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Patent 2461890 Summary

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(12) Patent: (11) CA 2461890
(54) English Title: WATER SOLUBLE NANOPARTICLES OF HYDROPHILIC AND HYDROPHOBIC ACTIVE MATERIALS
(54) French Title: NANOPARTICULES SOLUBLES DANS L'EAU CONSTITUEES DE MATERIAUX HYDROPHILES ET HYDROPHOBES
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 9/00 (2006.01)
  • A61K 9/51 (2006.01)
(72) Inventors :
  • GOLDSHTEIN, RINA (Israel)
(73) Owners :
  • SOLUBEST LTD. (Israel)
(71) Applicants :
  • SOLUBEST LTD. (Israel)
(74) Agent: SMART & BIGGAR
(74) Associate agent:
(45) Issued: 2010-12-14
(86) PCT Filing Date: 2002-09-27
(87) Open to Public Inspection: 2003-04-10
Examination requested: 2007-09-26
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/IB2002/004176
(87) International Publication Number: WO2003/028700
(85) National Entry: 2004-03-26

(30) Application Priority Data:
Application No. Country/Territory Date
09/966,847 United States of America 2001-09-28
10/256,023 United States of America 2002-09-26

Abstracts

English Abstract




This invention provides a soluble nano-sized particles formed of a core (Water-
insoluble lipophilic )compound or hydrophilic compound and an amphiphilic
polymer and which demonstrated improved solubility and/or stability. The
lipophilic compound within the soluble nano-sized soluble ("Solu-
nanoparticles") may consist of Phannaceutical compounds, food additives,
cosmetics, agricultural products and veterinary products. The invention)also
provides novel methods for preparing the nano-sized soluble Particles, as well
as a novel chemiCal reactor for manufacturing an inclusion complex comprising
the nano-sized soluble Particles.


French Abstract

L'invention concerne des particules nanométriques solubles formées d'un noyau (composé lipophile insoluble dans l'eau ou composé hydrophile) et d'un polymère amphiphile, lesdites particules présentant une meilleure solubilité et/ou stabilité. Ledit composé lipophile desdites particules nanométriques solubles peut se présenter sous la forme de composés pharmaceutiques, d'additifs alimentaires, d'agents cosmétiques, de produits agricoles et vétérinaires. L'invention concerne également de nouveaux procédés de fabrication desdites particules nanométriques solubles, ainsi qu'un nouveau réacteur chimique destiné à la fabrication d'un complexe d'inclusion contenant lesdites particules nanométriques solubles.

Claims

Note: Claims are shown in the official language in which they were submitted.



CLAIMS:

1. A hydrophilic dispersion, comprising a water-
insoluble or water soluble active compound and an
amphiphilic polymer which wraps said active compound to form
a water-soluble nano-sized molecular entity in which non-
valent bonds are formed between said active compound and
said amphiphilic polymer such that said bonds fixate said
active compound within said polymer, in which nano-sized
molecular entity the active compound is in the amorphous or
partially crystalline state and wherein said amphiphilic
polymer does not form rigid matrices nor cross-linked
polymers.


2. The hydrophilic dispersion as recited in claim 1,
wherein said non-valent bonds include electrostatic forces,
Van der Waals forces and hydrogen bonds.


3. The hydrophilic dispersion as recited in claim 1,
wherein said amphiphilic polymer is selected from natural
polysaccharides, modified polysaccharides, polyacrylic acid,
polyethylene imine, polymethacrylic acid, polyethylene
oxide, polyvinyl alcohol, polyacetylene, polyisoprene or
polybutadiene.


4. The hydrophilic dispersion as recited in claim 1,
wherein said active compound is selected from: peptides,
polypeptides, nucleotides and co-ferments, vitamins,
steroids, porphyrins, metal-complexes, purines, pyrimidines,
antibiotics or hormones.


5. The hydrophilic dispersion as recited in claim 1,
wherein said active compound is a pharmaceutical compound.

32


6. The hydrophilic dispersion as recited in claim 5,
wherein said pharmaceutical compound is a chemotherapeutic
agent, or an antibiotic agent.


7. The hydrophilic dispersion as recited in claim 1,
wherein said nano-sized molecular entity is in the range of
from approximately 10 to approximately 1000 nanometers in
size.


8. A hydrophilic dispersion, comprising a water-
insoluble or water soluble active compound and an
amphiphilic polymer which wraps said active compound to form
a water-soluble nano-sized molecular entity in which non-
valent bonds are formed between said active compound and
said amphiphilic polymer and said active compound is in the
amorphous or partially crystalline state and wherein said
molecular entity is an inclusion complex.


9. Nano-sized particles, comprising a water-insoluble
or water soluble active compound in an amorphous or
partially crystalline state wrapped within an amphiphilic
polymer such that non-valent bonds are formed between said
active compound and said amphiphilic polymer, and said
amphiphilic polymer does not form rigid matrices or cross-
linked polymers.


10. The nano-sized particles as recited in claim 9,
wherein said non-valent bonds include electrostatic forces,
Van der Waals forces and hydrogen bonds.


11. The nano-sized particles as recited in claim 9,
wherein said active compound wrapped in said amphiphilic
polymer is fixated within said polymer.


12. The nano-sized particles as recited in claim 9,
wherein said particles are substantially spherical.

33


13. The nano-sized particles as recited in claim 9,
wherein said amphiphilic polymer is selected from: natural
polysaccharides, polyacrylic acid, polyethylene imine,
polymethacrylic acid, polyethylene oxide, polyvinyl alcohol,
polyacetylene, polyisoprene or polybutadiene.


14. The nano-sized particles as recited in claim 9,
wherein said active compound is a pharmaceutical compound.

15. The nano-sized particles as recited in claim 14,
wherein said pharmaceutical compound is a chemotherapeutic
agent, or an antibiotic agent.


16. The nano-sized particles as recited in claim 9,
wherein said particles are in the range of from
approximately 10 to approximately 1000 nanometers in size.

17. The hydrophilic dispersion as recited in claim 1,
wherein said nano-sized molecular entity is bioavailable in
the human body.


18. The nano-sized particles as recited in claim 9,
wherein said active compound is selected from: peptides,
polypeptides, nucleotides and co-ferments, vitamins,

steroids, porphyrins, metal-complexes, purines, pyrimidines,
antibiotics, hormones or chemotherapeutic agents.


19. A hydrophilic inclusion complex consisting
essentially of nano-sized particles of a water-soluble
compound surrounded by and entrapped within an amphiphilic
polymer, wherein said inclusion complex is water-soluble.

20. The hydrophilic inclusion complex as recited in
claim 19, wherein said water-soluble compound interacts with
said amphiphilic polymer via the formation of non-valent
bonds.


34


21. The hydrophilic inclusion complex as recited in
claim 20, wherein said non-valent bonds are electrostatic
forces, Van der Waals forces or hydrogen bonds.


22. The hydrophilic inclusion complex as recited in
claim 19, wherein said amphiphilic polymer does not form
rigid matrices.


23. The hydrophilic inclusion complex as recited in
claim 19, wherein said active compound entrapped within said
amphiphilic polymer is fixated within said polymer.


24. The hydrophilic inclusion complex as recited in
claim 19, wherein said active compound entrapped within said
amphiphilic polymer is in the amorphous or partially
crystalline state.


25. The hydrophilic inclusion complex as recited in
claim 19, wherein said hydrophilic inclusion complex is
bioavailable.


26. The hydrophilic inclusion complex as recited in
claim 19, wherein said water-soluble active compound is a
pharmaceutical compound.


27. A hydrophilic dispersion comprising a water-
insoluble or water soluble active compound and an
amphiphilic polymer which wraps said active compound to form
a water-soluble nano-sized molecular entity in which non-
valent bonds are formed between said active compound and
said amphiphilic polymer such that said bonds fixate said
active compound within said polymer, in which nano-sized
molecular entity the active compound is in the amorphous or
partially crystalline state and wherein said amphiphilic
polymer does not form rigid matrices.




28. Nano-sized particles comprising a water-insoluble
or water soluble active compound in an amorphous or
partially crystalline state wrapped within an amphiphilic
polymer such that non-valent bonds are formed between said
active compound and said amphiphilic polymer, and said
amphiphilic polymer does not form rigid matrices.


36

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02461890 2009-10-29
51580-1

WATER SOLUBLE NANOPARTICLES OF HYDROPHILIC
AND HYDROPHOBIC ACTIVE MATERIALS

Field of the Invention
The invention is in the field of nanoparticles. More particularly, the
invention
relates to soluble nano-sized particles ("solu-nanoparticles") and methods of
producing
solu-nanoparticles that render insoluble compounds solubilized in a medium
otherwise not
soluble.

Background of the Invention
Two formidable barriers to effective drug delivery and hence to disease
treatment, are solubility and stability. To be absorbed in the human body, a
compound has
to be soluble in both water and fats (lipids). Solubility in water is,
however, often
associated with poor fat solubility and vice versa.
Over one third of drugs listed in the U.S. Pharmacopoeia and about 50% of
new chemical entities (NCEs) are insoluble or poorly insoluble in water. Over
40% of
drug molecules and drug compounds are insoluble in the human body. In spite of
this,
lipophilic drug substances having low water solubility are a growing drug
class having
increasing applicability in a variety of therapeutic areas and for a variety
of pathologies.
There are over 2500 large molecules in various stages of development today,
and over
5500 small molecules in development. Each of the existing companies focusing
on these
large and small molecules has its own restriction and limitations with regard
to both large
and small molecules on which they focus.
Solubility and stability issues are major formulation obstacles hindering the
development of therapeutic agents. Aqueous solubility is a necessary but
frequently
elusive property for formulations of the complex organic structures found in
pharmaceuticals. Traditional formulation systems for very insoluble drugs have
involved
a combination of organic solvents, surfactants and extreme pH conditions.
These
formulations are often irritating to the patient and may cause adverse
reactions. At times,
these methods are inadequate for solubilizing enough of a quantity of a drug
for a
parenteral formulation. In such cases, doctors may administer an "overdosage",
such as
for example with poorly soluble

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vitamins. In most cases, this overdosage does not cause any harm since the
unabsorbed
quantities exit the body with urine. Overdosage does, however waste a large
amount of the
active compound.
The size of the drug molecules also plays a major role in their solubility and
stability
as well as bioavailability. Bioavailability refers to the degree to which a
drug becomes
available to the target tissue or any alternative in vivo target (i.e.,
receptors, tumors, etc.) after
being administered to the body. Poor bioavailability is a significant problem
encountered in
the development of pharmaceutical compositions, particularly those containing
an active
ingredient that is poorly soluble in water. Poorly water soluble drugs tend to
be eliminated

from the gastrointestinal tract before being absorbed into the circulation. It
is known that the
rate of dissolution of a particulate drug can increase with increasing surface
area, that is,
decreasing particle size
Recently, there has been an explosion of interest in nanotechnology, the
manipulation
on the nanoscale. Nanotechnology is not an entirely new field; colloidal sols
and supported
platinum catalysts are nanoparticles. Nevertheless, the recent interest in the
nanoscale has
produced, among numerous other things, materials used for and in drug
delivery.
Nanoparticles are generally considered to be solids whose diameter is varies
between 1-1000
run.
Although a number of solubilization technologies do exist, such as liposomes,

cylcodextrins, microencapuslation, and dendrimers, each of these technologies
has a number
of significant disadvantages.
Phospholipids exposed to aqueous environment form a bi-layer structure called
liposomes. Liposomes are microscopic spherical structures composed of
phospholipids that
were first discovered in the early 1960s (Bangham et al., J. Mol. Biol. 13:
238 (1965)). In
aqueous media, phospholipid molecules, being amphiphilic, spontaneously
organize
themselves in self-closed bilayers as a result of hydrophilic and hydrophobic
interactions.
The resulting vesicles, referred to as liposomes, therefore encapsulate in the
interior part of
the aqueous medium in which they are suspended, a property that makes them
potential
carriers for biologically active hydrophilic molecules and drugs in vivo.
Lipophilic agents
may also be transported, embedded in the liposomal membrane. Liposomes
resemble the bio-
membranes and have been used for many years as a tool for solubilization of
biological active
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molecules insoluble in water. They are non-toxic and biodegradable and can be
used for
specific target organs.
Liposome technology allows for the preparation of smaller to larger vesicles,
using
unilamillar (ULV) and multilamillar (MLV) vesicles. MLV are produced by
mechanical
agitation. Large ULV are prepared from MLV by extrusion under pressure through
membranes of known pore size. The sizes are usually 200 nm or less in
diameter, however,
liposomes can be custom designed for almost any need by varying lipid content,
surface
change and method of preparation.
A number of companies such as Elan, Corp., Dublin, Ireland; Endorex Corp.,
Lake
Forest, IL; Advanced Drug Deliveries Technologies, Muttenz, Switzerland; The
Liposome
Company, Inc., Princeton, New Jersey (a subsidiary of Elan, Corp.); and
Mibelle AG, Buchs,
Switzerland, offer contract research and production -facilities to the
industry for the
preparation of Liposome inclusion complexes or inclusion moieties.
As drug carriers, liposomes have several potential advantages, including the
ability to
carry a significant amount of drug, relative ease of preparation, and low
toxicity if natural
lipids are used. However, common problems encountered with liposomes include:
low
stability, short shelf-life, poor tissue specificity, and toxicity with non-
native lipids.
Additionally, the uptake by phagocytic cells reduces circulation times.
Furthermore,
preparing liposome formulations that exhibit narrow size distribution has been
formidable

challenge under demanding conditions, as well as a costly one. Also, membrane
clogging
often results during the production of larger volumes required for
pharmaceutical production
of a particular drug.
Cyclodextrins are crystalline, water soluble, cyclic, non-reducing
oligosaccharides
built from six, seven, or eight glucopyranose units, referred to as alpha,
beta and gamma
cyclodextrin respectively, which have long been known as products that are
capable of
forming inclusion complexes. The cyclodextrin structure provides a molecule
shaped like a
segment of a hollow cone with an exterior hydrophilic surface and interior
hydrophobic
cavity.
The hydrophilic surface generates good water solubility for the cyclodextrin
and the
hydrophobic cavity provides a favorable environment in which to enclose,
envelope or entrap
the drug molecule. This association isolates the drug from the aqueous solvent
and may

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increase the drug's water solubility and stability. For a long time most
cyclodextrins had been
no more than scientific curiosities due to their limited availability and high
price.
As a result of intensive research and advances in enzyme technology,
cyclodextrins
and their chemically modified derivatives are now available commercially,
generating a new
technology: packing on the molecular level. Companies such as Cyclolab Ltd.,
Budapest,
Hungary; Cydex, Inc., Overland Park, Kansas; and Cyclops, Inc., Reykjavik,
Iceland, have
been involved in the development and manufacture of cyclodextrins.
Cyclodextrins are, however, fraught with disadvantages. An ideal cyclodextrin
would
exhibit both oral and systemic safety. It would have water solubility greater
than the parent
cyclodextrins yet retain or surpass their complexation characteristics. The
disadvantages of
the cyclodextrins, however, include: limited space available for the active
molecule to be
entrapped inside the core, lack of pure stability of the complex, limited
availability in the
marketplace, and high price.
Microencapsulation is a process by which tiny parcels of a gas, liquid, or
solid active
ingredient (also referred to herein and used interchangeably with "core
material") are
packaged within a second material for the purpose of shielding the active
ingredient from the
surrounding environment. These capsules, which range in size from one micron
(one-
thousandth of a millimeter) to approximately seven millimeters, release their
contents at a
later time by means appropriate to the application.
There are four typical mechanisms by which the core material is released from
a
microcapsule: (1) mechanical rupture of the capsule wall, (2) dissolution of
the wall, (3)
melting of the wall, and (4) diffusion through the wall. Less common release
mechanisms
include ablation (slow erosion of the shell) and biodegradation.
Microencapsulation covers several technologies, where a certain material is
coated to
obtain a micro-package of the active compound. The coating is performed to
stabilize the
material, for taste masking, preparing free flowing material of otherwise
clogging agents etc.
and many other purposes. This technology has been successfully applied in the
feed-addition
industry and to agriculture. The relatively high production cost needed for
many of the
formulations is, however, a significant disadvantage.
In the cases of nanoencapsulation and nanoparticles (which are advantageously
shaped
as spheres and hence, nanospheres), two types of systems having different
inner structures are
possible:

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a) a matrix-type system composed of an entanglement of oligomer or

polymer units, defined as nanoparticles or nanospheres and
b) a reservoir-type system, consisting of an oily core surrounded by a
polymer wall, defined as a nanocapsule.
Depending upon the nature of the materials used to prepare the nanospheres,
the
following classification exists:
a) amphiphilic macromolecules that undergo a cross-linking reaction
during preparation of the nanospheres;
b) monomers that polymerize during preparation of the nanoparticles;
c) hydrophobic polymers, which are initially dissolved in organic solvents
and then precipitated under controlled conditions to produce
nanosparticles.
Problems associated with the use of polymers in micro- and nanoencapsulation
include: the use of toxic emulgators in emulsions or dispersions,
polymerization or the
application of high shear forces during emulsification process, insufficient
biocompatibility
and biodegrability, balance of hydrophilic and hydrophobic moieties, etc.
These
characteristics lead to insufficient drug release.
Dendrimers are a class of polymers distinguished by their highly branched,
tree-like
structures. They are synthesized in an iterative fashion from ABn monomers,
with each
iteration adding a layer or "generation" to the growing polymer. Dendrimers of
up to ten
generations have been synthesized with molecular weights in excess of 106 kDa.
One
important feature of dendrimeric polymers is their narrow molecular weight
distributions.
Indeed, depending on the synthetic strategy used, dendrimers with molecular
weights in
excess of 20 kDa can be made as single compounds.
Dendrimers, like liposomes, display the property of encapsulation; being able
to
sequester molecules within the interior spaces. Because they are single
molecules, not
assemblies, drug-dendrimer complexes are expected to be significantly more
stable than
liposomal drugs. Dendrimers are thus considered as one of the most promising
vesicles for
drug delivering systems. However, dendrimer technology is still in the
research stage, and it
is speculated that it will take years before the industry will apply this
technology as a safe and
efficient drug delivery system.

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What is needed is a safe, biocompatible, stable and efficient drug delivery
system that
comprises nano-sized particles of an active ingredient for enhanced
bioavailability and which
overcomes the problems inherent in the prior art.

Summary of the Invention
Lipophilic and hydrophilic compounds that are solubilized in the form of nano-
sized
particles, or "nanoparticles", can be used in pharmacology, in the production
of food
additives, cosmetics, and agriculture, as well as in pet foods and veterinary
products, amongst
other uses.
The present invention provides nanoparticles and methods for the production of
soluble nanoparticles and, in particular, inclusion complexes of water-
insoluble lipophilic and
water-soluble hydrophilic organic materials. The present invention also
provides an
apparatus for producing these soluble nanoparticles using the novel method of
production.
Soluble nanoparticles, referred to as "solu-nanoparticles" in accordance with
the
present invention are differentiated by the use of water soluble amphiphilic
polymers that are
capable of producing molecular complexes with lipophilic and hydrophilic
active compounds
or molecules (particularly, drugs and pharmaceuticals). The solu-nanoparticles
formed in
accordance with the present invention render insoluble compounds soluble in
water and
readily bioavailable in the human body.
In accordance with the present invention, the solu-nanoparticles are comprised
of
polymers having an active compound or molecule wrapped and fixated or secured
within the
polymer. The solu-nanoparticles involve the active compound or molecule, which
is linked
with the polymer by non-valent bonds and form a polymer-active compound as a
distinct
molecular entity. The outer surface of the solu-nanoparticles is comprised of
a polymer that
carries the drug molecule to the target destination. The complex may be nano-
level in size,
and no change occurs in the drug molecule itself when it is enveloped, or
advantageously
wrapped, by the polymer. The solu-nanoparticle remains stable for long periods
of time, may
be manufactured at a low cost, and may, improve the overall bioavailability of
the active
compound.
The polymer used in the formation of these complexes is selected from the
group of
amphiphilic polymers that demonstrate hydrophilic-lipophilic balance (HLB) so
that the sum
total HLB of the complex allows for water solubility with stable solutions of
nano-emulsions
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or nano-suspensions . The amphiphilic polymer is selected using an algorithm
that takes into
account the molecular weight, the dimensions (in three directions), the
surface polarity and
the solubility in non-aqueous solvents of the lipophilic or hydrophilic
compound. Unlike
prior art inclusion complexes, the inclusion complex of the present invention
imposes no
limitations upon the size of the core compound that can be used. The
conditions during the
process of forming the nano-soluparticles are such that they do not lead to
the destruction of
the molecular composition of the core active lipophilic or hydrophilic
compound or to the
loss of its physiological or biological activity. With regard to the process
of preparing the
inclusion complexes of the present invention, the process temperature is
always lower than

the temperature at which the lipophilic compound is losing its physiological
or biological
activity, or the temperature at which the lipophilic composition changes its
chemical
composition.
Depending upon the polymer used in the formation of the solu-nanoparticles,
drugs
and pharmaceuticals as the active compound within the complex, are able to
reach specific
areas of the body readily and quickly. The polymer and active compound
selected will also
provide solu-nanoparticles capable of multi-level, multi-stage and/or
controlled release of the
drug or pharmaceutical within the body.
A significant advantage and unique feature of the complex (inclusion or other)
of
present invention is that no new bonds are formed and no existing bonds are
destroyed during
.the formation of the inclusion complex. Additionally, existing conditions
during the addition
of the active compound into the formulation of this complex assures the
creation of soluble
nanoparticles. Furthermore, the ingredients used in the preparation of the
complex are
inexpensive, abundant, non-toxic and safe for use in the surrounding
environment.

In another aspect of the present invention, a novel chemical reactor apparatus
is
provided for carrying out the method of forming the solu-nanoparticles in
accordance with the
present invention. The chemical reactor of the present invention provides for
continuous
circulation of a "carrier" between the polymer solution and the active
compound during the
production of the complex of the present invention. This ensures a high
uniformity of the
emulsion or the suspension formed during the process. The design of the
chemical reactor
allows all of the processes to occur in the same vessel, thus ensuring high
purity in the final
product and also simplifying the process and reducing the labor required.

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According to one aspect of the present invention,
there is provided a hydrophilic dispersion, comprising a
water-insoluble or water soluble active compound and an
amphiphilic polymer which wraps said active compound to form

a water-soluble nano-sized molecular entity in which non-
valent bonds are formed between said active compound and
said amphiphilic polymer such that said bonds fixate said
active compound within said polymer, in which nano-sized
molecular entity the active compound is in the amorphous or

partially crystalline state and wherein said amphiphilic
polymer does not form rigid matrices nor cross-linked
polymers.

According to another aspect of the present
invention, there is provided a hydrophilic dispersion,
comprising a water-insoluble or water soluble active

compound and an amphiphilic polymer which wraps said active
compound to form a water-soluble nano-sized molecular entity
in which non-valent bonds are formed between said active
compound and said amphiphilic polymer and said active
compound is in the amorphous or partially crystalline state
and wherein said molecular entity is an inclusion complex.
According to still another aspect of the present
invention, there is provided nano-sized particles,

comprising a water-insoluble or water soluble active

compound in an amorphous or partially crystalline state
wrapped within an amphiphilic polymer such that non-valent
bonds are formed between said active compound and said
amphiphilic polymer, and said amphiphilic polymer does not
form rigid matrices or cross-linked polymers.

According to yet another aspect of the present
invention, there is provided a hydrophilic inclusion complex
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consisting essentially of nano-sized particles of a water-
soluble compound surrounded by and entrapped within an
amphiphilic polymer, wherein said inclusion complex is
water-soluble.

According to a further aspect of the present
invention, there is provided a hydrophilic dispersion
comprising a water-insoluble or water soluble active
compound and an amphiphilic polymer which wraps said active
compound to form a water-soluble nano-sized molecular entity

in which non-valent bonds are formed between said active
compound and said amphiphilic polymer such that said bonds
fixate said active compound within said polymer, in which
nano-sized molecular entity the active compound is in the
amorphous or partially crystalline state and wherein said
amphiphilic polymer does not form rigid matrices.

According to yet a further aspect of the present
invention, there is provided nano-sized particles comprising
a water-insoluble or water soluble active compound in an
amorphous or partially crystalline state wrapped within an

amphiphilic polymer such that non-valent bonds are formed
between said active compound and said amphiphilic polymer,
and said amphiphilic polymer does not form rigid matrices.
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The above description sets forth rather broadly the more important features of
the
present invention in order that the detailed description thereof that follows
may be better
understood, and in order that the present contributions to the art may be
better appreciated.
Other objects and features of the present invention will become apparent from
the following
detailed description considered in conjunction with the accompanying drawings.
It is to be
understood, however, that the drawings are designed solely for the purposes of
illustration
and not as a definition of the limits of the invention, for which reference
should be made to
the appended claims.

Brief Description of the Drawings
The invention will be better understood by reference to the appended figures
in which:
FIG. 1 is a schematic drawing of a chemical reactor for the manufacture of
nano-sized
soluble particles in accordance with the present invention;
FIG. 2 illustrates the concentrations for both control and complexed
clarithromycin
testing material observed until 216 hours post application;
FIG. 3 is a chart comparing the pharmacokinetics constants of the tested
clarithromycin in nano-particle complex compared to published data of
commercial
clarithromycin;
FIG. 4 is a chart comparing the PK constants of clarithromycin in nano-
particle
complex with published studies with commercial clarithromycin;
FIG. 5 illustrates a complexed Clarithromycin particle having a size of
approximately
190nm;
FIG. 6 is an SEM micrograph illustrating the consistent spherical complexed
Clarithromycin particles prepared according to the method of the present
invention;

FIG. 7 illustrates the comparison of the solubility of Erythromycin and
Clarithromycin
alone and as part of the inclusion complex in accordance with the present
invention;
FIG. 8 illustrates the X-ray diffraction comparison of intact Erythromycin
compared
with the inclusion complex of Erythromycin in accordance with the present
invention.
FIG. 9 illustrates the X-ray diffraction comparison of intact Clarithromycin
compared
with the inclusion complex of Clarithromycin in accordance with the present
invention; and
FIG. 10 is an X-ray spectrum of 6 month old clarithromycin complexed sample
(bottom trace) compared to the commercially available Clarithromycin (upper
trace).
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Detailed Description of the Invention
The nanoparticles of the present invention comprises an insoluble or soluble
active
compound or core, wrapped within a medium soluble amphiphilic polymer. A
variety of
different polymers can be used for any selected active (lipophilic or
hydrophilic) compounds.
The polymer, or groups of polymers, is selected according to an algorithm that
takes into
account various physical properties of both the active lipophilic or
hydrophilic compound and
the interaction of this compound within the resulting active compound /polymer
nano-
soluparticle .
As used herein, the terms "lipophil", "lipophilic molecule" and "lipophilic
compound" are used interchangeably and are all intended to refer to the same
thing. The
molecules and compounds referred to herein as lipophilic molecules and
lipophilic
compounds have a hydrophilic-lipophilic balance (HLB) of less than 6, and fall
within the
HLB International scale, which ranges from 0-20. Hydrophilic molecules have a
hydrophilic-

lipophilic balance (HLB) of more than 6. HLB is discussed in greater detail
herein below.
More particularly, the ingredients of the composition of the present invention
comprise the active (lipophilic or hydrophilic) compound (preferably a
lipophil) and the
polymer to provide a molecular entity. The lipophil may be any organic
molecule or
compound that is insoluble in the water and is preferably a drug or
pharmaceutical
composition. The lipophilic compound can be small or large, simple or complex,
heavy or
light and may comprise a variety of functional groups. The polymer or polymers
used to
make up the complex may be selected from the group of polymers approved for
human use
(i.e. biocompatible and FDA-approved). Such polymers comprise, for example,
but are not
limited to: natural polysaccharides, polyacrylic acid and its derivatives,
polyethylene imine
and its derivatives, polymethacrylic acid and its derivatives, polyethylene
oxide and its
derivatives, polyvinyl alcohol and its derivatives, polyacetylene derivatives,
polyisoprene
derivatives and polybutadiene derivatives.
As recited, the polymer or groups of polymers used in the formation of the
nano-
soluparticles of the present invention are selected according to an algorithm
that takes into
account various physical properties of the active compounds and the polymer or
polymers, as
well as their future interaction in the resulting complex. The algorithm is
utilized in this
manner to select the optimal polymer(s) and to assess properties such as pH,
ionic force,

9


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temperature and various solvent parameters. More specifically, the amphiphilic
polymer is
selected using the algorithm that assesses the molecular weight, dimensions
(in three
directions) and the solubility of the lipophilic or hydrophilic compound in
non-aqueous
solvents. The algorithm also takes into consideration the following properties
of the polymer
itself in selecting a polymer for the active molecule/polymer interaction in
the formation of
the complex: molecular weight, basic polymer chain length, the length of the
kinetic unit, the
solubility of the polymer in water, the overall degree of solubility, the
degree of polymer
flexibility, the hydrophilic-lipophilic balance, and the polarity of the
hydrophilic groups of the
polymer.
This system comprises a selected polymer that is soluble in water, and has a
hydrophilic-lipophilic balance (HLB) that assures solubility of the complex
including the
lipophil or hydrophil and the polymer. The carrier is a non-aqueous solvent
(or group of
solvents) of the water-insoluble lipophilic compound or of the water soluble
hydrophilic
compound, having a boiling point temperature lower than that of water, and
more specifically
having a boiling point temperature lower that that of destruction of non-
valent bonds creating
the complex (at that pressure at which the process of complex creation is
being carried out).
The creation of the complex does not involve the formation of any valent bonds
(which may
change the characteristics or properties of the active compound). In the
complex of the
present invention, weak, non-covalent bonds, such as H-bonds and Van der Waals
forces
form during the creation of the inclusion complex. The formation of non-valent
bonds
preserves the structure and properties of the lipophilic compound, which is
particularly
important when the active compound is a pharmaceutical. As used herein, "non-
valent" is
intended to refer to non-covalent, non-ionic and non-semi-polaric bonds and/or
interactions.
Following the selection of the active compound, a determination is made of its
requisite
properties for construction of a geometrical model. A polymer suitable for
complexation
with the given compound is then selected. The main properties of the polymer
include its
HLB (hydrophilic-lipophilic balance), the length and the flexibility of its
polymer chain, and
also the state of polarity of the hydrophilic groups. The HLB of the polymer
is selected in
such a way that after combining to it the active compound the summary HLB of
the complex
renders the complex soluble. At this stage a geometrical model of the complex
is constructed
and determination is made of the length of the fragment of the polymer chain
needed for the
complex. The HLB is calculated following the building of a virtual complex on
a computer


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
screen. To this end existing computer programs for animation of molecular
structures are
used. The HLB can be calculated as a ratio of hydrophilic and lipophilic
groups on the
surface of the virtual complex. The molecular weight of the complex is easily
computed and
its geometry is determined. More precisely, total HLB of the complex in
accordance with the

present invention can be calculated after the virtual construction of the
complex on the
computer screen of a computer system upon which the aforementioned algorithm
has been
loaded as software. The algorithm that determines the summary HLB thus plays a
major role
in the selection of components from which the complex is formed. The
parameters and
library information pertaining to active compounds and polymer molecules are
stored in the

computer program for calculation of the summary HLB of the complex to be
formed.
A determination of the weight correlation of the "amphiphilic polymer to
active
molecule" is then made. This determination is essential to the generation of
the geometric
model. The correlation is made based on the total length of the polymer chain,
length of the
fragment needed to create the complex, molecular mass of the active compound
and

molecular mass of the fragment:
Formula:
MfxNf Mf MP MP (g-mol)
Nc _ --------- _ --- x ---- _ ------------
MI Ml Mf Ml (g-mol)
wherein:
N0 - the weight ratio of the "amphiphilic polymer to lipophilic compound".
Mf- the molecular mass of the polymer fragment.
Ml - molecular mass of the lipophilic compound.
Mp - molecular mass of the polymer.
Nf_ the quantity of the polymer fragments capable of participating in the
complex creation.
Next, the physical parameters of the water solvent for the polymer are
evaluated. At
this stage determination is made of the pH required to create the complex, the
necessary ionic
force and the required carrier for the lipophilic compound. Use of the above
components
creates optimal conditions for controlling the flexibility of the polymer
chain.
The carrier non-aqueous solvent is then selected. The purpose of this solvent
to
transfer the active compound into a very weak (low concentration) solution
such that the
11


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molecules of the dissolved compound practically do not react with one another.
This solution
is then delivered into the zone of reaction in the chemical reactor (discussed
in detail infra)
for the creation of nano-dispersions, such as a nano-emulsion (having a liquid
core material)
or nano-suspension (having a solid core material).
As used herein, the term "suspension" generally refers to a dispersion of fine
particles
in a liquid.
As used herein, the term "emulsion" generally refers to a mixture of two
normally
unmixable liquids in which one is colloidally suspended in the other (defining
a dispersed
phase). The particle sizes of the dispersed phase in an emulsion generally lie
between a few

hundred nanometers and a few tens of micrometers

Unlike known processes for the preparation of nano-sized particles where
polymers
are used for stabilization of the dispersion formed, only some of the
aforementioned
amphiphilic polymers (with previously calculated hydrophilic-lipophilic
balance HLB) are
used in these dispersion stabilizations. Additionally, specific conditions are
selected for the
dynamic three dimensional conformation of the amphiphilic polymer in the
dispersion, which
serves as the creator of the complex and fixator of the core active compound,
as opposed to
acting as a viscosifier (i.e., for increasing the viscosity). Previously
calculated HLB provides
for the necessary solubilization of the active compound.
Specific conditions created for the amphiphilic polymer in the "nano-
dispersion"
formation, results in two factors: (1) the provision of free rotation of the
kinetic segments of
the polymer chain around the chemical bonds, thus connecting these segments,
and (2) the
provision of non-valent interaction of the lipophilic functional groups of the
amphiphilic
polymer and the lipophilic groups of the compound intended for solubilization.
These
specific conditions include: the pH parameter of the dispersive medium, the
ionic forces of
the dispersive medium, the components composition of the dispersive medium,
the
temperature of the complex formulation, the process duration, and the
mechanical
components of the process. Each of these specific conditions will be discussed
in more detail
below.


12


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The pH parameter of the dispersion medium.
If the composition of the amphiphilic polymer includes ionogenic functional
groups,
the polymer could be soluble or at a pH higher than the iso-electric point
(polyacids) or lower
than iso-electric point (polybases) depending on the polarity of these groups.
In both of these cases the iso-electric point could be determined with a high
degree of
accuracy on the curve of "viscosity of the polymer solution-pH of the polymer
solution".
These two types of polymers could participate in the complex creation only
within the pH
range where their solutions are viscous liquids. For polymers with non-
ionogenic functional
groups, the clearly defined iso-electric point does not exist and for this
reason these polymers

could participate in the complex creation in a wide pH range.
Ionic force of the dispersive medium.
Under the influence of the ions of the water-soluble salts in the polymer
solution, the
geometry of the amphiphilic polymer chains changes. This factor is used for
creation of

stereo-specific conditions of non-covalent interaction between lipophilic
groups of the
polymer and the lipophil itself. Nonetheless, many polymers react so actively
on the
appearance of the salts (a "salting out" process of the polymer), that it is
not always possible
to utilize this factor in the reaction of complex creation.
Competition exists between the ions and the polymer for water molecules and
the ions
take water from the hydrate shells of the polymer. As a result of decreasing
hydrate shell, the
polymer coils to a globule. The greater the ionic activity, the greater the
polymer coiling to
the globule.
Components composition of the dispersive medium.
With the help of the composition of the solvents it is possible to flexibly
control the
geometry of the macromolecules. However, for the purpose of solubility
(solubilization) of
pharmaceuticals, food additives and cosmetics compounds, only biologically
safe solvents,
such as glycerol, ethylene glycol and less often ethyl alcohol, iso-butanol
and
dimethylsulfoxide could be used. Additive solvents decrease the dissolving
capacity of water.
This is, similar to salts addition, i.e. the uncoiled polymeric chain
transforming to a loose or
compact globule. Thus, options for this methods are limited.

13


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Temperature of the complex formation.
With the changes of the temperature of the polymer solution, the hydration
conditions
of the polymer molecule and accordingly its configuration in the solution
drastically changes.
With the raising of the temperature, hydration shells surrounding the polymer
molecule start

to detach and the linear macromolecule starts to take on globular form. At the
same time, the
flexibility of the macromolecule increases. As a result, additional positive
conditions for
complex creation are created.

The process duration
Because of the non-valent interaction during creation of the inclusion, the
limiting
phase of the process consists of the diffusion of the lipophilic compounds and
macromolecules to each other, for each reaction system exists at a minimum
time for complex
creation. If less time is allowed, the system remains two-phased. This two-
phased nano-
dispersion is thermodynamically unstable. The subsequent step of evaporating
the carrier

leaves particles of the dispersed phase in sizes ranging from 1-1000 nm. The
polymer
molecule in its solution then covers and entraps the active compounds,
creating particles. The
carrier is evaporated thus forming stable nanoparticles.

The mechanical component of the process
Mixers, dispersers, homogenizers and other equipment provide maximum
dispersing
of the active compound in the water-polymer solution and accelerate creation
formation of an
emulsion or suspension with nano-dimension sized particles in a dispersed
phase. An
advantageous and novel chemical reactor for forming the nano-emulsion or nano-
suspension
and the nano-soluparticles complex of the present invention is discussed in
detail herein

below.
The combined effect of the above conditions aids in achieving specifically
selected
dimensions and proportions for the complex, the maximum dispersing of the
active
compound and the optimal conditions for the non-valent interaction of the
polymer and these
compounds during complex formation.
As recited above, the preparation of the complex in accordance with the
present
invention requires a number of calculations and procedures to be performed
prior to

14


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commencing the process of preparing the complex. Some calculations and
procedures, which
are determined using an algorithm on a computer system, include:
(a) calculating the composition and properties of the components for preparing
the
complex, which comprises an active compound, an amphiphilic polymer, and
carrier solvent;
(b) calculating the weight ratio of the amphiphilic polymer to the active
compound;
(c) evaluating the physical parameters of the water solvent for the
amphiphilic
polymer;
(d) determining the proper non-aqueous solvent;
(e) creating a geometric model of the complex.
The algorithm is not limited to these calculations and maybe programmed to
make additional
calculations and determinations as necessary depending upon the properties and
characteristics of the complex to be made.
As recited, the production of the molecular complex consisting of an active
compound
and an amphiphilic polymer according to the present invention, requires the
dispersal of the
active compound to nano-particle size. The nano-sized particles assure an
almost immediate
interaction between the dispersed nano-sized particles of the active compound
and the
polymer molecules. In accordance with the process of the invention it is also
necessary to
prevent reverse aggregation (coacervation) of the nano-particles, and to
assure an immediate
interaction between the dispersed nano-particles of the active compound and
the polymer
molecules. This assures the formation of a stable complex (inclusion or
other). The size of
the active compound is determined by constructing its geometrical model
(taking into account
length of the connections and angles between these connections), and
thereafter transferring
the compound into a spherical configuration or other geometric shapes. The
diameter of this
sphere is the deciding measuring size of the active compound. There is a need
to take into
account that lipophils with long chain structures, as a rule, assume a shape
having a globular
configuration.
In accordance with the present invention, during the process of forming the
soluble
nano-sized particle or "solu-nanoparticle", a polymer is added to an aqueous
solvent,
preferably water, to form a polymer solution in a first vessel of a chemical
reactor.
Additionally, ingredients may be added to adjust the pH and ionic force level
of this solution
as needed based on the parameters determined via the algorithm used to select
the active
compound and polymer. An active compound, which is advantageously an insoluble
lipophil,



CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176

is placed in a second vessel of the chemical reactor. The active compound (or
core) may be
of any size, dimension or weight, and may comprise any of a variety of
functional groups. A
solution of the insoluble lipophilic or hydrophilic compound in a non-aqueous
solvent (or
mixture of solvents) is referred to as the "carrier". The velocity of pouring
or adding the
carrier to the polymer solution is regulated by one or more regulating taps,
which ensure that
the lipophil solution being added to the polymer solution has a concentration
below 0.1%.
The lipophil solution is formed when the polymer solution is heated and steam
from
the heated polymer solution condenses and dissolves the lipophil, present in
the second
vessel. The lipophil solution (in carrier) is then mixed with the polymer
solution to form a
dispersed phase in emulsion or suspension. Within the chemical reactor, the
emulsion is fed
into an area of turbulence caused by a disperser (more precisely a nano-
disperser) that causes
the formation of nano-sized lipophil molecules within the emulsion or
suspension. The area
of turbulence is referred to as the "action zone" or the "zone of
interaction". The emulsion or
suspension being fed into the area of turbulence has a Reynolds number of Re
>10,000. The
emulsion thus becomes a "nano-emulsion" or "nano-suspension" having particles
in the range
of approximately 1 to approximatelyl000 nm. The particle production can also
be extended
to include small micron sized particles. Within the nano-emulsion or nano-
suspension there
exists a dispersion medium comprised of the polymer solution, and a dispersed
phase
comprising the solution of the lipophil in the carrier. This two-phased nano-
emulsion or
nano-suspension is, however, unstable. Evaporating the carrier leaves
particles of the
dispersed phase in sizes ranging from approximately 1 to approximately 1000
nanometers.
The polymer molecule in the polymer solution then surrounds or envelopes, and
more
appropriately wraps, the active compounds that had remained in the particles
of the dispersed
phase after evaporation of the carrier, thus forming a homogeneous nano-sized
dispersion of

water-insoluble lipophilic compound wrapped by -a hydrophilic polymer in an
inclusion
complex. The remaining carrier is then evacuated by vacuum evaporation or
other
appropriate drying techniques (e.g., lyophilization, vacuum distillation). As
a result of the
algorithm used to select the optimal active compound and polymer for the
formation of the
emulsion or suspension and resulting complex, no free polymer generally
remains after the
evaporation of the carrier. Following evaporation of the carrier, the stable
inclusion complex
is comprised of amorphous and/or partially crystalline or crystalline active
entities. It is

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known by those skilled in the art that the amorphous state is preferred for
drug delivery as it
may indeed enhance bioavailability.
In an advantageous and preferred embodiment of the invention, the polymer
molecule
in the polymer solution "wraps" the active compound via non-valent
interactions (e.g.

electrostatic forces, Van der Waals forces, H-bonds) between the polymer and
active
compound such that the non-valent interactions fixate the active compound
within the
polymer which thus reduces the molecular flexibility of the active compound
and polymer.
The invention further comprises a novel chemical reactor designed for the
production
of the nano-soluparticles in accordance with the present invention. As
illustrated in FIG. 1,
the chemical reactor 10 comprises a first vessel 12 and a second vessel 14. In
accordance
with the present invention, a polymer solution 16 comprised of the selected
polymer in water
is prepared having a concentration, pH and ionic properties in accordance with
previously
determined parameters. Distilled water vessel 52 contains distilled water
indicated by "W"
and is positioned in cover 18. The distilled water in distilled water vessel
52 is transferred to
a polymer vessel 54 to which an estimated quantity of the selected polymer is
added. This
polymer solution formed in polymer vessel 54 is transferred to first vessel 12
via the action of
peristaltic pump 42 as indicated by directional arrow "X". Polymer solution 16
is added into
first vessel 12 via an opening in cover 18 with the assistance of peristaltic
pump 42. A non-
aqueous solvent ("carrier") is added to the polymer solution 16 in first
vessel 12.
The active compound is added to second vessel 14, which is connected to first
vessel
12 via reverse tube 20 so as to permit fluid communication between second
vessel 14 and first
vessel 12. A carbon dioxide (C02) balloon 56 with a pressing reducing valve 58
may provide
a feed of CO2 gas into second vessel 14. The feed of carbon dioxide acid gas
CO2 in an

organic solution improves following operational characteristics:
(a) lowering of boiling point of a solvent;
(b) lowering of density of a solvent;
(c) lowering of a thermal capacity of a solvent; and
(d) initiation of effect of "explosion" of microdrips of an organic
solution hitting in a polymeric solution (cavitation).
Factors (a) - (d) promote faster and complete removal of an organic solvent
from a
water-polymeric solution. Factor (d) promotes a more complete dividing of
microdrips of an
organic solution

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A nano-disperser 22 is positioned within first vessel 12 to create high shear
and
turbulence in the solution and to effect dispersal of the solution (in the
carrier) that enters first
vessel 12 from second vessel 14 via reverse tube 20. The nano-disperser 22
creates nano-
sized lipophilic particles within the polymer solution 16 in first vessel 12.
The nano-disperser
22 is also commonly referred to as a dispergator or homogenizer. A first
condenser 24
connected to a vacuum pump 26 extends into first vessel 12. A second condenser
28 is
connected to second vessel 14. Taps 30A, 30B, 30C are provided at various
locations on the
chemical reactor to control first and second condensers 24, 28, as well as to
regulate the flow
of solutions and vapors between said first vessel 12 and said second vessel
14.
An electrical heater 32 is positioned below first vessel 12 to heat solution
16 therein.
First vessel 12 is heated above the boiling point of the carrier, which is
lower than the boiling
point of polymer solution 16. An electric thermometer 34 extends into first
vessel 12 to
control and monitor the temperature of solution 16 within first vessel 12. A
magnetic mixer
and heater 36 is positioned below second vessel 14 to heat and mix the
lipophilic compound

with the carrier solvent in second vessel 14.
As a result of the heating, the vapors of the non-water solvent (carrier) in
first vessel
12 rise up through a steam pipe 38, enter second vessel 14 and condense
therein. In second
vessel 14, the active compound slowly dissolves in the non-aqueous solvent and
the resulting
lipophilic solution flows via reverse tube 20 back into the first vessel 12.
An opening 40 of
reverse tube 20 is arranged in such a way that the lipophilic solution enters
first vessel 12 in
the area close to the nano-disperser 22, referred to as the "action zone" or
"reaction zone",
and has a turbulent flow with a Reynolds number of Re>10,000. The Reynolds
number is a
measurement of the smoothness of flow of a fluid. A high Reynolds number
implies that the
flow is turbulent, while a low Reynolds number implies that the flow is
laminar. The
emulsion or suspension is formed here. In the action zone, the nano-disperser
operates in the
range of approximately 10,000 and up revolutions per minute.
Screen 44 prevents this turbulent flow from entering the air space 46 of the
first vessel
12 above the liquid phase. The process is continued until the entire active
compound
transfers into the polymer solution 16. The non-water solvent is removed from
both vessels
12, 14 via cooler 48 into a condensate container 50. The removal of the non-
water solvent is
illustrated by the arrow indicated by the letter "C". The leftover non-aqueous
solvent in first
vessel 12 is removed with the assistance of vacuum pump 26. The result is a
homogeneous

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nano-sized dispersion of water-insoluble lipophilic compound wrapped by a
hydrophilic
polymer in an inclusion complex.
Depending upon the previously determined protocol for a given reaction system
regime, the temperature is then lowered to ambient while simultaneously
reducing the
rotation speed to zero.
In an exemplary embodiment of the present invention, nano-sized soluble
particles of
macrolide drugs Erythromycin and Clarithromycin were prepared as inclusion
complexes in
accordance with the present invention and are described further herein in the
Examples. The
invention is not, however, limited to the formation nano-sized particles of
Erythromycin and

Clarithromycin. Other pharmaceuticals and classes of drugs are contemplated by
the present
invention, such as, for example, analgesics, anti-inflammatory agents,
anthelmintics,
antianginal agents, anti-arrhythmic agents, antibiotics (including
penicillins), anticoagulants,
antidepressants, antidiabetic agents, antiepileptics, antigonadotropins,
antihistamines,
antihypertensive agents, antimuscarinic agents, antimycobacterial agents,
antineoplastic
agents, immunosuppressants, antithyroid agents, antiviral agents, anti-
neoplastic agents and
chemotherapeutic agents, anxiolytic sedatives (hypnotics and neuroleptics),
astringents, beta-
adrenoceptor blocking agents, blood products and substitutes, cardiacinotropic
agents,
contrast media, corticosterioids, cough suppressants (expectorants and
mucolytics), diagnostic
agents, diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonian
agents),
haemostatics, immunosuppressive cyclic oligopeptides, immunological agents,
lipid
regulating agents, muscle relaxants, parasympatbomimetics, parathyroid
calcitonin and
biphosphonates, prostaglandin, radio-pharmaceuticals, sex hormones (including
steroids),
anti-allergic agents, stimulants and anorexics, sympathomimetics, thyroid
agents, vasidilators
and xanthines. Preferred drug substances include those intended for oral
administration,
intravenous administration, mucosal administration and pulmonary
administration. A
description of these classes of drugs and a listing of species within each
class can be found in
Martindale, The Extra Pharmacopoeia, Twenty-Ninth Edition, The Pharmaceutical
Press,
London, 1989.
Although the present invention has been described with reference to use in the
human
body, the invention is not limited in this respect and inclusion complexes can
be formed in
accordance with the present invention for use in veterinary pharmaceuticals
and other
products as well.

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Examples
Example 1. Experimental Procedure for the Production of the Inclusion Complex

A. Preparation of polymer solution.
500 ml of distilled water are transferred from distilled water vessel 52 to
polymer
vessel 54. To polymer vessel 54 is added an estimated quantity of polymer,
which was
chosen for creation of Inclusion Complex with lipophilic compound. At temp. 20-
25 C the
contents of polymer vessel 54 is mixed at velocity of 30-60 miri
1(rotation/min). up to
complete dissolution of the polymer and creation of transparent or opalescent
solution.

B. Loading compounds in the reactor
The polymer solution, prepared in polymer vessel 54 is transferred into first
vessel 12
by pump 42. In the same vessel the carrier solvent is loaded. Lipophilic
compound is placed
into second vessel 14.
C. Starting the reactor
The nano-disperser 22 is activated at velocity 500-800 miri 1. The cooling
water is
entered into first and second condensers 24 and 28. The heater (thermostat) 32
is activated
for temperature 5-10 C above the boiling point of carrier solvent. The
magnetic mixer 36 is
activated at velocity 5-10 min- 1.

D. Synthesis of the complex
After reaching the designated temperature, the carrier solvent starts
evaporation from
first vessel 12. Its vapor reaches second vessel 14 through steam pipe 38. At
this moment the
nano-disperser 22 is accelerated to a velocity of 8,000-10,000 miri 1. The
solution of active
compound in the carrier solvent then moves from second vessel 14 to first
vessel 12 through
reverse tube 20. The solution exits through opening 40 and reaches to the zone
of the most
active action of nano-disperser 22. The temperature in the heater 32 is raised
another 5-10 C
in order the concentration of active compound in the carrier solvent remained
within the

range 0.02-0.1%. The process lasts until all lipophilic compound passes from
second vessel
14 to first vessel 12.



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E. Removing the carrier solvent
The velocity of the nano-disperser reduced to 200-300 min 1. Tap 30A is opened
on
the conduit connecting first vessel 12 with cooler 48 and condensate container
50. The
carrier solvent is distilled off to condensate container 50. After the solvent
is transferred, tap
30A is closed together with tap 30B on cooler 28. Tap 30C, which is positioned
on the
conduit connecting first vessel 12 with vacuum-pump 26, is then opened. The
temperature in
the heater 32 is then reduced to 30-35 C, the vacuum-pump 26 is activated and
the remnants
of the solvent are evacuated for 1-2 hours. The vacuum-pump 26 is then
deactivated, all taps
30A, 30B and 30C are opened and the velocity of the nano-disperser is reduced
to 30-60 min"
1.

F. Completing the experiment
The solution of the inclusion complex is taken from first vessel 12 and was
analyzed.
The results are indicated in table 1.
Table 1. Combination of water-phase, polymer, and active compound and
the process temperature used for the preparation of selected nano-
emulsions or nano-suspensions and their stability (pre-formulation
level) determined via length of time (days).
Solvent (water Polymer Carrier active compound Process Stability
phase) Temp (days)
C
Water Carragenan Hexane Vaseline(Oleum vaselini), 65 30
+0.9% NaCI hydrocarbons' mixture

Solvent Xantan Diethyl Nut oil& almond oil 60 300
"Quartasolum" ether (Oleum Amigdalarum)
(NaCl, 1:1, aromatic esters'
KCI,NaHCO3, mixture, triglycerides,
CH3COONa) aromatic nitriles and
vitamins

Distilled water Polyacryl- Diethyl Oregano oil, phenols 55 100
+ ethanol amide ether and polyphenols,
(10%) complicated aromatic
esters' mixture

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Distilled water Starch Benzene Simethicone, 70 30
+ glycerol olygo(dimethylsiloxan)s
(10%) mixture
(Antifoaming medication)

Solvent Agar-agar Diethyl Pine Oil (raw material 60 100
"Quartasolum" ether for Camphora), mixture
of camphora, turpentine
and other terpens and
terpenoids
Example 2. Modification of polysaccharide
Distilled water with polysaccharide in varying amount was put into the vessel.
After
that a citric acid was added until the designated pH 2 at mixing was attained.
Xl signifies the
amount of polysaccaride in water, X2 signifies the pH value of water solution
of
polysaccharide
The obtained suspension is heated for approximately 10 - 20 minutes with
continuous
mixing at room temperature up till to 70-95 degrees C up till a homogeneous
opaque mass is
obtained. The obtained mass is put in an autoclave on time X3 and exposed in
an autoclave
at temperatures 160-180 C. Under these conditions the network structures of a
polysaccharide partially or completely are transformed to linear weakly
branched
macromolecules and which dissolve in water. Upon termination of the autoclave
time the
cooling below 100 C is effected and a solution of polymer is obtained. A
solution of
polyethylene glycol - 400 (PEG-400) in an amount X4 (% in relation to
polysaccharide) is
added. The obtained mixture is put in an autoclave and heated up a temperature
of 160 - 180
C during timeX5. At the end of the autoclave time cooling below 100 C is
effected and the
modified polymer is obtained. Turbidity and viscosity of the solution were
measured. The
observed data is shown in table 2.


Table 2. Modified starch based on potato starch

C p.st.,% pH Ti min PEG-400, T2 min Turbidity
Xi X2 X3 X4 % X5 FTU

4 2 30 0 0 370
22


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176

4 2 40 0 0 310
4 2 50 0 0 245
4 2 60 0 0 190
4 2 80 0 0 145
4 2 100 0 0 95
4 2 120 0 0 63
4 2 150 0 0 26
4 2 180 0 0 4
8 2 150 0 0 65
4 2 30 25 40 272
4 2 40 50 40 14
4 2 40 50 60 21
4 2 30 75 60 7
4 2 50 75 40 4
4 3 30 25 60 126
4 3 50 25 40 130
4 3 50 75 60 143
4 3 30 75 40 400
4 3.5 40 25 60 129
4 3.5 60 25 45 100
4 3.5 40 25 90 63
8 2 150 0 - 270
8 2 150 50 60 210
4 2 150 50 60 71
4 3 40 100 40 7
4 2 40 100 40 4
4 2 50 25 60 3
4 2 80 75 60 23

Example 3. Creation of solu nano-particles wrapped in modified polysaccharide
(parts
by weight)

In distilled water a polysaccharide is dissolved, initially heated at 160 -
180 degrees C
up to molecular masses (5-10)x 10(4) and is modified by the polyethylene
glycol PEG-400.
Conditions of modification: ratio "polysaccharide - polyethylene glycol PEG-
400" ratio from
2:1 up to 4:1, acidic environment with pH 2 - 5 created by a citric acid,
temperature 160-180
C, time of modification 60-180 min. Solution of a modified polysaccharide is
put in a

reactionary vessel, heated up to 60 C mixing by a homogenizer at speed 10,000
and up
rev/min.
Simultaneously a solution of macrolide in an organic solvent is prepared.
Allowing
the solution of a polysaccharide to reach given temperature 60 C, then it
start to add a

23


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
solution of macrolide with speed about 1 ml / sec.. Speed of a homogenizer is
increased to 10
thousand rev/min and up. The macrolide interacts with the polymer creating
nanoparticles,
and the organic solvent is evaporated. The organic solvent is condensed in a
direct
condenser. After all the macrolide has entered interaction with the polymer
and has
solubilized as an inclusion complex " macrolide- polymer ", the organic
solvent was vacuum
evaporated with continuous mixing, and the solution of the complex was cooled
to 30-35 C.
Turbidity (table 3) and viscosity are measured in the cooled solution. The
presence of
a crystalline phase and particles sizes of the complex were measured


Table 3. Complexes with Clarithromycin

Product Solution components Store Time Turbidity (FTU)
number (days) at room
temper ture
Hydrolyzed polysaccharide - 5%, 0 29
39 clarithromycin -1 %, 4 27
pH 5.0 10 35
28
26 30
Hydrolyzed polysaccharide - 0 38
37 4%,Clarithromycin - 2%, 5 40
pH 4.5 21 36
27 43
Hydrolyzed polysaccharide - 0 36
40 4%,Clarithromycin - 2%, 1 36
pH 5.5 7 37
17 36
Hydrolyzed modified (50% PEG) 0 40
42 polysaccharide - 6%, clarithromycin - 1 40
1%,pH4.5 6 39
16 41
22 40
Hydrolyzed modified (25% PEG) 0 21
34 polysaccharide - 3.75%, clarithromycin - 10 20
1%,pH4.5 16 19
26 20
Hydrolyzed modified (50% PEG) 0 46
36 polysaccharide - 6%, clarithromycin - 15 48
1.5%,pH5.0 21 47
49
24


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
Hydrolyzed modified (30% PEG) 0 26
38 polysaccharide - 5.2%, clarithromycin - 4 28
1.7%,pH5.5 10 27
20 26
Hydrolyzed modified (50% PEG) 0 38
43 polysaccharide - 12%, clarithromycin - 1 37
2.5%, pH 6.5 5 39
15 37
25 38
Hydrolyzed modified (50% PEG) 0 36
46 polysaccharide - 6%, clarithromycin - 1 40
2.5%, pH 5.0 3 44
9 48
50
17 47
Hydrolyzed polysaccharide - 3.75%, 0 32
47 clarithromycin - 1.5%, pH 4.5 1 31
6 35
14 32
Hydrolyzed polysaccharide - 3%, 0 25
48 clarithromycin -1.5%, 1 25
pH 5.0 2 26
8 25
Hydrolyzed polysaccharide - 8%, 0 70
50 clarithromycin - 1%, 1 77
pH 5.0 4 79
6 78
Hydrolyzed modified (25% PEG) 0 64
51 polyssacharide - 5%, clarithromycin - 1 62
3%, pH 5.0 2 65
3 61
10 64
Stable Turbidity = stable nano-dispersion.

Example 4. In Vitro Microbiological Results with Clarithromycin in Nano-
Particle Complex
5
The microbiological activity of complexed Clarithromycin prepared in Example 5
at
various concentrations were tested and compared directly to un-complexed
Clarithromycin at
the same concentrations. The testing method used was that of the accepted agar-
filled petri-
dish tests. The test microbe used was Micrococcous luteus, which is sensitive
to macrolide
10 antibiotics. Small filter paper cut discs were impregnated with specific
solution
concentrations of the tested antibiotics. Diameters of the zones of
bacteriostatic activity were
measured versus time. Concentrations were varied significantly for both
control and



CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
complexed testing material and observed until 216 hours post application.
These results are
illustrated in FIG. 2.
This test further demonstrated that the complexed clarithromycin shown to have
the
same microbiological activity as commercial clarithromycin while using 1/10 of
the amount
(concentration). Furthermore, for identical concentrations of drug, the
Clarithromycin

microbiological activity ceased at approximately 48 hours, while that of the
complexed
Clarithromycin continues significantly till approximately 216 hours of current
measurements
and we are continuing measurements. while that of the complexed Clarithromycin
continued
significantly until approximately 216 hours of current measurements. It was
also observed

that the difference in microbiological activity for complexed Clarithromycin
having
concentration differences of an order of magnitude between them is vastly
greater than the
corresponding differences noted with Clarithromycin alone.

Example 5. In vivo Studies with Clarithromycin Inclusion Complex
Rats received clarithromycin in nano-particle complex according to the present
invention by gavages 150 mg/kg. Blood samples were collected in time intervals
through the
jugular catheter.
Values of time 0 were the control baseline for each animal. Following oral
administration of clarithromycin in nano-particle complex, it was determined
that the drug
reached its maximum plasma value 4 hours following administration. The first
absorption
phase was rapid -- up to 1 hour and continued until maximum at 4 hours. The
clearance was
significantly slow in comparison to published data with the commercial
clarithromycin. The
circulating half-life was in the range of 2 hours. The Area Under the Curve
(AUCo-24hours) of
the clarithromycin complex in accordance with the present invention was
significantly higher
54.2 microg*h/ml in comparison to published data with the same dose of the
commercial
Clarithromycin in rats AUCo-24hours = 32.54 microg*h/ml with same oral dose of
150mg/kg. It
is thus believed that: complexed clarithromycin in accordance with the present
invention
exhibits either enhanced bio-availability or intestinal slow release following
oral
administration.
The clarithromycin complex exhibited the same range of circulating half-life
i.e., 2
hours in comparison to the commercial drug following IV bolus administration.
It possesses a
significant higher AUC after oral administration support the assumption of
bioavailability
enhancement or slow release properties.

26


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176

A comparison of the pharmacokinetics constants of the tested clarithromycin in
nano-
particle complex compared to published data of commercial clarithromycin is
illustrated in
FIG. 3. PK constants of clarithromycin in nano-particle complex in comparison
with
published studies with commercial clarithromycin is illustrated in FIG. 4.

Example 6. Physical Measurements and Characteristics of Clarithromycin and
Erythromycin in Nano-Particle Complex

1. Particle Size and Distribution
Complexes of Erythromycin or Clarithromycin plus polymer in aqueous solutions
have shown that the technology of the present invention allows the creation of
drug-polymer
dispersions with controllable nano-particle sizes, ranging from single
nanometers up to 1000
net, with a highly uniform size distribution.
A complex of Clarithromycin prepared according to the method of the present
invention showed identical dispersion spectra after 5 weeks time. FIG. 5
illustrates a
complexed Clarithromycin particle having a size of approximately 190 net. Size
measurements of the Erythromycin and Clarithromycin complexes have been
performed using
"ALV-Particle Sizer", which has a resolution of from 3-3000 rim. FIG. 6 is an
SEM
micrograph illustrating the consistent spherical complexed Clarithromycin
particles prepared

according to the method of the present invention.
2. Solubility
Erythromycin, an antibiotic practically insoluble in water, has been
reformulated into
thereto-dynamically stable nano-dispersions, with controllable size
distribution of the
particles in the dispersed phase. The resulting new formulation has 8% (w/v)
active drug,
which is 40 times higher than the solubility of the original drug in water
(0.2%). Moreover,
drug particles with a highly uniform size of complexes (over 95%) were
achieved. The
erythromycin was released from the inclusion complex in sufficient
concentration under
physiological conditions. No existing technologies of solubilization were
used, e.g.
surfactants, liposome, capsulation, etc. A comparison of the solubility of
Erythromycin and
Clarithromycin alone and as part of the inclusion complex in accordance with
the present
invention is illustrated in FIG. 7.

27


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
3. Stability
Observations were made of transparent aqueous solution of inclusion complexes
for
non-occurrence of phase separation and maintenance of particle size and size
distribution.

The following observations and results were obtained:
(a) Over the 75 days, the tests of the reformulated 8% Erythromycin showed
no phase separation and maintenance of particle size and size distribution.
(b) The stability of the complexed Clarithromycin in accordance with the
present invention was observed for 12 weeks at room temperature and 4 weeks at
35 C and
they found to be stable.
(c) Freeze-drying and subsequent rehydration of complexed Clarithromycin,
retained particle size of the drug-polymer complexes. For more than 30 days
there was no
aggregation and the nano-dispersion were stable.

4. X-Ray Diffraction Results and Characterizations

From the X-ray diffraction measurements it was found that the reformulation of
the
crystalline drug Erythromycin into nano-dispersions was accompanied by its
conversion into
an amorphous form material.
FIG. 8 and FIG. 9 illustrate the X-ray diffraction comparisons of intact
Erythromycin
and intact Clarithromycin compared with the inclusion complex of Erythromycin
and
Clarithromycin respectively in accordance with the present invention.

The comparison of known spectra of Erythromycin (FIG. 8) and Clarithromycin
(FIG. 9) with inclusion complexes in accordance with the present invention
were conducted.
The known spectrum of Erythromycin (FIG. 8) as a dry powder shows a well-
defined
crystalline pattern.
In comparison, the Erythromycin inclusion complex (FIG. 8) demonstrates that
the
majority of peaks derived from crystalline Erythromycin are not present, and
the few
remaining peaks have been drastically reduced in height. This spectrum is
undoubtedly
related to that of the known Erythromycin, however it is indicative that
another "form" is
now present after complexation.
When observing the average scattering angles in the spectra of both complexed
Erythromycin and Clarithromycin one can clearly see that certain peaks have
been "flattened"
28


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
showing widened virtually base line peaks. This phenomenon is indicative of an
amorphous
state.

These results show that complexation of Erythromycin and Clarithromycin using
the
technology of the present invention reduces crystallinity of the uncomplexed
drugs, as the
crystal lattices are unable to form, due to fixation of the drugs within the
inclusion polymer
on the basis of Van der Waals and hydrogen bonds. It is known that the
amorphous state is
preferred for drug delivery as it may indeed enhance bioavailability.

The X-ray spectrum of FIG. 10 depicts a 6 month old clarithromycin complexed
sample (bottom trace) compared to the commercially available Clarithromycin
(upper trace).
This specific complexed sample is identical to that appearing in FIG. 9 and in
the
microbiological tests discussed in Example 6. This validates the technological
ability to
prepare uniquely complexed drug conjugates in accordance with the present
invention that
demonstrate significantly stabilized amorphous states.
The present inventors believe that such stabilization of amorphous or
partially
amorphous drug states within the inclusion complex may well increase the
chances of greater
bioavailability as has been documented in the literature. Taken together with
other
parameters attained using the process and apparatus of the present invention,
such as very
accurate size control, the process lends itself easily to significantly
increased bioavailabilities.

Example 7. Controlled release from the Erythromycin Inclusion Complex.
Reformulation of the drug in inclusion complexes represents a new avenue to
achieve
controlled release systems that would deliver the drug at a specific rate and
pattern. To
examine the experimental controlled release pattern of Erythromycin from the
inclusion
complex, a dialysis method was been performed. In this method, the drug-
polymer nano-
dispersions were placed within a dialysis membrane bag. Such a membrane allows
the
diffusion of only molecules and ions of sizes less than 3000 Da, while
maintaining the nano-
dispersions. Dialysis was performed for 24 hours at a room temperature and
with a constant
stirring. Samples from the external buffer were taken periodically for the
analysis of drug
release. The concentration of Erythromycin released from the Inclusion Complex
was
detected by measuring the O.D. (optical density). After 24 hours of
incubation, the
concentration of Erythromycin in the external fluid was 25% of the initial
concentration of
Erythromycin in the inclusion complex (initial concentration is 4 mg/ml
(8%w/v)). The

29


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
released concentration also reflects the maximum solubility of erythromycin in
a serum-
modeled solution. Thus, this result indicates that the nano-dispersion has a
capability to
sustain the release of Erythromycin.

Example 8. In vitro Human Cellular Compatibility Study
Erythrocytes were separated from WBC of a fresh donor, and suspended in
isotonic
buffer. In a water and lyses buffer treatment erythrocytes were suspended in
the indicated
buffer. Hemolytic reactions were carried at 37 C with shaking (40 rpm) in a
total volume of
1 ml. An aliquot of 250 l was removed at 4 hr, and the rest was collected at
18 hr. Aliquots

were centrifuged at 250g for 5 min., and supernatant was read at 540 nm. The
results of this
test found the complexed Clarithromycin to be compatible with human blood.

Example 9. Suspension polymerization in nano-soluparticles
Using the chemical reactor of the present invention as illustrated in FIG. 1,
caprolactam is dissolved in ethyl ether. An amylose was modified by Urea up to
an amidation
degree 10 % and after that a solution of a modified amylose was prepared. The
polymer
solution was transferred into first vessel 12 and the caprolactam solution was
transferred into
second vessel 14. Nano-disperser 22 and heater 32 were activated. The heater
(thermostat)
32 was activated for 50-55 C. Caprolactam solution was then transferred from
second vessel
14 to first vessel 12 through reverse tube 20. After the all caprolactam
solution was fed
through reverse tube 20, the temperature of the reaction mixture was reduced
to 25-35 C and
evaporated from the reactor.
At the polymerization of Polycaprolactam (nylon - 6,6) the obtained "solution"
is
sprayed in a vacuum column with temperature 260-280 C. Polymer as fine
homogenous
powder (with uniform size of particles) is taken from the bottom of the
string. A molecular
weight of polymer is determined by a viscosity method.
The results are indicated in table 4.

Table 4
Concentratio Caprolactam Concentration Amylose Speed of Interaction MW
n of Solution of Amylose solution homogenizer time
Caprolactam (g) modified in (g) (rev/min)
in ethyl ether water
% %
2 2,000 4 250 20,000 150 120


CA 02461890 2004-03-26
WO 03/028700 PCT/IB02/04176
3 1,300 4 250 18,000 115 100
4 1,000 4 250 12,000 80 90
800 4 250 16,000 60 90
Equivalents
5 Thus, while there have been shown and described and pointed out fundamental
novel
features of the invention as applied to preferred embodiments thereof, it will
be understood
that various omissions and substitutions and changes in the form and details
of the disclosed
invention may be made by those skilled in the art without departing from the
spirit of the
invention. It is the intention, therefore, to be limited only as indicated by
the scope of the

claims appended hereto.
It is to be understood that the drawings are not necessarily drawn to scale,
but that
they are merely conceptual in nature.

31

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date 2010-12-14
(86) PCT Filing Date 2002-09-27
(87) PCT Publication Date 2003-04-10
(85) National Entry 2004-03-26
Examination Requested 2007-09-26
(45) Issued 2010-12-14
Deemed Expired 2013-09-27

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2004-03-26
Application Fee $400.00 2004-03-26
Maintenance Fee - Application - New Act 2 2004-09-27 $100.00 2004-08-31
Maintenance Fee - Application - New Act 3 2005-09-27 $100.00 2005-09-14
Maintenance Fee - Application - New Act 4 2006-09-27 $100.00 2006-09-05
Maintenance Fee - Application - New Act 5 2007-09-27 $200.00 2007-09-24
Request for Examination $800.00 2007-09-26
Maintenance Fee - Application - New Act 6 2008-09-29 $200.00 2008-09-18
Maintenance Fee - Application - New Act 7 2009-09-28 $200.00 2009-08-07
Final Fee $300.00 2010-08-10
Maintenance Fee - Application - New Act 8 2010-09-27 $200.00 2010-09-22
Maintenance Fee - Patent - New Act 9 2011-09-27 $200.00 2011-09-16
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
SOLUBEST LTD.
Past Owners on Record
GOLDSHTEIN, RINA
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Abstract 2004-03-26 2 76
Claims 2004-03-26 13 566
Drawings 2004-03-26 8 178
Description 2004-03-26 31 1,774
Representative Drawing 2004-03-26 1 34
Cover Page 2004-06-03 1 53
Claims 2007-09-26 5 157
Description 2007-09-26 33 1,875
Description 2009-10-29 33 1,865
Representative Drawing 2010-11-25 1 17
Cover Page 2010-11-25 2 52
Assignment 2004-03-26 6 293
Prosecution-Amendment 2004-03-26 1 23
Prosecution-Amendment 2007-09-26 9 302
Fees 2007-09-24 1 35
Fees 2007-09-24 1 35
Prosecution-Amendment 2009-05-13 2 44
Prosecution-Amendment 2009-10-29 3 110
Correspondence 2010-08-10 1 38