be prophylactically and/or therapeutically
effective. The amount of the composition to be delivered, generally
in the range of from 0.5 .mu.g/kg to 100 .mu.g/kg of nucleic acid
molecule per dose, depends on the subject to be treated. Doses for
other pharmaceuticals, such as physiological active peptides and
proteins, generally range from about 0.1 .mu.g to about 20 mg,
preferably 10 .mu.g to about 3 mg. The exact amount necessary will
vary depending on the age and general condition of the individual
to be treated, the severity of the condition being treated, the
particular preparation delivered, the site of administration, as
well as other factors. An appropriate effective amount can be
readily determined by one of skill in the art.
[0107] Thus, a "therapeutically effective amount" of the present
particulate compositions will be sufficient to bring about
treatment or prevention of disease or condition symptoms, and will
fall in a relatively broad range that can be determined through
routine trials.
C. EXPERIMENTAL
[0108] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0109] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
EXAMPLE 1
[0110] The following experiment was conducted to investigate the
possibility of using freeze-dried DNA as an alternative to
DNA-coated metal particles in the biolistic transfer of genetic
material. In particular, powdered DNA plasmids as well as
DNA-coated tungsten particles as controls were delivered ex vivo to
male human fibroblast HT1080 cells using a needleless syringe
apparatus as follows.
[0111] Clone 123 is a small plasmid of .about.11 kb which contains
the .beta.-galactosidase marker gene so that transient
transformation can be measured with the chromogenic indicator
X-Gal. Plasmids were bulked with a carbohydrate excipient,
trehalose. Trehalose was selected as the excipient because of its
stabilizing properties (Colaco et al. (1992) Bio/Technology
10:1009). The trehalose was dissolved in distilled water and
filter-sterilized prior to adding the DNA to the solution. Three
different solutions of DNA sugar were made up with the proportions
shown below in Table 1.
1TABLE 1 Preparation 1 2 3 Clone 123 800 .mu.g 160 .mu.g 80 .mu.g
(2.7 .mu.g/.mu.L) (296.3 .mu.L) (59.3 .mu.L) (29.6 .mu.L) Trehalose
10 mg 10 mg 10 mg (100 mg/15 ml H.sub.2O) (1.5 ml) (1.5 ml) (1.5
ml) Payload 0.1 mg 0.5 mg 1.0 mg (for 8 .mu.g DNA)
[0112] The plasmid/sugar solutions were then freeze-dried (using
solid CO.sub.2 and isopropanol to freeze the solution prior to
vacuum drying), and the freeze-dried DNA-trehalose trehalose solid
milled to form microparticles using an agate mortar and pestle.
[0113] As a control, DNA-coated tungsten particles were made by
coating tungsten microprojectiles (19.35.times.10.sup.3
kb.m.sup.-3) of 1.014 .mu.m median diameter (M-17, GTE/Sylvania,
Towanda, Pa., USA) with forty micrograms of Clone 123 plasmid DNA,
giving five payloads of 8 .mu.g DNA, using a derivative of known
methods for coating microparticles. Potter et al. (1984) Proc.
Natl. Acad. Sci. USA 81:7161, Klein et al. (1987) Nature 327:70,
and Williams et al. (1991) Proc. Natl. Acad Sci. USA 88:2726.
[0114] More particularly, prior to coating, the tungsten particles
were sterilized and brought into suspension. A 50 mg sample of
1.048 .mu.m (median diameter) tungsten microprojectiles (M-17,
GTE/Sylvania, Towanda, Pa., USA) was weighed into a 1.5 cc
Eppendorf tube and then sterilized in 100% ethanol (EtOH). In order
to disperse the microprojectiles (disrupt particle aggregates), the
sterilized solution was sonicated thoroughly by contacting the
outside of the Eppendorf tube with the probe of a sonicator. The
dispersed tungsten particles were centrifuged and the supernatant
removed. The tungsten particles were resuspended in 1 cc sterile
distilled water and centrifuged for two cycles, and then stored in
1 cc sterile distilled water until coating.
[0115] The plasmid DNA was absorbed to the tungsten
microprojectiles by adding 20 .mu.L of the DNA (1 mg/mL) to 40
.mu.L of the suspension of tungsten particles prepared above. The
suspension was vortexed to ensure adequate mixing of the reagents.
The following reagents were then added, in the order given, with
vortexing after each addition: 253 .mu.L CaCl.sub.2 (2.5 M); 50
.mu.L spermidine (0.10 M, stored frozen); and 207 .mu.L sterile
distilled H.sub.2O. The final mixture was vortexed for 10 minutes
at 4.degree. C. The DNA-coated tungsten microprojectiles were then
centrifuged at 500 G for 5 minutes. After centrifugation, all
supernatant was carefully removed and 100 .mu.L of 70% EtOH added.
The coated particles were again centrifuged, all supernatant
removed, and the final preparation resuspended in 30 .mu.L of 100%
EtOH.
[0116] The above method resulted in suitable quantities of
DNA-coated tungsten particles to allow for 4 to 5 deliveries by the
needleless syringe. The above-noted reagent quantities can, of
course, be varied to provide different loadings of DNA in
accordance with known methods. The volume and molar concentration
(M) of the stock solutions used to coat tungsten with DNA are given
below in Table 2.
2 TABLE 2 Component Quantity (.mu.L) Tungsten particles (50 mg/mL)
40 Clone 123 (2.7 .mu.g/.mu.L) 14.8 CaCl.sub.2 (2.5 M) 253
Spermidine (0.1 M) 50 distilled H.sub.2O 212.2
[0117] For transformation, 6 cm diameter culture dishes were seeded
with 5.times.10.sup.5 male human fibroblast HT1080 cells 24 hours
before transfection. Two replicate dishes were prepared for each of
the treatments, and two negative control plates were also
prepared.
[0118] The microparticles and the tungsten-coated particles were
then delivered to cells using a needleless syringe as described
above. The syringe included a 4.5 mL reservoir chamber with plunger
type valve, a helium gas reservoir, a Mach 2 nozzle and 12 .mu.m
Mylar sheet hand-punched into 6 mm diameter diaphragms.
[0119] In particular, mylar diaphragms were first sterilized by
singly layering between pieces of filter paper, stacked one atop
the other, wrapped in aluminum foil and sealed completely with
autoclave tape to ensure that no water entered the filter
paper/diaphragm stack during the autoclave process. This was placed
inside a beaker covered with aluminum foil and placed in an
autoclave chamber.
[0120] Two 12 .mu.m Mylar diaphragms of 6 mm diameter were used in
the membrane cassette. One milligram and one-half milligram
payloads of the freeze-dried DNA powder were loaded onto the lower
membrane in the cassette. This payload was then covered with the
other pieces of the cassette and the remaining diaphragm. Only
preparations 2 and 3 were used in the experiment because of the
difficulty in weighing out small masses accurately. Another five of
the cassettes were each loaded with 5 .mu.L of the DNA/tungsten
particle suspension. All the above quantities gave a mass of about
8 .mu.g of DNA being delivered in each shot regardless of particle
formulation used.
[0121] Referring now to FIG. 1, a delivery apparatus 2 was
assembled which contained a needleless syringe 4 loaded with a
cassette as described above. The needleless syringe 4 was arranged
on a ring stand 6 using a standard tube clamp 8 to hold the syringe
in position relative to a culture dish 10 seeded with the HT1080
cells 12. The distance between the downstream terminus 14 of the
needleless syringe 4 and the cells 14 in the culture dish 10 was
measured to affix a target distance, generally indicated at d. In
order to optimize the parameters for delivery of the freeze-dried
DNA, either the target distance d was varied over a constant
delivery pressure, or the delivery pressure was varied over a
constant target distance. In particular, the DNA preparations were
fired from a target distance ranging from 20 to 60 mm using helium
driver gas pressures ranging from 30 to 50 bar.
[0122] After transformation, cells were incubated for 2 days,
stained, and then transient assays with X-gal were performed to
determine transformation efficiencies using previously described
methods. Murray, E. J. (ed) (1991) Methods in Molecular Biology:
Gene Transfer and Expression Protocols, Vol. 7, Humana Press,
Clifton, N.J. Specifically, transformation efficiency was assessed
by counting the number of blue-stained cells. The delivery
parameters and transformation results are depicted below in Table
3. Blast effect was rated from 1 point for quite small (diameter of
dead cell zone being approximately 5-8 mm) to five points for very
large (diameter of cell zone being greater than 30 mm). As can be
seen, transformation by the plasmid/trehalose powder preparation of
the present invention was on the same order as that observed for
the metallic particles.
[0123] As shown in FIG. 2, optimal transformation results were seen
with the particulate plasmid/trehalose preparation when delivered
using 30 bar pressure at a target distance of 60 mm. More
particularly, FIG. 2 provides a direct comparison of the
transformation efficiency obtained by delivery of the particulate
nucleic acid preparation (both preparations 2 and 3) with
historical and contemporary deliveries of DNA-coated tungsten
particles. Referring now to FIG. 3, data obtained for deliveries at
30 bar are depicted in a graph which presents transformation
efficiency as a function of target distance. As can be seen, the
optimal target distance for the number 2 and 3 preparations
(referred to as OBS Formulation #2 and OBS Formulation #3,
respectively) was not reached; however, transformation efficiency
did substantially increase with increased target distances.
Further, when deliveries were carried out at the maximum distance
tested (60 mm), transformation efficiencies obtained with the
particulate DNA formulations (#2 and #3) were appreciably better
than those observed with the DNA-coated tungsten controls.
3TABLE 3 Target Blue Cell Blast Shot Formulation Distance Pressure
Count effect A1 Tungsten 60 30 224 2 A2 Tungsten 60 30 596 2 A3
Tungsten 60 30 575 2 A4 Tungsten 60 30 581 2 A5 Tungsten 60 30 --
-- B1 #3 trehalose 20 30 155 3 B2 #3 trehalose 20 30 227 3 C1 #3
trehalose 40 30 654 2 C2 #3 trehalose 40 30 643 2 D1 #3 trehalose
40 50 394 4 D2 #3 trehalose 40 50 175 5 E1 #3 trehalose 60 30 1416
1 E2 #3 trehalose 60 30 1654 1 F1 #3 trehalose 60 50 408 3 F2 #3
trehalose 60 50 486 3 G1 #2 trehalose 20 30 166 3 G2 #2 trehalose
20 30 180 3 H1 #2 trehalose 20 50 129 4 H2 #2 trehalose 20 50 53 4
J1 #2 trehalose 40 30 347 2 J2 #2 trehalose 40 30 546 2 K1 #2
trehalose 40 50 377 4 K2 #2 trehalose 40 50 198 4 L1 #2 trehalose
60 30 1451 1 L2 #2 trehalose 60 30 1164 1 M1 #2 trehalose 60 50 409
3 M2 #2 trehalose 60 50 336 3
EXAMPLE 2
[0124] The following studies were carried out to assess the ability
to deliver a powdered nucleic acid composition to test subjects in
vivo using the methods of the invention.
[0125] Plasmid Vector Construct: The pGREEN-1 vector construct,
which contains the Green Fluorescent Protein (GFP) gene under the
control of a CMV promoter, was used so that gene expression could
be assessed directly by UV microscopy of histological sections from
treated tissue samples.
[0126] Powdered Nucleic Acid Compositions: A powdered nucleic acid
composition was prepared as follows. A mixture was formed by
combining pGREEN-1 vector plasmid with trehalose sugar to obtain a
1 .mu.g:1 mg (w/w) DNA-sugar composition. This composition was
lyophilized, compressed, ground, and then sieved, using the
techniques described hereinabove. The resulting condensed nucleic
acid composition had an average particle size ranging from about
38-75 .mu.m.
[0127] Administrations: C57BL/10 mice were treated with 1 mg of the
particulate composition via needleless injection. The composition
was delivered to a suitably prepared target skin surface, and
histological sections were taken from the target site 24 hours
after administration. GFP expression was determined directly using
UV microscopy. As a result of the administrations, GFP expression
was seen in the treated skin tissue, confirming successful in vivo
delivery of the powdered nucleic acid composition to the target
skin, and the subsequent transfection of host cells and expression
of the GFP gene therefrom.
[0128] In another study, plasmids containing either a human Growth
Hormone (hGH) or .beta.-galactosidase (.beta.-Gal) expression
cassette were lyophilized with trehalose excipient to form nucleic
acid formulations, which were compressed, ground, and then sieved,
using the above-described techniques. The resulting condensed
nucleic acid compositions had an average particle size ranging from
about 38-75 .mu.m.
[0129] Female pigs (weighing 20-25 kg) were anesthestised with
halothane, and the belly skin was clipped to reveal a suitable
target site. The above powdered nucleic acid compositions were
individually administered to the prepared target site in 0.1 .mu.g
(hGH) or 1 .mu.g (.beta.-Gal) doses via a needleless injection
device (delivery pressure of 60 bar). The target sites were
biopsied 24 hours after treatment, and histological sections were
analyzed for human growth hormone or .beta.-Gal expression.
Although no hGH expression was seen within the detection limits of
the assay, a moderate degree of .beta.-Gal expression was seen in
the treated sites. The lack of detectable hGH expression in this
study is due, presumably, to the low loading density of the nucleic
acid (0.1 .mu.g) in the composition.
EXAMPLE 3
Densification of Recombinant Human Growth Hormone (rhGH)
[0130] Lyophilized recombinant human growth hormone powder
(Genotropin.RTM., available from Pharmacia, Piscataway, N.J.) was
obtained and reprocessed using the method of the invention.
Particularly, approximately 30 mg of Genotropin was compacted under
pressure using a Carver Laboratory Pellet Press (Model 3620,
available from Carver, Inc., Wabash, Ind.). The pressure of
compaction was 15,000 lbs/in.sup.2, which was applied for
approximately 45 seconds. A pellet was obtained which was ground
using mortar and pestle until visually broken up. The resulting
reduced pellet was then sieved using a 53 .mu.m sieve (Endecott,
London). Particles having a size greater than 53 .mu.m were
selected and appropriate dosages thereof were measured into drug
cassettes for delivery from a needleless syringe.
EXAMPLE 4
Visual Assessment of rhGH Particle Penetration
[0131] Lyophilized recombinant human growth hormone (rhGH) powder,
and densified rhGH particles prepared as described in Example 3
were administered to porcine subjects using a needleless syringe.
The degree of particle penetration was visually ascertained as
follows.
[0132] Genotropin.RTM. 36 IU lyophilized powder was milled gently,
weighed into individual doses of approximately 0.8 mg powder and
loaded into a needleless syringe device for delivery. Densified
Genotropin.RTM. was prepared as described in Example 3 and
approximately 0.8 mg of the densified particles were loaded into a
needleless syringe device for delivery.
[0133] Porcine subjects were prepared by clipping a sufficient area
on the hindquarters. The lyophilized powder and densified particles
were delivered to the--porcine skin under high velocity. Upon a
side-by-side comparison, it was observed that a higher proportion
of the densified particles penetrated the skin as evidenced by the
visual presence of the lyophilized powder remaining largely on the
surface of the skin while substantially no densified particles were
observed to remain on the surface of the skin.
EXAMPLE 5
Serum Levels of Transdermally-Delivered rhGH
[0134] In order to determine the efficiency with which densified
rhGH is delivered using a needleless syringe system, the following
study was carried out. Three groups of 5 healthy New Zealand White
rabbits were prepared by clipping the fur from the flank area to
expose a sufficient area for delivery of lyophilized recombinant
human growth hormone powder (rhGH) or densified rhGH by needleless
syringe.
[0135] Approximately 0.8 mg of lyophilized Genotropin.RTM. powder
was reconstituted into 1.8 mL of a suitable buffer without
preservatives (e.g., sterile phosphate buffered saline (PBS)) to
provide a Genotropin.RTM. solution having a concentration of 20
IU/mL. 1 mL dosages were withdrawn by syringe aim gently mixed with
1 mL of buffer to provide an injection solution having a
concentration of 10 IU/mL.
[0136] Each animal in the first group were given 0.1 mL/kg of the
injection solution by subcutaneous needle and syringe injection,
and the injection site was observed to ensure that there was no
leakage of the injected solution after administration. Venous blood
samples were taken from the marginal ear vein of the right ear of
each animal at 0, 30 minutes, 1, 2, 4, 6, 12 and 24 hours after
administration. Serum levels of rhGH were ascertained using an
immunoassay with labeled anti-rhGH antibodies. The mean serum
levels of subcutaneously delivered Genotropi.RTM. (.circle-solid.)
found in the animals of Group 1 at each time point are depicted in
FIG. 4.
[0137] Approximately 2 mg of lyophilized Genotropin.RTM. powder was
loaded into a needleless syringe. The lyophilized powder was
administered to each animal in the second group by needleless
injection at high velocity. Venous blood samples were taken from
the marginal ear vein of the right ear of each animal at 0, 30
minutes, 1, 2, 4, 6, 12 and 24 hours after administration. Serum
levels of the administered rhGH were ascertained using an
immunoassay with labeled anti-hGH antibodies. The mean serum levels
of transdermally injected lyophilized Genotropin.RTM. powder
(.tangle-solidup.) found in the animals of Group 2 at each time
point are depicted in FIG. 4.
[0138] Approximately 2 mg of densified Genotropin.RTM. particles
prepared as in Example 3 was loaded into a needleless syringe. The
densified particles were administered to each animal in the third
group by needleless injection at high velocity. Venous blood
samples were taken from the marginal ear vein of the right ear of
each animal at 0, 30 minutes, 1, 2, 4, 6, 12 and 24 hours after
administration. Serum levels of the administered rhGH were
ascertained using an immunoassay with labeled anti-rhGH antibodies.
The mean serum levels of transdermally injected densified
Genotropin.RTM. particles (.box-solid.) found in the animals of
Group 3 at each time point are depicted in FIG. 4.
[0139] As can be seen, markedly increased blood serum levels of the
densified Genotropin.RTM. particles administered by needleless
syringe were obtained as compared to the lyophilized
Genotropin.RTM. powder.
EXAMPLE 6
Determination of Optimum Conditions for Needleless Syringe Delivery
of rhGH
[0140] In order to determine optimum conditions for delivery of
rhGH using a needleless syringe delivery system, the following
study is carried out. One group of 8 healthy New Zealand White
rabbits (2.+-.0.25 kg) are prepared by clipping the fur from the
flank area to expose a sufficient area for delivery of Lyophilized
recombinant human growth hormone powder (rhGH) or densified rhGH by
needleless syringe, sub-cutaneous (SC) or intravenous (IV)
injection. The animals are weighed at the start of the study and on
a weekly basis throughout the study to determine appropriate
Genotropin.RTM. dosages. The animals remain in one large group to
obtain statistically-significant data.
[0141] For an initial needleless syringe injection series,
Genotropin.RTM. 36 IU lyophilized powder is milled gently and
filled into a glass vial. The milled lyophilized powder is weighed
into individual dosages and loaded into needleless syringe devices
at approximately 0.8 mg powder/kg. The injection is conducted in
the first week of the study, and multiple venous blood samples (1
mL whole blood) are taken from the marginal ear vein at times 0, 30
minutes, 1, 2, 4, 6, 12, 24 and 48 hours after administration. The
animals are individually-housed at all times with food and water
available ad libitum.
[0142] Blood samples are handled and processed as follows: each
venous blood sample is allowed to clot at ambient temperature for
approximately 30 minutes and then left for an additional 30 minutes
at approximately 4.degree. C. Clotted samples are centrifuged for
10 minutes and the serum is aspirated and stored at -20.degree. C.
for analysis.
[0143] In the second week of the study, (approximately 1 week
following the initial needleless injection), the animals are
administered a Genotropin.RTM. formulation prepared as follows: 36
IU (approximately 30 mg) of the lyophilized Genotropin.RTM. powder
is reconstituted into 1.8 mL of a suitable buffer without
preservatives (e.g., sterile phosphate buffered saline (PBS)) to
provide a Genotropin.RTM. solution having a concentration of 20
IU/mL. 1 mL dosages are withdrawn by syringe and gently mixed with
1 mL of buffer to provide an injection solution having a
concentration of 10 IU/mL. Each animal is given an IV injection of
0.1 mL/kg of the solution in the left ear.
[0144] Following IV injection, multiple venous blood samples are
taken from the marginal ear vein of the right ear at times 0, 5,
10, 30 minutes, 1, 2, 4, 6 and 12 hours after injection.
[0145] In the third week of the study, the animals are administered
0.1 mL/kg of a buffered Genotropin.RTM. solution (prepared as
above) by sub-cutaneous injection. The injection site is observed
to ensure that there is no leakage after administration. Following
the SC injections, multiple venous blood samples are taken from the
marginal ear vein of the right ear at times 0, 30 minutes, 1, 2, 4,
6, 12, 24 and 48 hours after administration.
[0146] In the fourth week of the study, approximately 0.8 mg
powder/kg of densified Genotropin.RTM. particles (prepared as
described in Example 3) are administered to each animal using a
needleless syringe, and multiple venous blood samples are taken
from the marginal ear vein of the right ear at times 0, 30 minutes,
1, 2, 4, 6, 12, 24 and 48 hours after administration.
[0147] Serum rhGH levels are determined as previously described and
pharmacokinetic variables are calculated for each drug
administration technique. It is expected that needleless syringe
administration of the densified Genotropin.RTM. particles will
result in achieving and maintaining in vivo therapeutic levels of
the administered drug.
EXAMPLE 7
Bio-Activity of Densified rhGH Delivered in vivo to Growth
Hormone-Deficient Rats
[0148] To evaluate the bio-activity of recombinant human growth
hormone that has been densified using the method of the present
invention the following study is carried out.
[0149] Dwarf or hypophysectomized (growth hormone-deficient) rats
are administered pharmaceutical preparations containing either:
densified Genotropin.RTM. particles; lyophilized Genotropin.RTM.
powder; or placebo at approximately 4 IU rhGH per animal/week by
daily subcutaneous (SC) injection. In particular, on 5 successive
days, fur from the peritoneal region of the animal subjects is
clipped prior to administration of the densified rhGH, the
lyophilized rhGH or the placebo by SC injection. Body weight and,
if desired, bone size and length are monitored on a daily
basis.
[0150] Bio-activity of the densified rhGH particle formulation is
determined by measuring body weight change over time. It is
expected that the densified rhGH particle formulation will retain
sufficient bio-activity.
EXAMPLE 8
Densification of Commonly Used Excipients
[0151] Finely ground powders of pharmaceutical grade mannitol and
lactose were obtained and reprocessed using the method of the
invention. Particularly, approximately 30 to 50 mg of mannitol or
lactose were compacted under pressure using a Carver Laboratory
Pellet Press (Model 3620, available from Carver, Inc., Wabash,
Ind.). The pressure of compaction was 10,000 lbs/in.sup.2 which was
applied for approximately 30 seconds. The resulting compacted
pellets were ground using mortar and pestle until visually broken
up, and then sieved to select for particles having a size greater
than about 50 .mu.m using the methods described in Example 3. In
both the mannitol and the lactose preparations, a significant size
reduction was observed when the compacted particles were compared
against like weights of the non-densified starting materials.
EXAMPLE 9
Quantification of Densified Excipients
[0152] Pharmaceutical grade trehalose and mannitol excipients were
obtained and processed according to the method of the invention.
Both excipient preparations were processed in several different
ways, and absolute density, envelope density, and average particle
size of the resultant preparations were measured as described
below.
[0153] Trehalose 45H3830 (Sigma) and mannitol K91698380-703 (Merck)
were either sieved, or freeze dried, compacted, ground and then
sieved. A range of the resulting preparations were then analyzed
for particle size and density measurements.
[0154] Portions of each sugar excipient were sieved to obtain
preparations having reduced particle sizes. Sieving was carried out
using stainless steel sieves for two hours at 3 mm amplitude using
three sieve sizes (75 .mu.m, 53 .mu.m and 38 .mu.m) without
additional processing.
[0155] Alternatively, portions of each sugar excipient were
processed using the methods of the invention as follows. 40 g of
each of the sugars was dissolved in water, flash frozen, and then
freeze dried over night. Samples of each freeze dried preparation
were retained, and the remainder compacted in a 13 mm compression
die (15,000 lbs/inch.sup.2 for 45 seconds) into discs. The mannitol
discs were ground using mortar and pestle, and then sieved as
described above at 3 mm amplitude, using three sieve sizes. The
trehalose discs were first ground in a vibratory ball mill (Retsch
mill), then ground by mortar and pestle and sieved as above.
[0156] Samples from each of the above-described excipient
preparations were then analyzed for absolute and envelope density.
Absolute density was determined using helium pycnometry, and
envelope density was determined using a GeoPyc.TM. Model 1360
Envelope Density Analyzer (Micromeritics Instrument Corp.). The
results of the analysis are depicted below in Table 4.
4 TABLE 4 Trehalose Density Mannitol Density (g/cm.sup.3)
(g/cm.sup.3) Pretreatment Absolute Envelope Absolute Envelope
Sieved 1.5 0.5 1.5 0.5 Freeze Dried 1.5 0.3 1.5 0.3 Freeze 1.5 0.8
1.5 0.8 Dried, Compressed, Milled, Sieved
[0157] As can be seen in Table 4, none of the various processing
methods had a significant effect on the absolute density of the
powdered excipients. Further, as expected, the non-compacted,
freeze-dried sugars had a much lower envelope density than the
other preparations, and a concomitantly higher porosity
(measurement not shown). The density measurements for the trehalose
and mannitol samples clearly demonstrate that the methods of the
invention (compression, milling, sieving) lead to a significant
increase in envelope density relative to both the freeze-dried and
the sieved preparations. These results indicate that the novel
methods described herein can be used to provide densified
particulate pharmaceutical preparations that are suitable for
delivery via needleless injection techniques.
[0158] Accordingly, novel methods for DNA delivery as well novel
methods for densifying particulate pharmaceutical compositions, and
densified pharmaceutical compositions formed therefrom, have been
described. Although preferred embodiments of the subject invention
have been described in some detail, it is understood that obvious
variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
* * * * *