Note: Descriptions are shown in the official language in which they were submitted.
21137~
SOLID LIPID PARTICLES, PARTICLES OF BIOACTIVE AGENTS AND
METHODS FOR THE MANUFACTURE AND USE THEREOF
This invention relates to suspensions of particles of biodegradable lipids
5 solid at room temperature, preferably triglycerides, which can be used as
carriers for poorly water soluble drugs or other bioactive agents, and to
suspensions of particles constituted by biologically active agents such as
drugs, insecticides, fungicides, pesticides, herbicides and fertilizers, as well as
to the Iyophilisates thereof. Both systems can be prepared by a melt
10 emulsification process.
The properties of the solid lipid particles (SLPs) include biodegradability,
avoidance of toxicologically active residues from the production process,
enhanced physicochemical stability with regard to coalescence and drug
leakage, modified surface characteristics, controlled release of incorporated
15 substances and modified biodistribution. The particles can be prepared by an
emulsification process of molten material creating liquid droplets which form
crystalline, anisometrical particles on cooling. The anisometrical particles areof micron and submicron size, predominantly in the size range from 50 to 500
nm. The described suspensions have several advantages over other drug
20 carrier systems deriving from the solid biodegradable matrix being
predominantly present in a ,~-polymorphic modification (e.g. ~ 2)l and not
in an amorphous or a-crystalline state.
The preparation of micron and submicron particles consisting of poorly
water-soluble bioactive agents (PBAs) by emulsification of the molten
25 substance presents a novel process to reduce the particle size and/or to
modify the surface characteristics of powdered substances which can be
accomplished by inexpensive techniques and by the use of physiologically
acceptable additives only. The suspensions of the particles are an easy to
handle product from the security point of view. The particles of bioactive
30 agents provide for the modified biodistribution and bioavailability of the
formulated drugs or other bioactive substances which implies a modification
of the extent and rate of dissolution and absorption, the circulation time, the
site of action and the way of disposition of the drug or other bioactive
- ~ 13795
substance. A reduction in particle size below the micrometer range provides
for the direct intravenous administration of particles made from poorly water
soluble drugs without the need of a carrier vehicle.
5 FIELD OF THE INVENTION
The present invention is in the area of administration forms and delivery
systems for drugs, vaccines and other biologically active agents such as
insecticides, fungicides, pesticides, herbicides and fertilizers. More specifically,
10 the invention is related to the preparation of suspensions of colloidal solid lipid
particles (SLPs) with the lipid matrix being in a stable polymorphic modification
and of suspensions of micron and submicron particles of bioactive agents
(PBAs); as well as to the use of such suspensions or the Iyophilisates thereof
as delivery systems primarily for the parenteral but also for the peroral, nasal,
15 pulmonary, rectal, dermal and buccal administration of preferably poorly water-
soluble bioactive substances, particularly drugs; and to their use in cosmetic,
food and agricultural products. These suspension systems provide for the
controlled release of incorporated or constituting substances as well as for themodified biodistribution and bioavailability of incorporated or constituting drugs
20 which implies a modification of the extent and rate of dissolution and
absorption, the circulation time, the site of action and the way of disposition of
the drug.
BACKGROUND OF THE INVENTION
The parenteral, in particular the intravenous administration of water-
insoluble or poorly water-soluble substances such as drugs or other biological
materials often presents a problem to the formulator. Since the diameter of
the smallest blood capillaries is only a few microns, the intravenous application
30 of larger particles would lead to capillary blockage. Solid drug substances are,
however, commonly disintegrated by milling and grinding thereby generating
particles from a few millimeters down to the micrometer size range which are
too large to be injected directly as an aqueous suspension. As a consequence,
~1~37~
intravenous administration systems containing suspended particles of water
insoluble drugs are not commercially available due to the risk of embolism. A
further decrease in particle size is expensive, ineffective or even impossible by
conventional techniques. Additionally, the reduction of solids to submicron-
5 sized powders brings about heavy difficulties in handling of these dry productssuch as an increased risk of dust explosions and cross-contamination
problems in a factory environment. Moreover, such systems present a risk to
health for persons exposed to the possible inhalation and absorption of potent
bioactive materials. Up to now, the only possibilities to administer poorly water-
10 soluble substances by the intravenous route are the use of co-solvents or thedevelopment of carrier systems which incorporate such substances in vehicles
with hydrophilic surfaces.
Basic requirements of an ideal drug carrier system imply
biodegradability, nontoxicity and nonimmunogenicity. Moreover, the carrier
15 should be suitable for the intended route of administration e.g. with regard to
particle size. Often a controlled release of the incorporated bioactive materialis desired, for example when constant serum levels should be maintained over
a long period of time or when the drug exhibits only a low therapeutic index.
Furthermore, carrier systems can be employed to prolong the half-life
20 of certain substances which are unstable due to rapid enzymatic or hydrolyticdegradation in biological milieu. On the other hand, the incorporation of drug
in the carrier material also presents an opportunity to protect the host from the
drug in case of non-selective toxic substances such as antitumour agents.
In many cases drug carrier systems are developed with the object to
25 deliver drugs to site-specific targets under circumvention of uptake by the
reticuloendothelial system (RES). The rationale for such a drug targeting is an
enhancement of the drug s therapeutic efficacy by an increase of the drug
concentration at the target site with a simultaneous decrease at non-target
sites thereby rendering possible a reduction of the administered dose. Thus
30 the toxicity of drugs, e.g. anticancer agents, can be diminished leading to a decrease of side effects.
The prerequisite of a successful site-specific delivery implies a certain
selectivity of the carrier system for the target tissue as well as the accessibility
21~373~
of the desired target site. Targeting by the intravenous route of application isgenerally connected to an avoidance or at least reduction of carrier uptake by
the RES except for the cases where a direct targeting to cells of the RES is
desired. Clearance of colloidal particles by the RES has been described to
depend on particle size as well as on particle surface characteristics such as
surface charge and surface hydrophobicity. In general, small particles are
cleared less rapidly from the blood stream than large particles whereas
charged particles are taken up more rapidly than hydrophilic non-charged
particles. Due to these facts, approaches to drug targeting are the modificationof surface characteristics and the reduction of particle size.
Moreover, a small particle size is also required for the targeting of drugs
to extravascular sites since extravasation is only feasible through a receptor-
mediated uptake by phagocytosistpinocytosis or where the endothelial wall is
fenestrated. These fenestrations can be found e.g. in the sinusoids of liver,
spleen and bone-marrow and show diameters of up to approximately 150 nm.
From the manufacturing point of view, the ideal drug carrier system
should be preparable without complications by easy-to-handle techniques in
a reproducible manner and possibly at low production costs. The formulation
should exhibit sufficient stability during preparation as well as on storage.
In recent years, several colloidal systems have received special interest
for their potential application as drug carriers among them being liposomes,
lipid emulsions, microspheres and nanoparticles. However, all of the systems
mentioned possess a certain number of drawbacks which so far have
prevented the break-through of any such system as a widespread
commercially exploited drug carrier.
Drug carrier systems in the micrometer size range are represented by
microspheres consisting of a solid polymer matrix, and microcapsules in which
a liquid or a solid phase is surrounded and encapsulated by a polymer film.
Nanoparticles consist, like microspheres, of a solid polymer matrix, however
their mean particle size lies in the nanometer range. Both micro- and
nanoparticles are generally prepared either by emulsion polymerization or by
solvent evaporation techniques. Due to these production methods, micro- and
nanoparticles bear the risk of residual contaminations from the production
21 1379~
process like organic solvents such as chlorinated hydrocarbons, as well as
toxic monomers, surfactants and cross-linking agents which can lead to
toxicological problems. Moreover, some polymeric materials such as polylactic
acid and polylactic-glycolic acid degrade very slowly in vivo so that multiple
administration could lead to polymer accumulation associated with adverse
side effects. Other polymers such as polyalkylcyanoacrylates release toxic
formaldehyde on degradation in the body.
Drug carrier systems for parenteral administration which are based on
lipids are liposomes and submicron lipid emulsions. Although such systems
consist of physiological components only thus reducing toxicological problems,
there are a number of disadvantages associated with these lipid carriers.
Liposomes are spherical colloidal structures in which an internal
aqueous phase is surrounded by one or more phospholipld bilayers. The
potential use of liposomes as drug delivery systems has been disclosed inter
alia in the U.S. Pat. Nos. 3,993,754 (issued Nov. 23, 1976 to Rahmann and
Cerny), 4,235,871 (issued on Nov 25, 1980 to Papahadjopoulos and Szoka),
and 4,356,167 (issued Oct 26, 1982 to L. Kelly). The major drawbacks of
conventional liposomes are their instability on storage, the low reproducibilityof manufacture, the low entrapment effficiency and the leakage of drugs.
According to the IUPAC definition, in an emulsion liquids or liquid
crystals are dispersed in a liquid. Lipid emulsions for parenteral administration
consist inter alia of liquid oil droplets, predominantly in the submicron size
range, dispersed in an aqueous phase and stabilized by an interfacial film of
emulsifiers. Typical formulations are disclosed in the Jap. Pat. No. 55,476/79
issued on May 7, 1979 to Okamota, Tsuda and Yokoyama. The preparation
of a drug containing lipid emulsion is described in WO 91102517 issued on
March 7, 1991 to Davis and Washington. The susceptibility of these lipid
emulsions towards the incorporation of drugs is relatively high due to the
mobility of drug molecules within the internal oil phase since diffusing
molecules can easily protrude into the emulsifier film causing instabilities which
lead to coalescence. Furthermore, release of incorporated drugs from lipid
emulsions is relatively fast so that the possibilities for a sustained drug release
are limited.
21137~
Fountain et al. (U.S. Pat. No. 4,610,868 issued Sept. 9, 1986)
developed lipid matrix carriers which are described as globular structures of
a hydrophobic compound and an amphiphatic compound with diameters from
about 500 nm to about 100,000 nm. The hydrophobic compound can be liquid
5 or solid. The preparation techniques employ, however, organic solvents and
are thus associated with the problem of complete solvent removal.
So-called lipospheres disclosed by Domb et al. (U.S. Pat. Appl. No.
435,546 lodged Nov 13, 1989; Int. Appl. No. PCT/US90/06519 filed Nov. 8,
1990) are described as suspensions of solid, water-insoluble microspheres
10 formed of a solid hydrophobic core surrounded by a phospholipid layer.
Lipospheres are claimed to provide for the sustained release of entrapped
substances which is controlled by the phospholipid layer. They can be
prepared by a melt or by a solvent technique, the latter creating toxicological
problems in case the solvent is not completely removed.
A slow release composition of fat or wax and a biologically active
protein, peptide or polypeptide suitable for parenteral administration to animals
is disclosed in U.S. Pat. Appl. No. 895,608 lodged Aug 11, 1986 to Staber,
Fishbein and Cady (EP-A-0 257 368). The systems are prepared by spray
drying and consist of spherical particles in the micrometer size range up to
20 1,000 microns so that intravenous administration is not possible.
Problems with the formulation of water insoluble or poor.ly water soluble
substances are not restricted to the parenteral route of administration. Thus,
the peroral bioavailability of drugs is related to their solubility in the gastro-
intestinal tract (GIT) and it is generally found that poorly water so!uble drugs25 exhibit a low bioavailability. Moreover, the dissolution of drugs in the GIT is
influenced by their wettability. Substances with apolar surfaces are scarcely
wetted in media so that their dissolution rate is very slow.
In an attempt to improve the intestinal absorption of lipophilic drugs,
Eldem et al. (Pharm. Res. 8, 1991, 47-54) prepared lipid micropellets by spray-
30 drying and spray-congealing processes. The micropellets are described as
spherical particles with smooth surfaces. The lipids are, however, present in
unstable polymorphic forms and polymorphic phase transitions occur during
storage so that the product properties are constantly changing (Eldem, T. et
~11379~
al., Pharm. Res. 8, 1991, 178 -184). Thus, constant product qualities cannot
be assured.
Lipid nanopellets for peroral administration of poorly bioavailable drugs
are disclosed in EP 0 167 825 of Aug 8, 1990 to P. Speiser. The nanopellets
represent drug loaded fat particles solid at room temperature which are small
enough to be persorbed. Persorption is the transport of intact particles throughthe intestinal mucosa into the Iymph and blood compartment. The lipid
nanopellets are prepared by emulsifying molten lipids in an aqueous phase by
high speed stirring. After cooling to room temperature the pellets are dispersedby sonication.
OBJECT OF THE INVENTION
Considering the limitations of conventional drug carriers such as
liposomes, lipid emulsions, nanoparticles and microspheres as outlined above
there is an obvious demand for a carrier system for the controlled delivery of
poorly water-soluble bioactive substances which circumvents the drawbacks
of traditional systems particularly with regard to preparation, stability, toxicity
and modification of biodistribution.
The present invention introduces a new type of carrier system
characterized as non-spherically shaped particles composed of crystalline
lipids, preferably triglycerides, and physiologically acceptable additives as well
as a process for the manufacturing thereof. These carriers provide for the
controlled delivery of poorly water-soluble substances such as drugs or other
biological materials primarily by the parenteral but also by the peroral, nasal,pulmonary, rectal, dermal and buccal route of administration, and will
hereinafter be referred to as solid lipid particles (SLPs).
SLPs are characterized as lipidic particles of solid physical state in the
micro- and predominantly in the nanometer size range. The shape of the
particles is mainly anisometric which is a consequence of the matrix forming
lipids being present in a ~-polymorphic modification (e.g. ~ 2)~ and not
in an amorphous or a-crystalline state. The properties of the SLPs include: (1 )biodegradability and nontoxicity; (2) the ability to incorporate poorly water
~1137~ 5
soluble substances; (3) improved chemical and physical stability; (4) the
possibility to prepare a dry storage formulation; (5) control of release
characteristics of incorporated substances; and (6) modified surface
characteristics. As a result of these properties, SLPs overcome many of the
problems encountered with conventional drug carrier systems.
The present invention is supposed to bring about the following
advantages as derived from the characteristics of the SLPs described above:
(1) SLPs can be prepared of biodegradable, pharmacologically acceptabie
compounds only and are therefore non-toxic. Additionally, the
preparation of SLPs avoids the employment of organic solvents or any
other potentially toxic additives thus evading the contamination of the
product with residual impurities.
(2) SLPs possess an enhanced chemical stability as compared with
conventional lipid emulsions based on liquid triglyceride oils owing to
the lower degree of unsaturated fatty acids of solid triglycerides.
Moreover, SLPs exhibit a better physical stability due to the solid nature
of the lipid matrix which is expectedly more resistant to coalescence
than fluid emulsion droplets. Furthermore, the lipid matrix is present in
a stable ~-polymorphic modification (e.g. ~ " ~(32) Thus the product
properties will not change significantly during long-term storage due to
polymorphic transformations.
(3) Suspensions of SLPs can be Iyophilized by freeze-drying to provide a
water-free storage system that exhibits a good long-term stability. The
Iyophilized powder can be redispersed in water, buffer or solutions of
amino acids, carbohydrates and other infusion solutions directly before
use or can be processed into other pharmaceutical formulations.
(4) Due to their lipophilic nature SLPs are suited for the solubilization of
lipophilic and poorly water soluble substances by entrapment into the
lipid matrix. Compared to lipid emulsions SLPs are supposed to be less
sensitive to the incorporation of drugs or other bioactive materials, since
the mobility of drug molecules is drastically reduced in the crystalline
matrix thereby hindering the drug to diffuse into the emulsifier film or to
recrystallize and thus perturb the stabilizing shield of the particles.
9 211379~
(5) Drug release from the lipid carrier can be controlled e.g. by the
composition of the lipid matrix, by the choice of stabilizing agents as
well as by the size of SLPs. Drug leakage is hindered by the solid state
of the carrier due to the restricted drug diffusion.
5 (6) Drugs or bioactive substances exhibiting a short half-life due to
enzymatic or hydrolytic degradation can be protected from rapid
decomposition by incorporation within the lipid carrier since the
hydrophobic matrix prevents the access of water to the incorporated
drug - on storage as well as in body fluids.
10 (7) The incorporation into SLPs of drugs or other bioactive substances with
a low bioavailability due to poor solubility in the gastro-intestinal tract
(GIT) can enhance the bioavailability of such substances because these
are solubilized in the biodegradable lipid matrix and are thus present in
the dissolved state.
15 (8) Due to the anisometrical shape of SLPs the specific surface area is
larger than that of spherical particles of the same volume. Substances
with a low peroral bioavailability can be absorbed faster and to a higher
degree in the GIT when they are incorporated in anisometrical SLPs
than in spherical lipid particles of the same volume due to the larger
surface area of SLPs since the potential site of action for lipolytic
enzymes is larger.
(9) The surface characteristics of SLPs can be modified by variation of the
lipid composition, use of different stabilizers, exchange of surfactants
and/or adsorption of polymeric compounds. The modification of surface
characteristics brings about the possibility to modify the in vivo
distribution of the carrier and the incorporated substance. In case of
intravenous administration this implies a modified uptake by the RES
with the potential for drug targeting.
(10) Due to the submicron particle size of SLPs there is no risk of embolism
by parenteral administration. Since SLPs can be prepared down to a
particle size of about 50 nm, they possess the opportunity for
extravasation through fenestrations of the endothelial wall. Thereby
2 1 ~ 373~
drugs can be targeted to extravascular sites such as the bone marrow,
for example.
Furthermore, the present invention introduces a new type of delivery
sytem for the parenteral, peroral, nasal, pulmonary, rectal, dermal and buccal
administration of drugs or other bioactive substances as well as the process
for the manufacturing thereof. These formulations are suspensions of particles
formed by bioactive substances with modified surface characteristics and/or a
reduced particle size as compared to the powdered substance, and will
hereinafter be referred to as particles of bioactive agents (PBAs). The
preparation of PBAs can avoid the employment of any toxicologically active
additives such as organic solvents or toxic monomers, and can be
accomplished by easy-to-handle techniques.
PBAs can be used in the following fields of application:
15 a) as a parenteral delivery system with modified biodistribution forsparingly water soluble bioactive substances without the need of a
carrier vehicle;
b) as a delivery system according to a) for peroral, nasal, pulmonary,
rectal, dermal and buccal administration;
20 c) as a formulation for the peroral administration of drugs with a poor
bioavailability due to a low dissolution rate in the gastrointestinal tract;
d) as a delivery system for use in agricultural applications;
e) the Iyophilisate of formulations a) to d) as a reconstitutable powder with
an enhanced stability on storage;
Owing to the special characteristics of the present invention PBAs are
supposed to bring about the following advantages over conventional
pharmaceutical delivery systems:
1 ) The formulation of poorly water-soluble drugs or other bioactive
substances as micron and submicron particles avoids the need of a
carrier system for their parenteral application thereby circumventing the
disadvantages of conventional drug carriers like liposomes, lipid
emulsions, nanoparticles and microspheres.
21 ~ 3~3
-- 11
2) PBAs can be prepared by easy-to-handle techniques in a reproducible
way. There are no problems to foresee for the scaling up of the
manufacturing process.
3) Since the particles consist of the pure bioactive compound with only
small amounts of stabilizers, the drug load capacity of the drug particles
is high.
4) The release of drugs or other bioactive compounds from the formulation
can be controlled by the choice of amphiphatic compounds employed
to stabilize the particles.
10 5) The preparation of PBAs can avoid the use of toxicologically active
additives.
6) A water-free storage system with enhanced stability can be produced,
for example, by freeze-drying of the PBA dispersions.
7) The surface characteristics of PBAs can be modified by the choice of
amphiphatic compounds used as stabilizers as well as by the
attachment of so-called homing devices for the targeting of drugs, e.g.
monoclonal antibodies or carbohydrate moieties. The surface
modifications give rise to a modified bioavailability and biodistribution
with regard to the extent and rate of absorption, the circulation time, the
site of action and the way of disposition of the bioactive substance. The
modification of surface characteristics also provides the opportunity to
avoid or at least to reduce the uptake of intravenously administered
particles by cells of the RES.
8) Since the particles can be prepared with a size below 100 nm to 200
nm, they possess the opportunity for extravasation by fenestrations of
the endothelial wall. Thereby drugs can be targeted to extravascular
sites such as the bone-marrow, for example.
9) A reduction in particle size to the nanometer size range which is
generally not achievable by milling or grinding leads to an enormous
increase of the specific surface area of the particles. Since the peroral
bioavailability of drugs or other bioactive substances is related to the
specific surface area via the dissolution rate of the substance in the
~113~ '3
12
gastrointestinal tract, the submicron sized particles give rise to an
enhanced bioavailability of drugs poorly soluble in the GIT.
10) Hydrophobic substances can be formulated as PBAs with hydrophilicsurfaces. Hydrophilic surfaces provide for a good wettability of the
particles e.g. in the GIT facilitating dissolution of the compound. Thus
the bioavailability can be increased.
11) The process of manufacturing of PBAs involves inexpensive easy-to-
handle techniques only and provides a product which is safe with
respect to its handling. Since the particles are present in a liquid
dispersion there is no risk of dust explosions, cross-contamination or
inhalation of bioactive substances as often encountered with the
production of extremely fine powders.
DESCRIPTION OF THE INVENTION
The present invention relates to suspensions of micron and submicron
particles of biodegradable lipids solid at room temperature (solid lipid particles,
SLPs), to suspensions of particles of meltable bioactive substances (PBAs),
to the Iyophilisates thereof and to methods for the manufacturing thereof.
Solid lipid particles (SLPs) are of predominantly anisometrical shape
which is a consequence of the lipid matrix being present in a ,~-polymorphic
modification (e.g. ~ 2) or in a polymorphic state analogous to that of ~-
crystals of triglycerides, and not in an amorphous or a-crystalline-like state.
SLPs can be used as carrier systems primarily for the parenteral but also for
the peroral, nasal, pulmonary, rectal, dermal and buccal administration of
poorly water-soluble substances such as drugs or other biologically active
materials. The application of SLPs is, however, not restricted to the
administration of pharmaceuticals to humans or animals. SLPs can also be
used in cosmetic, food and agricultural products. SLPs are novel lipid
structures with properties that overcome many of the problems associated with
previously described carrier systems.
The matrix of SLPs is constituted by biocompatible hydrophobic
materials which are solid at room temperature and have melting points ranging
~113~3~
~_ 13
from approximately 30 to 120~C. The preferred matrix constituents are solid
lipids (fats) such as mono-, di- and trigtycerides of long chain fatty acids;
hydrogenated vegetable oils; fatty acids and their esters; fatty alcohols and
their esters and ethers; natural or synthetic waxes such as beeswax and
5 carnauba wax; wax alcohols and their esters, sterols such as cholesterol and
its esters, hard parafffins, as well as mixtures thereof. The carrier material must
be compatible with the agent to be incorporated.
Lipids are known to exhibit a pronounced polymorphism. Polymorphism
can be defined as the ability to reveal different unit cell structures in crystal,
10 originating from a variety of molecular conformations and molecular packings.Depending on the conditions, glycerides, for example, can crystallize in three
different polymorphic forms which are termed alpha (a), beta prime (~') and
beta (,~) according to the classification of Larsson (Larsson, K., 1966, Acta
Chem. Scand. 20, 2255-2260). These polymorphic modifications which are
15 characterized by a particular carbon chain packing can differ significantly in
their properties such as solubility, melting point and thermal stability.
Transformations take place from a to ~' to ~, the transition is monotropic. The
~-form is the thermodynamically most stable polymorph, whereas a is the least
stable and will transform more or less rapidly into the more stable polymorphs
20 ~' and ~ depending on the thermal conditions. This transformation is
accompanied by a change of physicochemical properties.
In the described suspensions of SLPs, the lipid matrix is predominantly
present in a stable polymorphic modification. Although on cooling down the
dispersed melt metastable polymorphs such as the a-form can occur
25 intermediately, a stable polymorph is formed within several hours or days after
preparation of the dispersions.
The suspensions of SLPs can be stabilized by amphiphatic compounds
such as ionic and non-ionic surfactants. Suitable stabilizers include but are not
limited to the following examples: naturally occuring as well as synthetic
30 phospholipids, their hydrogenated derivatives and mixtures thereof;
sphingolipids and glycosphingolipids; physiological bile salts such as sodium
cholate, sodium dehydrocholate, sodium deoxycholate, sodium glycocholate
and sodium taurocholate; saturated and unsaturated fatty acids or fatty
2 1 137~3
14
alcohols; ethoxylated fatty acids or fatty alcohols and their esters and ethers;alkylaryl-polyether alcohols such as tyloxapol; esters and ethers of sugars or
sugar alcohols with fatty acids or fatty alcohols; acetylated or ethoxylated
mono- and diglycerides; synthetic biodegradable polymers like block co-
5 polymers of polyoxyethylene and polyoxypropyleneoxide; ethoxylatedsorbitanesters or sorbitanethers; amino acids, polypeptides and proteins such
as gelatine and albumin; or a combination of two or more of the above
mentioned.
The aqueous phase in which the SLPs are dispersed can contain water
10 soluble or dispersable stabilizers; isotonicity agents such as glycerol or xylitol;
cryoprotectants such as sucrose, glucose, trehalose etc.; electrolytes; buffers;antiflocculants such as sodium citrate, sodium pyrophosphate or sodium
dodecylsulfate; preservatives.
Depending on the characteristics of the employed stabilizers, the
15 coexistence of other colloidal structures such as micelles and vesicles in
suspensions of SLPs cannot be ruled out.
Substances particularly suitable for the entrapment into SLPs are drugs
or other bioactive compounds which are poorly water-soluble, show a low
bioavailability, are badly absorbed from the intestinum, and/or will be rapidly
20 degraded in biological environment by chemical or enzymatical processes, as
well as low-specific active substances which are highly toxic at non-target
sites. In case it is desired to incorporate a relatively water soluble compound
into SLPs, it is necessary to decrease the water solubility of this compound
which can be achieved, for example, by using a water insoluble derivative of
25 the compound such as an acid or base, a complex, or a lipophilic precursor.
Drugs or bioactive agents which are particularly suited for incorporation
into SLPs are antibiotics such as fosfomycin, fosmidomycin and rifapentin;
antihypertensives such as minoxidil, dihydroergotoxine and endralazine;
antihypotensives such as dihydroergotamine; systemic antimycotics such as
30 ketoconazole and griseofulvin; antiphlogistics such as indomethacin,
diclofenac, ibuprofen, ketoprofen and pirprofen; antiviral agents such as
aciclovir, vidarabin and immunoglobulines; ACE inhibitors such as captopril
and enalapril; betablockers such as propranolol, atenolol, metoprolol, pindolol,
~1137~S
oxprenolol and labetalol; bronchodilators such as ipratropiumbromide and
sobrerol; calcium antagonists such as diltiazem, flunarizin, verapamil, nifedipin,
nimodipin and nitrendipin; cardiac glycosides such as digitoxin, digoxin,
methyldigoxin and acetyldigoxin; cephalosporins such as ceftizoxim, cefalexin,
cefalotin and cefotaxim; cytostatics such as chlormethin, cyclophosphamid,
chlorambucil, cytarabin, vincristin, mitomycin C, doxorubicin, bleomycin,
cisplatin, taxol, penclomedine and estramustin; hypnotics such as flurazepam,
nitrazepam and lorazepam; psychotropic drugs such as oxazepam, diazepam
and bromazepam; steroid hormones such as cortison, hydrocortison,
prednison, prednisolon, dexamethason, progesteron, pregnanolon, testosteron
and testosteronundecanoat; vasodilators such as molsidomin, hydralazin and
dihydralazin; cerebral vasodilators such as dihydroergotoxin, ciclonicat and
vincamin; lipophilic vitamins such as vitamins A, D, E, K and their derivates.
The bioactive substances can be located in the core of SLPs where
they are dissolved, solubilized or dispersed in the matrix, and/or in the
stabilizer layer(s) surrounding the particle matrix, and/or can be adsorbed to
the surface of SLPs. The bioactive substances can be dissolved or crystalline
or amorphous or a mixture of these crystallographic states.
SLPs can be prepared by an emulsification process which exhibit
certain similarities to the preparation of lipid(oil)-in-water emulsions but is
mainly characterized by its basic differences as will be outlined below. The
process is described as follows:
(1) The solid lipid or the mixture of lipids is melted.
(2) The stabilizers are added either to the lipid and to the dispersion
medium or to the dispersion medium only depending on their
physicochemical characteristics. The choice of stabilizers and the
admixture regime are not comparable with those applied for lipid (oil)-in-
water emulsions which is evident from the below examples. Stabilizers
may also be added or exchanged after homogenization e.g. by
adsorption of polymers or by dialysis of water-soluble surfactants.
(3) Drugs or other bioactive substances to be incorporated into the SLPs
may be melted together with the lipids if the physicochemical
~3~3 '~1
16
characteristics of the substance permits or may be dissolved, solubilized
or dispersed in the lipid melt before homogenization.
(4) The dispersion medium is heated to the temperature of the melt before
mixing and may contain e.g. stabilizers, isotonicity agents, buffering
substances, cryoprotectants and/or preservatives.
(5) The melted lipid compounds are emulsified in dispersion medium
preferably by high pressure homogenization, but emulsification is also
possible by sonication, high speed stirring, vortexing and vigorous hand
shaking. The way of homogenization determines the particle size of the
1 0 SLPs.
The basic differences to the preparation of lipid-in-water emulsions
beside the choice and admixture regime of the stabilizers is related to the
following steps:
(6) After homogenization the dispersion can be sterilized by standard
techniques such as autoclaving orfiltration through a 0.2 ,um sterile filter
provided the particles are small enough not to be retained by the filter.
These steps have to be performed before the system is cooled down
belowthe recrystallization temperature. Moreover, contaminationswhich
could lead to heterogenous nucleation should be avoided. It is therefore
advisable to remove particular contaminations from the dispersions by
filtration prior to cooling below the recrystallization temperature. The
pore size of the filter should be chosen large enough not to retain the
lipid particles.
(7) The dispersions are allowed to stand for cooling at room temperature
forming SLPs by recrystallization of the dispersed lipids. During cooling
the dispersion may be agitated by a magnetic stirrer, for example.
(8) In a subsequent step the dispersion medium is reduced in volume e.g.
by evaporation or can be removed by standard techniques such as
filtration, ultrafiltration orfreeze-drying thus yielding a water-free storage
system which can be reconstituted prior to use. The Iyophilized powder
can also be processed into other pharmaceutical, cosmetic, food or
agricultural formulations such as powders, tablets, capsules etc.
21~37~
17
SLPs are typically solid particles of anisometrical shape as
demonstrated by Figure 1 which shows a transmission electron micrograph of
a freeze-fractured specimen of the SLPs of Example 1. The anisometrical
particle shape results from crystallization of the lipid matrix in the ~-
polymorphic form. Solidification of the amorphous fat or crystallization of the
unstable a-polymorph generally reveals spherical particles. The presence of
the stable ~-form could be detected by differential scanning calorimetry (Fig.
2) and synchrotron radiation wide angle X-ray diffraction (Fig. 3).
The particle size of SLPs depends on the type and amount of emulsifier
and on the emulsification technique and conditions (see below). SLPs in the
nanometer size range are obtained by high pressure homogenization. The
particles show a relatively narrow particle size distribution with mean particlesizes by number of approximately 50 - 300 nm as determined by photon
correlation spectroscopy (PCS). The dispersions of SLPs are stable on storage
for several months. The long-term stability is thus similar to that of submicrono/w emulsions used for parenteral nutrition. Long-term stability data of other
solid lipid based carrier dispersions described in the patent literature such aslipospheres (Domb, A. et al., Int. Appl. No. PCT/US90/06519 filed Nov. 8,
1990) and lipid nanopellets (Speiser, P., Eur. Pat. No. 0167825 issued Aug
8,1990) could not be found. Domb describes phospholipid stabilized tristearate
lipospheres with a seven days stability as "exceptionally stable".
It turned out that the stabilization of SLP suspensions requires the
presence of a highly mobile surface active agent in the dispersion medium in
such a way that the amount of highly mobile stabilizers in the dispersion
medium is, after emulsification, suffficient to stabilize newly created surfacesduring recrystallization. Bile salts have proved to be very efficient in this
respect. SLPs stabilized by phospholipids alone tend to form semi-solid
ointment-like gels as shown on the transmission electron micrograph of Figure
4, whereas the addition of sodium glycocholate to the aqueous phase prevents
this gel formation (Siekmann, B. and Westesen, K.,1992, Pharm. Pharmacol.
Lett. 1, 123-126).
~7 A molar phospholipid to bile salt ratio between 2:1 and 4:1 turned out
to be most effective regarding the initial stabilization during homogenization as
21;~ 3 ~ ~ ~
18
well as the long-term storage stability of SLP dispersions. These
phospholipid/bile salt ratios are above the ratio of formation of mixed micellesand coincide with a swollen lamellar phase of mixed lecithin/bile salt layers inthe ternary phase diagram of the system lecithin/bile salVwater. The data
therefore suggest that stabilization is most effective if the bile salt is not bound
to mixed micelles and that during stabilization of SLPs the bile salt molecules
are inserted in the phospholipid layer at the surface of the particles.
SLPs can also be sterically stabilized by nonionic surfactants. Steric
stabilization of SLPs requires, however, a relatively high amount of surfactantswith lipid/surfactant ratios up to 1:1. It can be observed in general that the
stability of SLPs decreases with increasing lipid/surfactant ratio. The amount
of emulsifier required for surface stabilization of the dispersed particles is
higher than in conventional lipid emulsions, e.g such as used in parenteral
nutrition. This effect can be attributed to the crystallization of the molten lipids
after homogenization. Since the lipids recrystallize as anisometric particles,
there is a large increase in surface area as compared to the droplets of the
emulsified molten lipids or of conventional lipid emulsions, respectively. The
choice of stabilizers cannot be deduced from compositions and stabilization
mechanisms for oil-in-water emulsions but is dependent on the existence of at
least one highly mobile (micelle forming) stabilizer due to the formation
mechanism of the anisometrical particles. In fact, preparing SLPs with a
standard composition of lipid emulsions, e.g. 10% fat and 1.2% phospholipids,
results in unstable SLP dispersions. Although stated in the patent literature
(e.g. Domb et al. in U.S. Pat. No. 435,546 issued Nov 13, 1989 and Int. Appl.
No. PCT/US90/06519 filed Nov. 8, 1990), fine suspensions of solid lipids are
not equivalent to submicron lipid emulsions in that respect that the inner phaseis only replaced by solid fats instead of liquid ones. The physicochemical
properties of lipid suspensions such as SLPs differ substantially from that of
lipid emulsions. As a consequence of these differences, lipid suspensions
cannot be prepared and treated analogously to lipid emulsions. One basic
difference of SLPsis their much larger particle surface area as outlined above
so that SLPs require a higher amount of surfactants which additionally need
to be highly mobile to stabilize the new surfaces immediately that are created
21137~5
19
when the molten lipid recrystallizes. The second basic difference is that once
SLPs are formed by recrystallization of the molten lipid, any renewed melting
of the small particles may result in a destabilization of the dispersions if no
excess of mobile stabilizer is still present in the aqueous phase but not
5 adsorbed to particle surfaces where it is immobilized. A further requirement for
physicochemical stability of SLPs in contrast to oil-in-water emulsions is the
absence of particulate impurities which could promote heterogenous
nucleation. It is therefore advisable to remove particular contaminations from
the dispersions by filtration prior to cooling below the recrystallization
1 0 temperature.
The present invention also relates to suspensions of particles of
bioactive agents (PBAs). Sparingly water soluble substances such as drugs,
insecticides, fungicides, pesticides, herbicides, fertilizers, nutrients, cosmetics
etc., which are meltable in the temperature range from approximately 30 to
15 120~C can be formulated as PBAs by a procedure similar to the preparation
of SLPs as described above. The matrix of PBAsis constituted by the
bioactive agent itself.
PBAs present a novel type of delivery system and can be characterized
as predominantly submicron and/or micron particles of bioactive agents
20 suspended in an aqueous media stabilized by amphiphatic compounds. PBAs
possess modified surface characteristics which can be controlled by the choice
of amphiphiles and/or a reduced particle size of the matrix constituting
compound as compared to the powdered substance. These characteristics
give rise to a modified biodistribution and bioavailability of the formulated drugs
25 or other bioactive substances which implies a modification of the extent and
rate of dissolution and absorption, the circulation time, the site of action andthe way of disposition of the drug or other bioactive substance. The
physicochemical properties of PBAs depend strongly on the characteristics of
the bioactive agent they are formulated of, on the type and amount of
30 stabilizing agents as well as on the way of emulsification. Suspensions and
Iyophilisates of PBAs can be used for the peroral, nasal, pulmonary, rectal,
dermal, buccal and - depending on the particle size - also for the parenteral
administration of poorly water soluble drugs or other biologically active
21137~
compounds. Moreover, PBAs can also be employed in cosmetic, food and
agricultural products, in particular for the formulation of poorly water solubleherbicides and pesticides.
The matrix of PBAs is constituted by practically insoluble or sparingly
5 water soluble agents with melting points below preferably 100~C or which
melting points can be decreased to below 100~C by the addition of certain
adjuvants. Substances particularly suitable for the formulation as PBAs are
drugs or other bioactive materials which are poorly water soluble, show a low
bioavailability and/or are badly absorbed from the intestinum. Examples of
10 such substances comprise but are not limited to:
Anesthetics and narcotics such as butanilicaine, fomocaine,
isobutambene, lidocaine, risocaine, prilocaine, pseudococaine, tetracaine,
trimecaine, tropacocaine and etomidate; anticholinergics such as metixen and
profenamine; antidepressives, psychostimulants and neuroleptics such as
15 alimenazine, binedaline, perazine, chlorpromazine, fenpentadiol, fenanisol,
fluanisol, mebenazine, methylphenidate, thioridazine, toloxaton and
trimipramine; antiepileptics such as dimethadion and nicethamide; anitmycotics
such as butoconazole, chlorphenesin, etisazole, exalamid, pecilocine and
miconazole; antiphlogistics such as butibufen and ibuprofen; bronchodilators
20 such as bamifylline; cardiovascular drugs such as alprenolol, butobendine,
cloridazole, hexobendine, nicofibrate, penbutolol, pirmenol, prenylamine,
procaine amide, propatylnitrate, suloctidil, toliprolol, xibendol and viquidile;cytostatics such as asperline, chlorambucile, chlornaphhazine, mitotane,
estramustine, taxol, penclomedine and trofosfamide; hyperemic drugs such as
25 capsaicine and methylnicotinate; lipid reducers such as nicoclonate,
oxprenolol, pirifibrate, simfibrate and thiadenol; spasmolytics such as
aminopromazine, caronerine, difemerine, fencarbamide, tiropramide and
moxaverine; testosterone derivates such as testosterone enantate and
testosterone-(4-methylpentanoate); tranquilizers such as azaperone and
30 buramate; virustatics such as arildon; vitamin A derivates such as retinol,
retinol acetate and retinol palmitate; vitamin E derivates such as tocopherol
acetate, tocopherol succinate and tocopherol nicotinate; menadione;
cholecalciferol; insecticides, pesticides and herbicides such as acephate,
~137~
21
cyfluthrin, azinphosphomethyl, cypermethrine, substituted phenyl
thiophosphates, fenclophos, permethrine, piperonal, tetramethrine and
trifluraline.
As with SLPs, suspensions of PBAs can be stabilized by amphiphatic
5 compounds. Principally, the same ionic and nonionic sur~actants which can be
employed for the stabilization of SLPs are also suitable for the preparation of
PBA suspensions. The choice of the stabilizing agents depends on the
physicochemical properties of both the bioactive substance and the dispersion
medium as well as on the desired surface characteristics of the particles.
The aqueous phase in which the PBAs are dispersed should contain
water soluble (or dispersable) stabilizers; isotonicity agents such as glycerol
orxylitol; cryoprotectants such as sucrose, glucose, trehalose etc.; electrolytes;
buffers; antiflocculants such as sodium citrate, sodium pyrophosphate or
sodium dodecylsulfate; preservatives. Although water is the preferred
dispersion medium, the invention is, however, not restricted to aqueous
dispersions alone but can be extended to any other pharmacologically
acceptable liquid such as ethanol, propylene glycol and methyl-isobutyl-ketone,
or a mixture thereof.
Depending on the characteristics of the employed stabilizers, the
coexistence of other colloidal structures such as micelles and vesicles in
suspensions of PBAs cannot be ruled out.
Suspensions of PBAs are typically prepared by an emulsification
process similar to that of SLPs. The melted drug or bioactive substance or a
mixture of such compounds is emulsified in a pharmacologically acceptable
liquid immiscible with the melt preferably by high pressure homogenization.
Emulsification is also possible by sonication, high speed stirring, vortexing and
vigorous hand shaking. The liquid is heated to the temperature of the melt
before mixing and may contain e.g. isotonicity agents, buffering substances,
cryoprotectants andlor preservatives.
The stabilizing amphiphatic compounds are added either to the melt and
to the liquid or to the liquid only depending on their physicochemical
characteristics. Stabilizers may also be added or exchanged after
homogenization e.g. by the adsorption of polymers or by dialysis.
~ il37~S
22
The PBAs manufactured according to the above described process can
be categorized in two distinguishable groups.
The PBAs of the first group are characterized in that they are water-
insoluble at the temperature of emulsion preparation and will not be solubilized5 by the excess of stabilizers or form micelles by themselves the particle size of
PBAs remaining unchanged before and after cooling down to room
temperature.
The PBAs of the second group are characterized in that they are partly
water-soluble at the temperature of emulsion preparation and/or are able to
10 form mixed micelles by the excess of stabilizers and/or form micelles by
themselves leading to an increase of particle size after cooling down to room
temperature due to the precipitation of dissovled bioactive agent andlor due
to mass transport from smaller to larger particles e.g. in micelles and/or by
processes such as Ostwald ripening.
In a subsequent step the liquid phase can be removed by freeze-drying,
for example, producing a reconstitutable powder which can also be processed
into other pharmaceutical formulations.
PBAs are finely dispersed particles consisting of a matrix of bioactive
material which is surrounded by one or more layers rich in surfactant. The
particle size and the size distribution as well as the particle shape and the
surfactant coating depend on the properties and amounts of the matrix forming
bioactive substances and the stabilizing agents, the ratio of bioactive materialto amphiphatic compounds as well as on the way of emulsification.
EXAMPLES
Example 1: Method of preparation of tripalmitate SLPs
In a thermostatized vial 4.0 9 tripalmitate (tripalmitin, 99% pure, Fluka)
is heated to 75~C to melt the lipid. In the lipid melt 0.48 g soy bean lecithin
(Lipoid S100, Lipoid KG) is dispersed by probe sonication (MSE Soniprep 150)
until the dispersion appears optically clear. 0.16 9 sodium glycocholate
(glycocholic acid, sodium salt 99%, Sigma) and 4 mg thiomersal (Synopharm)
are dissolved in 36 ml bidistilled water. The aqueous phase is heated to 75~C
~1137~ ~
_ 23
and is added to the lipid melt. A crude dispersion is produced by probe
sonication for approximately 2 min. The crude dispersion is transferred to a
thermostatized high pressure homogenizer (APV Gaulin Micron Lab 40) and
is passed 5 times through the homogenizer at a pressure of 500 bar.
5 Homogenization with this equipment is accomplished by extrusion through a
small ring-shaped orifice. The homogenized dispersion was allowed to stand
at room temperature for cooling.
The importance of the admixture of highly mobile surfactants such as
bile salts with regard to the particle size distribution and the stability of SLP
dispersions is demonstrated below, e.g. in Examples 2, 5 to 7 and 13 to 15.
The mean particle size after preparation (by number) of the tripalmitate
SLPs determined by photon correlation spectroscopy (PCS, Malvern Zetasizer
3) is 205 nm. After 15 months of storage the particles show no visible signs
of aggregation, creaming, sedimentation or phase separation. A PCS
multiangle measurement (Malvern Zetasizer 3, detection at five different
angles: 50,70,90,110 and 130 degrees) reveals a monomodal particle size
distribution (by number) with a peak at 250 nm (Fig. 5).
At temperatures below the melting point of the lipid matrix tripalmitate
SLPs are predominantly anisometrical particles as demonstrated by Figure 1
which is a transmission electron micrograph of a freeze fractured specimen of
the tripalmitate SLPs of Example 1. Before preparation of the specimen the
sample was stored at room temperature for 5 months. The sample was freeze-
fractured at 173 K in a freeze-fracture unit BAF 400 (Balzers AG, CH-
Liechtenstein). Fast freezing was accomplished by slush into melting propane.
Shadowing of the sample was performed with platinum/carbon (layer thickness:
2 nm) at 45 degree and with pure carbon at 90 degree for replica preparation.
Replica were cleaned with a 1:1 (v/v) chloroformlethanol mixture. Replica on
uncoated grids were viewed with an electron microscope EM 300 (Philips, D-
Kassel).
In the anisometrical tripalmitate particles the glyceride is present in the
stable ,B-crystalline polymorph as indicated by thermoanalytical investigations.Figure 2 presents a differential scanning calorimetric (DSC) thermogram of
SLPs of Example 1 and of pure tripalmitate. The samples were accurately
~1137~
' ~ 24
weighed into standard aluminium pans. Thermograms were recorded from
20~C to 90~C at a scan rate of 10~C/min on a Perkin Elmer calorimeter DSC-7.
The detected transition peaks correspond to the melting of tripalmitate ,~-
crystals. The melting point of tripalmitate SLPs is shifted to a lower
5 temperature compared to that of pure tripalmitate due to the presence of
phospholipids and due to the small crystallite size.
Example 2: Method of preparation of isotonic tripalmitate SLPs
7.0 9 tripalmitate (tripalmitin, Fluka) is melted in a vial thermostatized
at 75~C. 840 mg soy bean lecithin (Lipoid S100, Lipoid KG) is dispersed in the
tripalmitate melt as described in Example 1. The aqueous phase containing
1.575 9 glycerol, 280 mg sodium glycocholate and 4 mg thiomersal is heated
to 75~C and is added to the lipid melt to a weight of 70 9. A crude dispersion
is produced by sonication for approximately 2 min. The crude dispersion is
transferred to a thermostatized high pressure homogenizer (APV Gaulin Micron
Lab 40) and is passed 10 times through the homogenizer at a pressure of 800
bar. The homogenized dispersion is allowed to stand at room temperature for
cooling.
The mean particle size by number of the isotonic tripalmitate SLPs
determined by PCS is 125.9 nm after preparation and 116.2 nm after 50 days
of storage, i.e. there was practically no particle growth. The slight deviationsof the values fall into the range of accuracy of the sizing method. The PCS
particle size distribution is compared to that of the commercially available lipid
emulsion for parenteral nutrition IntralipidTM in Figure 6. IntralipidTM is
composed of 10% soy bean oil, 1.2% fractionated phospholipids and 2.25%
glycerol dispersed in water for injection. It can be observed that the particle
size distribution of tripalmitate SLPs of Example 2 is significantly smaller andmore narrow than that of IntralipidTM.
Investigations by synchrotron radiation wide angle X-ray diffraction (Fig.
3) and differential scanning calorimetry revealed that the tripalmitate in SLPs
is present in the stable ~-polymorphic form at room temperature.
211 373J
Example 3: Preparation of hard fat SLPs by microfluidization
3.0 9 hard fat (WitepsolTM W35, Huls AG) is melted in a thermostatized
vial at 75~C. 1.8 9 soy bean lecithin (Phospholipon 100, Natterman) is
dispersed in the tripalmitate melt as described in Example 1. The aqueous
5 phase containing 375 mg sodium glycocholate and 4 mg thiomersal is heated
to 75~C and is added to the lipid melt to a weight of 100 g. A crude dispersion
is produced by ultra-turrax vortexing for approximately 2 minutes. The crude
dispersion is transferred to a microfluidizer (Microfluidics Microfluidizer M-
110T), a high pressure homogenizer of the jet-stream principle, which is
10 immersed in a thermostatized water bath (70~C). The dispersion is cycled
through the microfluidizer for 10 minutes and is allowed to stand at room
temperature for cooling.
The mean particle size of hard fat SLPs after preparation is 45.9 nm as
determined by PCS.
15During homogenization a sample for particle sizing was drawn each
minute in order to monitor the time course of homogenization. Figure 7
displays the mean particle size versus homogenization time. The mean particle
diameter is decreasing with time and levels off at the end of homogenization.
20Example 4: Long-term stability of hard fat SLPs prepared by microfluidization
The stability of hard fat SLPs was monitored over a period of one year.
During this time the sample was stored in a refrigerator at approximately +4~C.
After certain time intervals the particle size distribution of the sample was
determined by PCS. Figure 8 demonstrates that the mean particle size of hard
25fat SLPs is practically constant over the monitored period of one year.
Example 5: Preparation of unstable SLPs dispersions
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116, Huls AG) is
heated to 75~C to melt the lipid. In the lipid melt 0.48 9 soy bean lecithin
30(Lipoid S100, Lipoid KG) is dispersed by probe sonication (MSE Soniprep 150)
until the dispersion appears optically clear. 4 mg thiomersal is dissolved in
35.6 ml bidistilled water. The aqueous phase is heated to 75~C and is added
to the lipid melt. A crude dispersion is produced by probe sonication for
21~37~S
26
approximately 2 min. The crude dispersion is transferred to a thermostatized
high pressure homogenizer (APV Gaulin Micron Lab 40) and is passed 5 times
through the homogenizer at a pressure of 500 bar. The homogenized
dispersion was allowed to stand at room temperature for cooling. On storage
5 the SLP dispersion becomes a milky semi-solid, ointment-like gel.
3.0 9 hard fat (WitepsolT~ W35, Huls AG) is melted in a thermostatized
vial at 75~C. 1.8 9 soy bean lecithin (Phospholipon 100, Natterman) is
dispersed in the tripalmitate melt as described in Example 1. The aqueous
phase containing 4 mg thiomersal is heated to 75~C and is added to the lipid
10 melt to a weight of 100 9. A crude dispersion is produced by ultra-turrax
vortexing for approximately 2 minutes. The crude dispersion is transferred to
a microfluidizer (Microfluidics Microfluidizer M-110T) which is immersed in a
thermostatized water bath (70~C). The dispersion is cycled through the
microfluidizer for 10 minutes and is allowed to stand at room temperature for
15 cooling. On storage the SLP dispersion becomes a turbid semi-solid, ointment-like gel. A transmission electron micrograph of this gel is presented in Figure
4.
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116, Huls AG) is
heated to 80~C to melt the lipid. In the lipid melt 0.8 9 of a soy bean lecithin20 mixture (Lipoid S75, Lipoid KG) is dispersed by probe sonication (MSE
Soniprep 150) until the dispersion appears optically clear. 4 mg thiomersal is
dissolved in 35.6 ml bidistilled water. The aqueous phase is heated to 80~C
and is added to the lipid melt. A crude dispersion is produced by probe
sonication for approximately 2 min. The crude dispersion is transferred to a
25 thermostatized high pressure homogenizer (APV Gaulin Micron Lab 40) and
is passed 5 times through the homogenizer at a pressure of 800 bar. The
homogenized dispersion is filled in a glas vial and is allowed to stand at room
temperature for cooling. On cooling to room temperature the dispersion forms
semi-solid like fat aggregates at the wall of the glass vial. The dispersion
30 gelatinizes when shear forces are applied, e.g. by passing it through a
hypodermic syringe.
~1~3~
_ 27
Obviously, the use of phospholipids only as stabilizers - as found in
commercial parenteral oil-in-water emulsions - does not yield stable systems
in the case of SLP suspensions. Even the employment of phospholipids such
as Lipoid S75 which induce a considerably high negative net charge cannot
provide a sufficient stabilization. Electrostatic repulsion alone cannot be the
basic stabilization mechanism of SLPs as will be further outlined in Examples
6,7 and 13.
Example 6: Preparation of tripalmitate SLPs sterically stabilized by tyloxapol
A series of tripalmitate SLP dispersion stabilized by tyloxapol (Eastman
Kodak) were prepared with varying lipid/surfactant ratios. The SLP dispersion
were manufactured according to the following procedure: Tyloxapol was
dissolved in heated bidistilled water while the temperature was held below the
cloud point of tyloxapol (approx. 90 - 95~C). The tyloxapol solution of a
temperature of 80~C was added to the molten tripalmitate or, respectively,
tripalmitate/lecithin dispersion of the same temperature. A crude emulsion was
prepared by probe sonication for approximately 2 minutes. Then the crude
emulsion was passed 5 times through a high pressure homogenizer at a
pressure of 1200 bar. The homogenized dispersion was allowed to stand at
room temperature for cooling. All dispersions contained 2.5% glycerol and
0.01% thiomersal.
Table 1 gives the composition of the prepared SLP dispersions and their
mean particle size after preparation (by number) as determined by PCS. The
asterix (*) in the particle size column indicates that the dispersions displayeda bimodal size distribution with particle sizes considerably larger as indicatedby the mean particle size. It turns out that sterically stabilized SLP dispersions
require a high amount of surfactant in order to obtain homogenously sized
SLPs. In case of SLPs stabilized by tyloxapol and phospholipids, the ratio of
the surfactants needs to be optimized. In the present series a tyloxapol/lecithin
30 ratio of at least 1:1 turned out to yield homogenously sized SLPs. With
increasing ratio the mean particle size is decreasing. As with examples 1 to
3, the addition of a highly mobile surfactant which is able to form micelles is
required to obtain stable dispersions. The high amount of surfactant is needed
~1137~
28
in order to create a reservoir of surfactant in the phase that can provide
enough surfactant molecules at the moment when the molten lipids
recrystallize and form anisometrical particles with a large specific surface area.
5 Table 1: SLP dispersions sterically stabilized by tyloxapol
CGIII~OSj~;On (W~/0) Mean particle ske (nm)
TP TYI PL
2 - 138.0*
4 - 84.9
0.7 2 487.4
1 2 207.4
2 2 102.8
4.5 3 60.9
Abbre~Jiations: TP = tripalmitate, Tyl = tyloxapol, PL = phospholipids (Lipoid
S1 00).
Example 7: Preparation of tripalmitate SLPs sterically stabilized by
poloxamers
1.2 g soy bean lecithin (Lipoid S100, Lipoid KG) is dispersed in 4.0 9
molten tripalmitate (Dynasan 116, Huls AG) by probe sonication at a
temperature of 80CC. 1.8 9 poloxamer (PluronicT~ F68, BASF), 0.9 g glycerol
and 4 mg thiomersal are dissolved in 32.1 9 bidistilled water heated to 80~C.
The heated solution is added to the lipid melt and a crude dispersion is
prepared by 2 min. probe sonication. The crude dispersion is transferred to a
thermostatized high pressure homogenizer (APV Gaulin Micron Lab 40) and
is passed 5 times through the homogenizer at a pressure of 1200 bar. The
homogenized dispersion is allowed to stand at room temperature for cooling.
The poloxamer stabilized SLPs display a monomodal size distribution
with a mean particle size after preparation (by number) of 77.9 nm determined
by PCS. Due to the presence of an excess of highly mobile surfactant in the
aqueous phase the system is stabilized on recrystallization of the molten lipids
~11373~
29
and a gelation as found with systems stabilized by phospholipids only does not
occur.
Example 8: The influence of homogenization pressure on the mean particle
size of SLPs
SLPs of the following composition are prepared at different
homogenization pressures. The SLP dispersions are composed of 3%
tripalmitate (Dynasan 116, Huls AG), 1.5% tyloxapol, 1% soy bean lecithin
(Lipoid S100, Lipoid KG), 0.01% thiomersal and bidistilled water to 100% (by
weight). The lecithin is dispersed in the molten tripalmitate (80~C) by probe
sonication until the dispersion appears optically clear. Tyloxapol is dissolved
in warm water (80~C) containing thiomersal. The SLP dispersions are prepared
as described in Example 6.
Figure 9 displays the influence of homogenization pressure on the mean
particle size of the SLPs. With increasing pressure the particle size is
decreasing and the particle size distribution becomes more narrow.
Example 9: The influence of homogenization passes on the mean particle
size of SLPs
Tripalmitate SLPs composed of 3% tripalmitate (Dynasan 116, Huls
AG), 1.5% tyloxapol, 1% soy bean lecithin (Lipoid S100, Lipoid KG), 0.01%
thiomersal and bidistilled water to 100% (by weight) were prepared at a
pressure of 800 bar as described in Example 6. Samples for size
measurements were taken from the dispersion after preparation of the crude
emulsion and after each pass through the homogenizer.
Figure 10 presents the influence of the number of homogenization
passes on the mean particle size of SLPs which is decreasing with increasing
number of passes.
Example 10: Preparation of SLPs by probe sonication - Influence of sonication
time on the mean particle size of SLPs
In a sonication vial thermostatized at 80~C 1.20 9 tripalmitate is molten.
In the lipid melt 0.40 g soy bean lecithin (Lipoid S100) is dispersed by probe
30 211373~
sonication until the dispersion appears optically clear. 0.60 9 tyloxapol and 4
mg thiomersal are dissolved in bidistilled water heated to 80~C. The aqueous
phase is added to the lipid melt and an SLP dispersion is prepared by probe
sonication at 80~C. The sonicator operated at 50% of its maximum power. At
certain time intervals (1, 5, 10 and 15 min) samples were taken from the
dispersion for size measurements. After 30 minutes probe sonication was
stopped and the dispersion was allowed to stand at room temperature for
cooling.
The influence of sonication time on the mean particle size of the SLPs
is displayed in Figure 11. With increasing sonication time the mean particle
size is decreasing and the size distribution becomes more narrow.
Example 11: Preparation of SLPs by stirring
An SLP dispersion composed as in Examples 9 and 10 was prepared
1~ by use of a heated magnetic stirrer (Pyro-Magnestir, Lab-Line). The lecithin
was dispersed in the tripalmitate as described before. The heated aqueous
phase was added to the melt. A dispersion was produced by stirring the
mixture for 30 minutes at a temperature of 80~C. The dispersion was allowed
to stand at room temperature for cooling.
The mean particle size after preparation (by volume) of the SLP
dispersion was 59.5 ,um determined by laser diffractometry (Malvern
Mastersizer MS20). The maximum particle size measured was 250 ,um. In
contrast to high pressure homogenization and probe sonication, stirring
produces relatively large particle in the micrometer size range.
Example 12: Influence of the matrix constituent on the mean particle size of
SLPs
SLP dispersions composed of 10% matrix constituent, 1.2% soy bean
lecithin (Lipoid S100), 0.4% sodium glycocholate, 2.25% glycerol and 0.01%
thiomersal in bidistilled water to 100% were prepared as described in Example
1. Five different matrix constituents were employed: the waxes cetylpalmitate
and white bees-wax and the triglycerides trilaurate, trimyristate and tripalmitate.
2113~
~ 31
Table 2 presents the PCS mean particle sizes of the different SLP
dispersions and the melting points of the matrix constituents.
Table 2 Influence of matrix constituents
Matnix MeKinq point (~C)Mean size of SLPs (nm)
Cetylpalmitate 45.5 141.0
White bees-wax 62.5 195.3
Trilaurate 45.0 137.2
Trimyristate 56.5 161.1
Tripalmitate 63.0 209.2
The mean particle size of SLPs is increasing with the melting point of
15 the matrix constituent.
Example 13: Influence of emulsifier type and amount on the mean particle
size and stability of SLPs
Tripalmitate SLP dispersions with different types and amounts of
20 emulsifiers were prepared as described in Example 2. The composition of the
different batches is given in Table 3.
The mean particle size of the different batches of SLPs is presented in
Figure 12. The mean particie size depends on the type and amount of
emulsifier.
Table 3 Compositions of SLP batches (in w%)
Batch No. TP PL SGC Plu
10% 1.2%
2 10% 1.2% 0.4%
3 10% 2.4% 0.4%
4 10% - - 1.8%
10% - - 3.6%
21137~.~
32
Abbreviations: TP = tripalmitate, PL = phospholipids (Lipoid S100), SGC =
sodium glycocholate, Plu = Pluronic F68.
The combination of phospholipids and bile salts is most effficient with
regard to the mean particle size and the stability. The system stabilized by
phospholipids only gelatinized and formed an ointment-like semi-solid on
storage. The systems stabilized by Pluronic F68 tended to gelatinize when
shear forces were applied, i.e. when the particles were forced to get closer to
each other. Obviously, the steric stabilization by poloxamers is not suffficientin this case. As a result, the optimum stabilization is that by a surfactant
combination of emulsifiers that are present in and act from the lipid side (suchas phospholipids) and of emulsifiers that constitute a reservoir of highly mobile
surfactant molecules in the dispersion medium (such as bile salts, tyloxapol
and poloxamers). Though repulsion forces represent an important factorforthe
long-term stability, the basic mechanism of SLP stabilization is the high
mobility of the excess of surfactant which provides for the immediate surface
coverage of newly created surfaces during recrystallization of the molten lipids.
Example 14: Effect of bile salt as co-emulsifier of phospholipid
stabilized SLPs
Phospholipid stabilized SLP dispersions employing different matrices
(tripalmitate, hard fat) are prepared with or without the addition of bile salt
(sodium glycocholate) to the aqueous phase according to the method
described in Example 1. Emulsification of the crude dispersions is performed
by high pressure homogenization (APV Gaulin Micron Lab 40) under different
homogenization conditions. The following dispersions were prepared:
21137~S
_~ 33
Com~Gsdio" Homoqenization CGI Id;t~GI15
7.0 9 TP, 0.84 9 PL, 62.2 9 H2O 3 x 500 bar
7.0 9 TP, 0.84 9 PL, 0.28 9 BS, 61,9 9 H2O 3 x 500 bar
7.0 9 TP, 0.84 9 PL, 62.2 9 H2O 10 x 1200 bar
7.0 9 TP, 0.84 9 PL, 0.28 9 BS, 61.9 9 H2O 10 x 1200 bar
7.0 9 HF, 0.84 9 PL, 62.2 9 H2O 3 x 500 bar
7.0 9 HF, 0.84 9 PL, 0.28 9 BS, 61.9 9 H2O 3 x 500 bar
7.0 9 HF, 0.84 9 PL, 62.2 9 H2O 10 x 1200 bar
7.0 9 HF, 0.84 9 PL, 0.28 9 BS, 61.9 9 H2O 10 x 1200 bar
Abbreviations: BS = bile salt; H2O = bidistilled water; HF = hard fat (Witepsol
W35); PL = phospholipids (Lipoid S 100); TP = tripalmitate
The mean particle size of the dispersions as determined by PCS after
preparation is presented in Figure 13. This example demonstrates the effect
of bile salts as co-emulsifier in the aqueous phase on the particle size of
phospholipid stabilized SLPs. It is clearly shown that the addition of bile salts
reduces the mean particle size of SLPs by up to 57%. Thus, by the use of
bile salts as co-emulsifier extremely fine dispersions can be obtained. The
effect of the bile salt can be attributed to the high mobility of this micelle
forming ionic surfactant which enables the surfactant molecules to immediately
cover freshly generated surfaces during the homogenization process. The
phospholipids which tend to form liquid crystalline structures in the aqueous
phase are not suffficiently mobile to provide the immediate stabilization of
freshly created particles so that instantaneous coalescence occurs in case that
there is no highly mobile co-surfactant in the aqueous phase.
Example 15: Preparation of trimyristate SLPs stabilized by a
lecithin/bile salt blend
In a thermostatized vial 7.0 9 trimyristate (Dynasan 114, Huls AG) is
melted at 70~C. 0.96 9 phospholipids (Lipoid S 100) are dispersed in the melt
by probe sonication. A solution of 280 mg sodium glycocholate and 4 mg
thiomersal in 61 ml bidistilled water is heated to 70~C and is added to the melt.
21137~ 3
34
A crude dispersion is prepared by probe sonication for approximately 2 min.
The crude dispersion is transferred to a high pressure homogenizer (APV
Gaulin Micron Lab 40) thermostatized at approximately 90~C and is passed 5
times through the homogenizer at a pressure of 500 bar. The homogenized
dispersion is allowed to stand at room temperature for cooling.
The mean particle size after preparation determined by PCS is 155.7
nm. In laser diffractometry (Malvern Mastersizer MS20) no particles above
11~m could be detected. The particle size distribution derived from laser
diffractometry is presented in Figure 14. This example demonstrates that the
use of bile salts as co-emulsifier of phospholipid stabilized SLPs efficiently
prevents the formation of particles larger than 1 ~Jm due to the rapid coverage
of freshly created surfaces during homogenization thereby minimizing
immediate coalescence.
Examples 16: Preparation of tripalmitate SLPs without ultrasonication
(method A)
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116, Huls AG) is
melted at 85~C. 0.96 9 lecithin (Lipoid S 100) is dissolved in ethanol. The
lecithin solution is added to the melt. The ethanol is evaporated at a
temperature of 85~C. 160 mg sodium glycocholate and 4 mg thiomersal are
dissolved in 35 ml bidistilled water. The solution is heated to 85~C and is
added to the melt. A crude dispersion is prepared by ultra-turrax vortexing for
approximately 2 min. The crude dispersion is transferred to a high pressure
homogenizer (APV Gaulin Micron Lab 40) thermostatized at approximately
90~C and is passed 10 times through the homogenizer at a pressure of 800
bar. The homogenized dispersion is allowed to stand at room temperature for
cooling.
Example 17: Preparation of tripalmitate SLPs without ultrasonication
(method B)
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116, Huls AG) is
melted at 85~CØ96 g lecithin (Lipoid S 100) is added to the melt. The mixture
is shaken until the lecithin is completely dispersed in the melt and the
21~3~S
_ 35
dispersion appears isotropic. 160 mg sodium glycocholate and 4 mg thiomersal
are dissolved in 35 ml bidistilled water. The solution is heated to 85~C and is
added to the melt. A crude dispersion is prepared by ultra-turrax vortexing for
approximately 2 min. The crude dispersion is transferred to a high pressure
homogenizer (APV Gaulin Micron Lab. 40) thermostatized at approximately
90~C and is passed 10 times through the homogenizer at a pressure of 800
bar. The homogenized dispersion is allowed to stand at room temperature for
cooling.
Example 18: Preparation of tripalmitate SLPs by dispersing
phospholipids in the aqueous phase
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116) is melted at
80~C. 0.96 9 phospholipids (Lipoid S 100) are dispersed in 35 ml of an
aqueous solution of 160 mg sodium glycocholate and 4 mg thiomersal by
stirring for approximately one hour. The phospholipid dispersion is heated to
80~C and is added to the tripalmitate melt. A crude dispersion is prepared by
probe sonication for approximately 2 min. The crude dispersion is transferred
to a high pressure homogenizer (APV Gaulin Micron Lab 40) thermostatized
at approximately 90~C and is passed 10 times through the homogenizer at a
pressure of 800 bar. The homogenized dispersion is allowed to stand at room
temperature for cooling.
Example 19: Preparation of tripalmitate SLPs stabilized by a highly
mobile surfactant
In a thermostatized vial 5.0 9 tripalmitate is molten at 80~C. 600 mg
sodium glycocholate is dissolved in 44.4 9 bidistilled water containing 0.01 %
thiomersal. The aqueous solution is heated to 80~C and is added to the melt.
A crude dispersion is prepared by sonication for approximately 5 minutes. The
crude dispersion is transferred to a thermostatized high pressure homogenizer
(APV Gaulin Micron Lab 40) and is passed 8 times through the homogenizer
at a pressure of 800 bar. The dispersion is allowed to stand at room
temperature for cooling.
211379~
36
The mean particle size (by number) of the SLP dispersion after
preparation is 96.8 nm determined by PCS. The size distribution is narrow and
monomodal.
This example demonstrates that it is possible to prepare small,
5 homogenously sized SLP by the use of one surfactant only, such as the bile
salt sodium glycocholate, provided the surfactant is highly mobile and
constitutes a reservoir of stabilizer in the aqueous phase in order to provide
for the stabilization of newly created surfaces during recrystallization of the
SLP matrix.
Example 20: Long-term stability of different SLP dispersions
Several different SLP dispersions are prepared according to the method
described in Example 2. All dispersions contain 2.25 % glycerol as isotonicity
agent and 0.01 % thiomersal as a preservative. The long-term stability of the
15 dispersions is judged from repeated size measurements (by PCS) over a
period of 18 months. The dispersions were stored at refrigeration
temperatures. For comparison a soybean oil emulsion system is included. The
composition of the dispersions and their mean particle sizes during storage are
summarized in Table 4.
Table 4
C ~ s- d ~ 'W~ ~S~ a ic'~- _e
~ a~ P~ S ~ P~ e~ a-a
10 % TP 1.2 % 0.4 % 125.9 121.6
10 % TP 2.4 % 0.4 % 104.5 111.2
10 % TP') 2.4 % 0.4 % 103.6 104.7
9,5 % TPt
0,5 % GMS 2.4 % 0.4 % 102.4 102.4
10 % SO 2.4 % 0.4 % 129.6 139.9
~1137~
37
Abbreviations: PL = Phospholipid (Lipoid S 100); SGC = sodium glycocholate;
TP = tripalmitate; GMS = glycerol monostearate; SO = soy bean oil.
') The SLP dispersion containts 5 % (related to fat phase) of the cardio-
protective drug ubidecarenone.
It is shown that the mean particle size of the dispersions remains practically
unchanged during storage for 18 months. The results thus demonstrate that
drug-free and drug-loaded SLP dispersions exhibit a long-term stability similar
to that of lipid emulsions.
Example 21: Sterile filtration of tripalmitate SLPs
40 ml of a crude SLP dispersion composed of 3% tripalmitate (Dynasan
116, Huls AG), 1.5% tyloxapol, 1 % lecithin (Lipoid S100), 2.25% glycerol and
0.01% thiomersal in bidistilled water to 100% was prepared according to the
15 method described in Example 6. The crude dispersion was passed 5 times
through a thermostatized homogenizer (APV Micron Lab 40) at a pressure of
1200 bar. Half the volume of the batch was allowed to stand at room
temperature for cooling, whereas the rest was filtered through a sterile syringefilter (Nalgene SFCA, 0.2 ,um pore size) before cooling down to the
20 recrystallization temperature of the molten lipids.
The particle size distribution of both samples was determined by PCS.
The mean particle size of the unfiltered sample was 56.7 nm, that of the sterilefiltered SLP dispersion was 53.2 nm, i.e. both samples have practically the
same mean particle size.
Example 22: Sterilization of tripalmitate SLPs by autoclaving
40 ml of a crude SLP dispersion composed of 3% tripalmitate (Dynasan
116, Huls AG),1.8% lecithin (Lipoid S100), 0.6% sodium glycocholate, 2.25%
glycerol and 0.01% thiomersal in bidistilled water to 100% was prepared
30 according to the method described in Example 2. The crude dispersion was
passed 10 times through a thermostatized homogenizer (APV Micron Lab 40)
at a pressure of 1200 bar.
~ 1 1 3 7 ~ -5
38
Before cooling down to the recrystallization temperature of the molten
lipids, the SLP dispersion was filled in an injection vial and was sterilized byautoclaving at 121~CI2 atm for 45 minutes. The autoclaved dispersion was
allowed to stand at room temperature for cooling. It showed no signs of
5 aggregation or phase separation and had a mean particle size of 65.9 nm
determined by PCS.
Example 23: Surface modification by adsorption of polymers
In a thermostatized vial 4.0 9 tripalmitate (Dynasan 116, Huls AG) is
molten at 80~C and 1.6 9 soy bean lecithin (Lipoid S100) is dispersed in the
melt by probe sonication. 35.25 9 of an aqueous solution of 0.01 % thiomersal
is heated to 80~C and is added to the melt. A crude dispersion is produced by
probe sonication which is then passed five times through a high pressure
homogenizer at a pressure of 1200 bar. The dispersion is filtered through a 0.2
~m syringe filter. The batch is divided into 3 parts of equal volume. One part
is diluted with the same amount of water and is stored at 90~C in order to
prevent gelation of the phospholipid stabilized dispersion on cooling down
below the recrystallization temperature. The other two parts of the batch are
incubated overnight with equal volumes of 6 % (w/w) poloxamer 407 (Pluronic
F127, BASF) and, respectively, 6 % (w/w) poloxamine 908 (Tetronic 908,
BASF) solution in such a way that the polymer solution is added to the SLP
dispersion prior to cooling below the recrystallization temperature of SLPs in
order to provide for the immediate availability of polymer molecules as soon
as new surfaces are created due to recrystallization. Both polymers have been
described in literature to modify the biodistribution of intravenously
administered colloidal particles.
The modification of the surfact properties of tripalmitate SLPs is
demonstrated by differences in the zetapotenial. Zetapotentials were
determined by laser Doppler anemometry in a microelectrophoresis cell
(Malvern Zetasizer 3). The results are summarized in Table 5.
~1137~
39
Table 5 Zetapotential of surface modified SLPs
cOmrOsition r~O (w~w~l Zetapotential
TP ¦ PL ¦ Polymer lmVl
5 % 2 % - -29.6
5 % 2 % 3 % F127 -1.9
5 % 2 % 3 % T908 -2.9
Abbreviations: TP = tripalmitate, PL = phospholipids, F127 = Pluronic F127,
T908 = Tetronic 908.
The incubation of SLPs with block copolymers of the poloxamer and
poloxamine type results in a decrease of the zetapotential. Due to the
adsorption of the polymers the surfaces become more hydrophobic. The
hydrophobicity of the surface is described to be one of the factors governing
the RES (reticuloendothelial system) activity and the biodistribution of colloidal
particles.
Example 24: Preparation of SLPs loaded with the cardio-protective drug
ubidecarenone
Three different types of SLPs containing the cardio-protective drug
ubidecarenone were prepared. The SLPs were composed as summarized in
table 6. All dispersions contained 2.25% glycerol and 0.01 thiomersal.
Batch 1 and 2 were prepared by dispersing lecithin in the molten matrix
constituent as described before. In this melt ubidecarenone was dissolved.
After addition of the aqueous phase containing sodium glycocholate, glycerol
and thiomersal, a crude dispersion was prepared by probe sonication. It was
transferred to a thermostatized homogenizer (APV Micron Lab 40) and passed
through the homogenizer ten times at a pressure of 800 bar. The dispersions
were allowed to stand at room temperature for cooling.
2113~
Batch 3 was prepared by dispersing lecithin in the molten matrix
constituent as described before. In this melt ubidecarenone was dissolved.
After addition of the aqueous phase containing tyloxapol, glycerol and
thiomersal, a crude dispersion was prepared by probe sonication. It was
5 transferred to a thermostatized homogenizer (APV Micron Lab 40) and passed
5 times through the homogenizer at a pressure of 1200 bar. The dispersion
was allowed to stand at room temperature for cooling.
Table 6 Ubidecarenone-loaded SLPs
Batch- Co,l~po~ion Mean particle ske
No
10% TP, 2.4% PL, 0.4% SGC, 1% Ubi80.2 nm
2 10% HF, 1.2% PL, 0.4% SGC, 1% Ubi78.9 nm
3 3% TP, 1.5% Tyl, 1% PL, 0.2% Ubi46.8 nm
Abbreviations: TP = tripalmitate, PL = phospholipids, SGC = sodium
glycocholate, Ubi = Ubidecarenone, HF = hard fat (Witepsol W35), Tyl =
Tyloxapol.
Example 25: Preparation of SLPs loaded with oxazepam
In a thermostatized vial 7.0 9 tripalmitate is molten at 80~C. 1.68 9
lecithin and 140 mg oxazepam are dispersed in the melt by probe sonication.
60 ml of heated aqueous phase containing 280 mg sodium glycocho!ate, 1.58
9 glycerol and 7 mg thiomersal is added to the melt and a crude dispersion
is prepared by probe sonication. The crude dispersion is homogenized by
passing 10 times through a thermostatized high pressure homogenizer at a
pressure of 800 bar. The dispersion is allowed to stand at room temperature
for cooling.
The dispersion of oxazepam loaded SLPs has a mean particle size after
preparation of 122.7 nm.
~ 137~
41
Example 26: Preparation and long-term stability of SLPs loaded with diazepam
In a thermostatized vial 4.0 9 tripalmitate is molten at 80~C. 0.96 9
lecithin and 120 mg diazepam are dispersed in the melt by probe sonication.
35 ml of heated aqueous phase containing 160 mg sodium glycocholate, 0.9
9 glycerol and 4 mg thiomersal is added to the melt and a crude dispersion
is prepared by probe sonication. The crude dispersion is homogenized by
passing 10 times through a thermostatized high pressure homogenizer at a
pressure of 800 bar. The dispersion is allowed to stand at room temperature
for cooling.
The dispersion of diazepam loaded SLPs has a mean particle size after
preparation of 104.6 nm. After 12 months of storage the mean particle size
determined by PCS was 113.9 nm. Precipitation of drug substance during
storage was not detected macroscopically. Investigations of the dispersion by
polarized light microscopy over the monitored period of 12 months did not
reveal the presence of drug crystals.
Example 27: Preparation of SLPs loaded with lidocaine
In a thermostatized vial 4.0 9 tripalmitate is molten at 80~C. 0.96 9
lecithin and 80 mg lidocaine are dispersed in the melt by probe sonication. 35
ml of heated aqueous phase containing 320 mg sodium glycocholate, 0.9 9
glycerol and 4 mg thiomersal is added to the melt and a crude dispersion is
prepared by probe sonication. The crude dispersion is homogenized by
passing 10 times through a thermostatized high pressure homogenizer at a
pressure of 800 bar. The dispersion is allowed to stand at room temperature
for cooling.
The dispersion of lidocaine loaded SLPs has a mean particle size after
preparation of 90.4 nm.
Example 28: Preparation and long-term stability of SLPs loaded with
prednisolone
In a thermostatized vial 4.0 9 tripalmitate is molten at 80~C. 0.48 9
lecithin and 80 mg prednisolone are dispersed in the melt by probe sonication.
36 ml of heated aqueous phase containing 160 mg sodium glycocholate, 0.9
2113~9~
_ 42
g glycerol and 4 mg thiomersal is added to the melt and a crude dispersion
is prepared by probe sonication. The crude dispersion is homogenized by
passing 10 times through a thermostatized high pressure homogenizer at a
pressure of 800 bar. The dispersion is allowed to stand at room temperature
5 for cooling.
The dispersion of prednisolone loaded SLPs has a mean particle size
after preparation of 118.3 nm. After 12 months of storage the mean particle
size determined by PCS was 124.2 nm. Precipitation of drug substance during
storage was not detected macroscopically. Investigations of the dispersion by
10 polarized light microscopy over the monitored period of 12 months did not
reveal the presence of drug crystals.
Example 29: Preparation of SLPs loaded with cortisone
Four different types of SLPs containing cortisone were prepared. The
15 SLPs were composed as summarized in table 7. All dispersions contained
2.25% glycerol and 0.01 thiomersal.
Batch 1 and 2 were prepared by dispersing lecithin in the molten matrix
constituent as described before. In this melt cortisone was dissolved. After
addition of the aqueous phase containing sodium glycocholate, glycerol and
20 thiomersal, a crude dispersion was prepared by probe sonication. It was
transferred to a thermostatized homogenizer (APV Micron Lab 40) and passed
10 times through the homogenizer. The dispersions were allowed to stand at
room temperature for cooling.
Batch 3 was prepared by dispersing lecithin in the molten matrix
25 constituent as described before. In this melt cortisone was dissolved. After
addition of the aqueous phase containing poloxamer (Pluronic F68), glycerol
and thiomersal, a crude dispersion was prepared by probe sonication. It was
transferred to a thermostatized homogenizer (APV Micron Lab 40) and passed
5 times through the homogenizer at a pressure of 1200 bar. The dispersion
30 was allowed to stand at room temperature for cooling.
Batch 4 was prepared by dispersing lecithin in the molten matrix
constituent as described before. In this melt cortisone was dissolved. After
addition of the aqueous phase containing tyloxapol, glycerol and thiomersal,
~1137~
43
a crude dispersion was prepared by probe sonication. It was transferred to a
thermostatized homogenizer (APV Micron Lab 40) and passed through the
homogenizer five times at a pressure of 1200 bar. The dispersion was allowed
to stand at room temperature for cooling.
s
Table 7 Cortisone-loaded tripalmitate SLPs
Batch-No. Composition Mean Pafficle size
10% TP, 1.2% PL, 0.4% SGC, 0.2% Cort 124.2 nm
2 3% TP, 1.8% PL, 0.6% SGC, 0.3% Cort 67.3 nm
3 10% TP, 4.5% Plu, 3% PL, 0.1% Cort 70.5 nm
4 3% TP, 1.5% Tyl, 1% PL, 0.1% Cort 48.5 nm
Abbreviations: TP = tripalmitate, PL = phospholipids, SGC = sodium
glycocholate, Cort = Cortisone, Plu = Pluronic F68, Tyl = Tyloxapol.
Example 30: Tripalmitate SLPs loaded with retinol (vitamin A)
In a thermostatized vial 1.0 9 tripalmitate (Dynasan 116, Huls AG) is
molten at 80~C. 60 mg retinol (vitamin A-alcohol >99 %, Fluka) is dissolved in
the melt. 300 mg soy bean lecithin (Lipoid S100) is dispersed in the melt by
probe sonication until the dispersion appears optically clear.450 mg poloxamer
407 (PluronicTM F127, BASF) is dissolved in 29.0 9 bidistilled water. The
aqueous phase is heated to 80~C and is added to the melt. A fine dispersion
is prepared by probe sonication for 20 min. The dispersion is filtered through
a 0.2 ,um syringe filter to remove metal shed from the ultrasound probe. The
dispersion is allowed to stand at room temperature for cooling.
The mean particle size by number after preparation of vitamin A-loaded
tripalmitate SLPs is 98.5 nm determined by PCS.
Example 31: Tripalmitate SLPs loaded with phytylmenadione (vitamin K3)
In a thermostatized vial 1.0 9 tripalmitate (Dynasan 116, Huls AG) is
molten at 80~C. 60 mg phytylmenadione (vitamin K3, Sigma) and 300 mg soy
bean lecithin (Lipoid S100) are dispersed in the melt by probe sonication until
211379~
44
the dispersion appears optically clear. 450 mg poloxamer 407 (PluronicTM
F127, BASF) is dissolved in 28.7 9 bidistilled water. The aqueous phase is
heated to 80~C and is added to the melt. A fine dispersion is prepared by
probe sonication for 20 min. The dispersion is filtered through a 0.2 ,um syringe
5 filter to remove metal shed from the ultrasound probe. The dispersion is
allowed to stand at room temperature for cooling.
The mean particle size by number after preparation of vitamin K3-loaded
tripalmitate SLPs is 86.8 nm determined by PCS.
10 Example 32: Preparation of tripalmitate SLPs loaded with estramustine
In a termostatized vial 7.0 9 tripalmitate (Dynasan 116, Huls AG) is
melted at 80~C. In the melt 1.68 9 soy bean lecithin (Lipoid S 100) is
dispersed by probe sonication until the dispersion appears optically clear. 40
mg estramustine are dissolved in the tripalmitate/lecithin dispersion. 0.42 9
sodium glycocholate and 1.58 9 glycerol are dissolved in 60 9 bidistilled water.The aqueous phase is heated to 80~C and is added to the melt. A crude
emulsion is prepared by probe sonication for approximately 2 min. The aude
emulsion is transferred to a thermostatized high pressure homogenizer (APV
Gaulin Micron Lab 40) and is passed 10 times through the homogenizer at a
pressure of 800 bar. The dispersion is allowed to stand at room temperature
for cooling.
Example 33: Physical state of different SLPs at body temperature
Two batches of SLPs from different matrix constituents were prepared
according to the method described in Example 2. Batch 1 was composed of
10% tripalmitate, 0.5% ubidecarenone, 1.2% soy bean lecithin (Lipoid S100,
Lipoid KG), 0.4% sodium glycocholate, 2.25% glycerol, 0.01% thiomersal and
bidistilled water to 100% (by weight). Batch 2 was composed of 10% hard fat
(WitepsolTM W35, Huls AG), 0.5% ubidecarenone, 1.2% soy bean lecithin
(Lipoid S100, Lipoid KG), 0.4% sodium glycocholate, 2.25% glycerol, 0.01%
thiomersal and bidistilled water to 100% (by weight).
The physical state of the matrix constituents was determined by
synchrotron radiation X-ray diffraction at 20~C and at 38~C. The samples were
~1137~5
placed in thermostatized sample holders. The diffraction patterns were
recorded for 180 seconds each. Figure 15a demonstrates that at room
temperature (20~C) both batches of SLPs are crystalline. The spacings
correspond to the ~-crystalline polymorphs. At body temperature (38~C) the
5 tripalmitate SLPs are still crystalline whereas there are no reflections detected
for the hard fat SLPs, i.e. they are amorphous and molten (Fig. 15b). The
different physical state of these SLPs at body temperature gives rise to a
different biopharmaceutical behaviour with respect to the release of
incorporated drugs or bioactive agents. SLPs molten at body temperature
10 display basically the release characteristics typical of conventional lipid
emulsions. Due to the free diffusion of drug molecules in the liquid lipid, the
drug can be released from the vehicle relatively fast. In contrast, SLPs which
are solid at body temperature give rise to sustained release of incorporated
drugs. Since the drug molecules are immobilized in the solid matrix, drug
15 release is not diffusion-controlled but depends on the degradation of the solid
lipid matrix in the body and is therefore delayed.
Example 34: Preparation of PBAs from miconazole
In a thermostatized vial 0.4 9 miconazole is melted at 90~C. 0.24 9
20 lecithin (Lipoid S100) is dispersed in the melt by probe sonication until thedispersion appears optically clear. 0.9 9 glycerol, 80 mg sodium glycocholate
and 4 mg thiomersal are dissolved in 38.5 ml bidistilled water and heated to
90~C. The aqueous phase is added to the miconazole/lecithin melt and a
crude dispersion is produced by probe sonication for 5 minutes. The crude
25 dispersion is transferred to a thermostatized high pressure homogenizer (APV
Gaulin Micron Lab 40) and is passed 10 times through the homogenizer at a
pressure of 800 bar. The PBA dispersion is allowed to stand at room
temperature for cooling.
On cooling the molten miconazole recrystallizes and forms a suspension
30 of miconazole microparticles. The mean particle size (by volume) of
miconazole PBAs is 21.8 ,um detennined by laser diffractometry. The sediment
of miconazole PBAs is easily redispersible by slight agitation.
21137~5
46
Example 35: Preparation of PBAs from ibuprofen
In a thermostatized vial 1.2 9 ibuprofen is melted at 85~C. 0.72 9
lecithin (Lipoid S100) is dispersed in the melt by probe sonication until the
dispersion appears optically clear. 0.9 9 glycerol, 240 mg sodium glycocholate
5 and 4 mg thiomersal are dissolved in 37 ml bidistilled water and heated to
85~C. The aqueous phase is added to the ibuprofen/lecithin melt and a crude
dispersion is produced by probe sonication for 5 minutes. The crude dispersion
is transferred to a thermostatized high pressure homogenizer (APV Gaulin
Micron Lab 40) and is passed 6 times through the homogenizer at a pressure
10 of 800 bar. The PBA dispersion is allowed to stand at room temperature for
cooling.
On cooling the molten ibuprofen recrystallizes and forms a suspension
of ibuprofen microparticles. The mean particle size (by volume) of ibuprofen
PBAs is 61.4 I m determined by laser diffractometry. The sediment of
15 ibuprofen PBAsis easily redispersible by slight agitation.
Example 36: Dissolution speed of ibuprofen PBAs
The dissolution speed of ibuprofen PBAs of Example 35 was measured
in a laser diffractometer (Malvern Mastersizer MS20) by monitoring the decay
20 of the so-called obscuration over a period of 10 minutes. The obscuration is
a measure for the reduction in intensity of unscattered laser light by a sample
and is related to the concentration of particles in the laser beam. In parallel the
particle size can be measured. For the measurement the ibuprofen PBA
sample was diluted with water and was dispersed by magnetic stirring in a
25 measuring cell which was placed in the laser beam line. Figure 16 presents
the decay of obscuration and particle size of a sample of ibuprofen PBAs.
Within 10 minutes the obscuration has decayed to zero, i.e. there is no
detectable amount of particles hinting at the complete dissolution of the PBAs.
The dissolution of the untreated raw substance ibuprofen could not be
30 measured by this technique since the substance is only poorly wettable in
water.
47 ~1137~
Example 37: Preparation of PBAs from lidocaine
In a thermostatized vial 1.2 9 lidocaine is melted at 80~C. 1.2 9
tyloxapol is dissolved in 37.6 ml bidistilled water and heated to 80~C. The
aqueous phase is added to the lidocaine melt and a crude dispersion is
5 produced by probe sonication for 2 minutes. The crude dispersion is
transferred to a thermostatized high pressure homogenizer (APV Gaulin Micron
Lab 40) and is passed 5 times through the homogenizer at a pressure of 1200
bar. The PBA dispersion is allowed to stand at room temperature for cooling.
On cooling the molten lidocaine recrystallizes in fine needles and forms
10 a suspension of lidocaine microparticles. Figure 17 shows a polarized
microscopic picture of the suspended lidocaine needles. The particle shape of
the raw material lidocaine-base (Synopharm) is different from that of lidocaine
PBAs as demonstrated by the polarized microscopic picture of Figure 18.
The mean particle size (by volume) of lidocaine PBAs is 174.2 ,um
15 determined by laser diffractometry. The maximum detected particle size is 400,um. The sediment of lidocaine PBAs is easily redispersible by slight agitation.The addition of water to PBAs leads to the rapid dissolution of the particles.
In contrast, the raw material lidocaine is only sparingly soluble in water and the
dissolution speed is much slower. The high dissolution speed of lidocaine
20 PBAs is a consequence of the modified surface properties and the finely
dispersed state of the particles. Due to the rapid dissolution a determination
of the dissolution speed according to the method described in Example 36 was
not possible.
25 Example 38: Preparation of PBAs from cholecalciferol (vitamin D3)
In a thermostatized vial 0.8 9 cholecalciferol is melted at 95~C. 120 mg
soy bean lecithin (Lipoid S100) is dispersed in the melt by probe sonication
until the dispersion appears optically clear. 40 mg sodium glycocholate is
dissolved in 379.2 ml bidistilled water and heated to 95~C. The aqueous phase
30 is added to the cholecalciferol/lecithin dispersion and a crude dispersion is produced by probe sonication for 5 minutes. The crude dispersion is
transferred to a thermostatized high pressure homogenizer (APV Gaulin Micron
~1 137~ :3
,_ 48
Lab 40) and is passed 8 times through the homogenizer at a pressure of 1200
bar. The PBA dispersion is allowed to stand at room temperature for cooling.
The mean particle size after preparation by number of cholecalciferol
PBAs is 325.1 nm determined by PCS.
Example 39: Preparation of PBAs from estramustine
In a thermostatized vial 2 g estramustine is melted at 1 05~C. In the melt
0.8 g soy bean lecithin (Lipoid S 100) is dispersed by probe sonication until the
dispersion appears optically clear. 0.2 g sodium glycocholate and 0.9 g
10 glycerol are dissolved in 36.1 9 bidistilled water. The aqueous phase is heated
to 95~C and is added to the melt. A crude emulsion is prepared by probe
sonication for approximately 5 min. The crude emulsion is transferred to a
thermostatized high pressure homogenizer (APV Gaulin Micron Lab 40) and
is passed 5 times through the homogenizer at a pressure of 1200 bar. The
15 dispersion is allowed to stand at room temperature for cooling.
