Note: Descriptions are shown in the official language in which they were submitted.
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t NON-IONIC SURFACTANT YESICLES AS THERAPEUTIC AGENT
The present invention relates to a method of
treating or preventing inflammatory and other conditions
associated with undesirably elevated levels of
cytokines, particularly proinflammatory cytokines and
other cytokines which when elevated induce an injurious
effect.
Cytokines are inducible, soluble proteins produced
by a variety of cells involved in immune inflammatory
responses including T cells, B cells and macrophages.
There are many different cytokines, including the
families of the interleukins, colony stimulating
factors, chemokines, interferons and tumour necrosis
factors. These are produced in response to a wide range
of stimuli including injury, infection, inflammation and
tumour states and serve a variety of functions,
including as immunoregulators, growth factors and
differentiation factors.
The immune system comprises an extremely complex
network with numerous interactions between cytokines and
host cells, other cytokines and regulatory molecules.
Each cell type of the immune system produces a distinct
repertoire of cytokines typical of that cell type, with
some overlap in production between the various cell
types since most cytokines are produced by more than one
cell type.
Cytokines act pleiotropically, i.e. they act on a
variety of target cells within the host, where their
effects are exerted by means of high affinity membrane
receptors, to produce an effect dependent upon the
particular target cell. This network therefore ensures
that a single cytokine interacts with more than one cell
type, that individual cytokines have multiple biological
activities, that several cytokines can act as factors
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W097/04768 PCT/GB96/018
mediating a common effect (often in a seemingly
synergistic manner) and that the effects of cytokines
can be various and widespread (as cytokine receptors are
present on multiple cells).
Cytokines may be induced extremely rapidly and,
when induced at normal levels, provide benefit to the
host by mediating the metabolic and biochemical changes
in response to challenges, including infection, injury
and inflammation which are essential to the body's
defence against such challenges and to the healing
process. However, the production of cytokines to
undesirably elevated levels can mediate some of the most
lethal and widespread chronically debilitating diseases
known to man, including sepsis, cachexia, rheumatoid
arthritis and asthma.
Of particular relevance in this regard are the
group of cytokines intrinsically linked to the
generation and maintenance of inflammation, the so-
called proinflammatory cytokines, which includes tumour
necrosis factor alpha (TNF-~), interleukin one alpha and
beta (IL-1~ and IL-1~), interleukin six (IL-6),
interleukin eight (IL-8), and interleukin-12 (IL-12) as
well as other cytokines which act as promoters of
allergic inflammation, including interleukin four (IL-4)
and interleukin five (IL-5). Overproduction of any of
these cytokines is associated with both acute and
chronic inflammatory pathologies.
The pathophysiological effects of these
proinflammatory cytokines, and the cytokine promoters of
allergic inflammation, are often characterised by the
exaggeration of the response they normally elicit, as
would be expected by overproduction. This may lead to
hyper-production of endothelial factors causing excess
vasodilation, leucocyte migration, endothelial
hyporesponsiveness, smooth muscle constriction,
diapedesis and further release of soluble factors or
stimulation of cells, e.g. macrophages and mast cells,
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capable of further inflammatory responses.
A variety of disease states are associated with
undesirably elevated cytokine levels, and often these
are exacerbated by synergy between the various
cytokines, which lead to a diversity of
pathophysiological effects. Examples of such diseases
include:
- Septic shock, a particular problem encountered
with hospitalised patients, often those with life-
threatening diseases, believed to be caused by infectionwith bacteria, where the cell wall component, the
endotoxin LPS, is responsible for initiating the
dlsease.
The destructive inflammatory response associated
with infection is the result of stimulation by LPS of
TNF-~ and the other proin~lammatory cytokines. TNF-
~has been shown to be a primary causative agent of
sepsis, (Glauser et al., Clinical Infectious Diseases
18, s205-216 (1994)). The acute or chronic
overproduction of TNF-~ has widespread effects on
various systems; it can result in disseminated
intravascular coagulation, an increase in vascular
adhesion molecules, and hence efflux of cells, and an
increase in prostagl~n~;n~ and leukotrienes, which can
effect the vascular tone and platelet aggregation.
TNF-~ mediates the progression of bacterial infection to
systemic inflammatory response syndrome (SIRS: also
known as sepsis), which can result from other insults
such as trauma, through severe sepsis (SIRS plus raised
temperature, tachycardia, lactic acidosis, perfusion
abnormalities and oliguria) to septic shock (severe
sepsis plus hypotension). This plethora of effects
takes the form of a cascade system that has TNF-~ as a
fulcrum with other proinflammatory cytokines such as IL-
1, IL-6 and IL-8 acting in association in a cumulative
process. In particular, IL-1 and IL-6 play critical
roles in mediating SIRS (Glauser et al., (1994);
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Dinarelli, Eur. Cytokine Netw., 5(6), 517-531 (1994);
Borden & Chin, J. Lab. Clin. Med., 126(6), 824-829
(1994))-
- The acute phase response (APR). This comprises
predetermined, well-orchestrated local and systemic
reactions resulting from infections, trauma, neoplasms
or other disorders which put a stress on homeostasis
(Borden ~ Chin, J. Lab. Clin. Med 123, 824-9 (1994)).
Local reactions at the site of injury include
aggregation of platelets and clot formation, dilation
and leakage of blood vessels and accumulation and
activation of granulocytes and monocytes~macrophages
that release a number of cytokines including I~-1, TNF-
~and IL-6 in particular. Systemic reactions include
fever, leukocytosis, activation of complement and
clotting cascades, and changes in concentrations of
"acute phase" plasma proteins generated by the liver.
Undesirably elevated levels of IL-6 have been noted in
patients with SIRS, burns injuries, trauma and after
organ transplantation, giving an early indication of
organ rejection in the latter case.
- Arthritic diseases, osteoarthritis (OA) and
rheumatoid arthritis (RA), which are controlled by a
complex cytokine network, in which the proinflammatory
cytokines IL-1, TNF-~ and IL-6 are of major importance
(Sipe et al., Mediators of Inflammation, 3, 243-256
(1994)). All three cytokines have been detected at
undesirably elevated levels in the synovial fluid,
synovium and cartilage from RA patients, whilst IL-1 and
IL-6 have been found in the latter tissues from OA
patients. IL-8 is also important in RA. IL-15 has also
been associated with RA.
Indeed certain therapies which reduce TNF-~ or IL-1
levels have been shown to have a positive benefit to the
swollen joints and overall pain in rheumatoid arthritis.
- Allergic inflammation.
Many allergies are associated with overproduction
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of IL-4 and IL-5 by Th2-type T helper cells, which are
involved in the pathogenesis of allergic inflammation in
humans (Kumar & Busse, 1995); Romagnami, Current Opin.
Immunology, 6(6), 838-846 (1994)) and which are
~ 5 associated with humoral immunity.
Both allergic and non-allergic asthma are
associated with undesirably elevated levels of IL-4 and
IL-5 (Kumar ~ Busse, Scientific American: Science &
Medicine, March/April, 38-47 (1995)). IL-6 and TNF-~
are also involved in the pathogenesis of asthma.
Other common allergies fundamentally linked to the
overproduction of the "allergic" inflammatory promoters
IL-4 and IL-5 and rapid mediators such as histamine
include, but are not limited to, pollen allergies (hay
fever), non-specific allergic rhinitis, house dust mite
allergies and animal dander allergies.
- Cachexia, a condition of severe weight loss and
tissue wasting which is associated with chronic invasive
diseases such as cancer and parasitic diseases as well
as HIV infection, characterised by continued lipid and
protein catabolism out of balance with nutritional
requirement and food intake in which TNF-~, IL-l and IL-
6 are all implicated as humoral mediators.
- Certain types of cancer, eg. B cell neoplasias
such as multiple myeloma, which have been shown to be
associated with increased levels of IL-6 (Akira et al.,
Adv. Immunology, 54 1-63, (1993)).
Other inflammatory diseases include ulcerative
colitis, inflammatory bowel disease, with which IL- 12
has been associated, and atherosclerosis.
Despite what might be perceived as an extremely
diverse range of symptoms exhibited by patients
suffering from these diseases, they are all
~ characterised by the underlying involvement of these
3 5 cytokines.
Current therapeutic strategies are based on
targeting the effect of individual cytokines (or second
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messengers) by seeking to block or interfere with the
interaction of the cytokine with its receptor using, for
example, a binding partner to one or other component
such as an antibody to the receptor or the cytokine.
Such strategies are extremely limiting in concentrating
only on a single mediator and are often short-lived.
Furthermore, removing or inactivating just one agent in
the complex network of interacting pathways between
cytokines could simply act as a switch between pathways
resulting in a different cytokine mediating the
inflammatory response.
In addition, treatment regimes which rely on
administration of antibodies to humans are not problem-
free and often the undesirable side effects of such
treatments outweigh any benefit.
There is accordingly a need for a new therapeutic
method capable of pleiotropically affecting multiple
cytokines. The present invention provides such a
method.
Thus viewed from one aspect, the present invention
provides a method for combating conditions associated
with undesirably elevated levels of one or more
cytokines which when elevated induce an injurious effect
comprising administering to a subject an effective
amount of non-ionic surfactant vesicles (NISV).
In a related aspect, the present invention provides
the use of NISV in the manufacture of an agent for use
in combating conditions associated with undesirably
elevated levels of one or more cytokines which when
elevated induce an injurious effect.
Such conditions include septic shock and severe
sepsis, cachexia as well as inflammatory conditions
including rheumatoid arthritis, asthma as well as
topical allergic conditions such as eczema and
psoriasis, bacterial endotoxaemia, SIRS, ulcerative
colitis, inflammatory bowel disease, Crohn's disease,
atherosclerosis, osteoporosis, diabetes, leukaemia,
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multiple myeloma, cystic fibrosis, pulmonary fibrosis,
acute meningococcal infections, alcoholic hepatitis,
various allergies, systemic lupus erythrymatosus,
multiple sclerosis and treatment of these disease
constitute particular aspects of the invention.
As used herein, the term 'combating' includes both
prophylaxis and therapy.
We have found that NISV are able to reduce
independently the levels of the proinflammatory
cytokines TNF-~, IL-l, IL-6 and IL-8, as well as the
cytokine mediators of allergic inflammation, IL-4 and
IL-5. This therapeutic activity at the cellular level
translates to an observable benefit at the physiological
level, in terms of management of those diseases which
are associated with raised levels of these cytokines.
In this context, the undesirably elevated cytokine
levels refers to levels greater than observed in
'normall subjects which do not exhibit any signs of
inflammation or responses characteristic thereof.
We have demonstrated the reduction in levels of
these cytokines in vitro and ex vivo in both human and
animal cells. We have demonstrated also a positive
therapeutic effect in animal models of cachexia.
In one aspect, the invention relates to the use of
NISV for reducing the level of TNF-~ produced by cells
in an inflammatory response. This is of particular
importanc~ in the therapy and prophylaxis of septic
shock, and of cachexia. The invention also relates to
the use of NISV for reducing the levels of one or more
of IL-l, IL-6, IL-8, IL-12 and/or IL-4 and/or IL-5
produced by cells involved in inflammatory and immune
processes.
NISV are known, for example as carriers e.g. for
drugs and also as components of cosmetics. With antigen
entrapped, such vesicles are also known as potent
;mmllnological adjuvants, as described in International
patent applications numbers WO93/19781 and WO95/09651.
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We are not aware however of any prior recognition of the
therapeutic potential of the vesicles themselves, and
particularly as an immunomodulating agent in the absence
of an antigen.
Thus, the present invention may be distinguished
from prior art therapies involving NISV in that the NISV
may be used as the sole therapeutically active agent,
rather than a carrier. The NISV are active without any
other biologically active agent being entrapped or
associated with them. In other words they may be used
"empty".
The vesicles used according to the invention may
comprise non-ionic surfactants alone and may optionally
include other components such as molecules which have
the ability to transport or facilitate the transport of
fats, fatty acids and lipids across mucosal membranes
for example bile salts as described in W095/09651.
Indeed compositions based on vesicles comprising
such molecules with these transporting capabilities are
new.
Examples of such vesicles are described in the
above-mentioned W095/09651 which also describes
preparative processes.
The invention is applicable to all types of NISV
vesicular structures, including unilamellar vesicles
(comprised of a single bilayer), multilamellar vesicles
(comprised of more than one bilayer) and multivesicular
vesicles, which may comprise unilamellar and/or
multilamellar vesicles.
Methods for preparing NISV are well known in the
art and described in the literature, including for
example in W093/19781 and W095/09651.
The non-ionic surfactant used to form the NISV may
be any pharmacologically acceptable material with the
appropriate surface active properties. Preferred
examples of such materials are glycerol esters. Such
glycerol esters may comprise one or two higher aliphatic
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acyl groups e.g. containing at least ten carbon atoms in
each acyl moiety. Glycerol monoesters are preferred,
particularly those containing a C12-C20 alkanoyl or
alkenoyl moiety, for example caproyl, lauroyl,
myristoyl, palmitoyl, oleyl or stearoyl. A particularly
preferred surfactant is l-monopalmitoyl glycerol.
Ether-linked surfactants may also be used as the
non-ionic surfactant of which the NISV according to the
invention are comprised. Preferred examples of such
materials are ether-linked surfactants based on glycerol
or a glycol preferably a lower aliphatic glycol of up to
4 carbon atoms, most preferably ethylene glycol.
Surfactants based on such glycols may comprise more than
one glycol unit, preferably up to 5 glycol units and
more preferably 2 or 3 glycol units, for example
diglycol cetyl ether or polyoxyethylene-3-lauryl ether.
Glycol or glycerol monoethers are preferred,
particularly those containing a Cl2-C20 alkyl or alkenyl
moiety, for example capryl, lauryl, myristyl, cetyl,
oleyl or stearyl.
The ethylene oxide condensation products usable in
this invention include those disclosed in W088/06882,
i.e. polyoxyethylene higher aliphatic ether and amine
surfactants. Particularly preferred ether-linked
surfactants are 1-monocetyl glycerol ether and diglycol
cetyl ether. However, for use in the present invention
it is necessary to select pharmacologically acceptable
materials, preferably those which are readily
biodegradable in the m~mm~l ian system. For this
reason, we prefer the aforementioned glycerol esters for
preparing vesicles to be administered by injection,
either subcutaneous, intramuscular, intradermal
intraperitoneal or intra-articular, or via the mucosal
route such as by oral, nasal, bronchial, urogenital,
rectal, intrapulmonary or ocular administration, oral
~m;n;stration being particularly preferred, especially
in the case of vesicles containing bile salts or
' , CA 02228298 1998-01-29
-- 10
equivalent compounds.
In one aspect, the invention provides a
pharmaceutical composition for combating conditions
associated with undesirably elevated levels of cytokines
which when elevated induce an injurious ef~ect
comprising NISV together with a pharmaceutically
acceptable carrier or excipient ln a form suitable for
intrapulmonary or intra-articular administration.
In the case of the aforementioned vesicles which
additionally comprise molecules which have the ability
to transport or facilitate the transport of fats, fatty
acids and lipids across membranes, (hereinafter
"transport enhancers") a variety of such molecules may
be used such as those described in W095/09651.
Cholesterol derivatives in which the C23 carbon atom of
the side chain carries a carboxylic acid, and
derivatives thereof are particularly preferred.
Amongst such derivatives are the "bile acids"
cholic acid and chenodeoxycholic acid, their conjugation
products with glycine or taurine such as glycocholic and
taurocholic acid, and derivatives including deoxycholic
and ursodeoxycholic acid, and salts of each of these
acids.
Also preferred as "transport enchancers" are
acyloxylated amino acids, preferably acyl carnitines and
salts thereof particularly those containing C620
alkanoyl or alkenoyl moieties, such as palmitoyl
carnitine. As used herein, the term acyloxylated amino
acid is intended to cover primary, secondary and
tertiary amino acids as well as ~, ~ & y amino acids.
Acylcarnitines are examples of acyloxylated y amino
acids.
These vesicles may, naturally, comprise more than
one type of "transport enhancer" in addition to the non-
ionic surfactants for example one (or more) differentbile salts and one (or more) acylcarnitines.
For effective vesicle formation, the non-ionic
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W O 97/04768 PCT/GB96/01861
surfactant may need to be admixed with an appropriate
hydrophobic material of higher molecular mass capable of
forming a bi-layer, particularly a steroid, e.g. a
sterol such as cholesterol. The presence of material
~ 5 such as cholesterol assists in forming the bi-layer on
which the physical properties of the vesicle depend.
The NISV may also incorporate a charge-producing
amphiphile, to cause the NISV to take on a charge.
Acidic materials such as higher alkanoic and alkenoic
acids (e.g. palmitic acid, oleic acid); or other
compounds containing acidic groups, e.g. phosphates such
as dialkyl, preferably di(higher alkyl), phosphates,
e.g. dicetyl phosphate or phosphatidic acid or sulphate
monoesters such as higher alkyl sulphates, e.g. cetyl
sulphate, may all be used for this purpose.
The steroid may e.g. comprise 20-120 percent by
weight of the non-ionic surfactant, preferably 60-100
percent. The amphiphilic material producing a charge
may e.g. comprise 1-30 percent by weight of the non-
ionic surfactant.
The charge-producing amphiphilic material
stabilises the structure of the vesicles and provides
effective dispersion.
The non-ionic surfactant and membrane-forming
hydrophobic material may be converted to NISV by
hydration in the presence of shearing forces. Apparatus
to apply such shearing forces is well known, suitable
equipment being mentioned e.g. in W088/06882.
Sonication and ultra-sonication are also effective means
to form NISV or to alter their particle size.
Essentially, the reagents to form the vesicles are
brought into contact conveniently at temperatures in the
range of 80 to 150~C to ~melt' the surfactants, and
~ conveniently in the presence of a suitable medium such
as a buffer or aqueous solution and mixed to ensure a
homogenous suspension. Such mi ~ ng may be by standard
well known techniques including vortexing or the use of
. CA 02228298 1998-01-29
homogenisation apparatus, such as is common in the
pharmaceutical industry.
An effective method for the production of NISV is
that dlsclosed by Collins et a~., J. Pharm. Pharmocol.
~2, Supp. 53P (1990). This involves melting a mixture
of the NIS, steroid, and amphiphile (if used) and
hydrating with vigorous mixing in the presence of
aqueous buffer. The suspension may then be extruded
several times through microporous polycarbonate
membranes at an elevated temperature sufficient to
maintain the NISV-forming mixture in a molten condition.
It is also possible to form NISV by rotary film
evaporation from an organic solvent, e.g. a hydrocarbon
or chlorinated hydrocarbon solvent such as chloroform.
The resulting thin fllm may then be hydrated optionally
in phosphate-buffered saline in the presence of any
material to be entrapped and optionally another
surfactant (Russell and Alexander, J. Immunol. 140, 1274
(1988)).
Where vesicles of specific size are required, these
may be prepared by sequential extrusion through
polycarbonate filters as described in Nayar et al.,
(Biochem. Biophys. Acta 986 200-206 (1989)) or by other
methods known in the art such as mixing and homogenising
the reagents under particular conditions, for example,
for different times and at different speeds, which may
be appropriately determined for each system and size
desired.
Without wishing to be bound by theory, it is
believed that the therapeutic agents of the invention
act at the level of the cells responsible for production
of the relevant cytokines, typically macrophages and
monocytes, as well as other immune cells. Down-
regulation of cytokine production at this level
represents a considerable advance over current therapies
which seek to block the activity of individual
cytokines, particularly since according to the invention
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WO 97/04768 PCT/GB96/01861
a single agent may be used to down regulate more than
one cytokine.
Viewed from another aspect, the present invention
provides a method of combating cachexia comprising
administering to a subject suffering from or liable to
cachexia an effective amount of NISV.
In a related aspect the present invention provides
the use of NISV in the manufacture of an agent for use
in the treatment or prophylaxis of cachexia.
In a further aspect the present invention provides
a method of treating septic shock comprising
administering to a subject suffering from or liable to
septic shock an effective amount of NISV.
In a related aspect, the present invention provides
the use of NISV in the manufacture of an agent for use
in the treatment or prophylaxis of septic shock.
Viewed from another aspect, the present invention
provides a method of combating arthritis comprising
administering to a subject suffering from or liable to
arthritis an effective amount of NISV.
In a related aspect the present invention provides
the use of NISV in the manufacture of an agent for use
in the treatment or prophylaxis of arthritis.
Viewed from another aspect, the present invention
provides a method of combating asthma comprising
administering to a subject suffering from or liable to
asthma an effective amount of NISV.
In a related aspect the present invention provides
the use of NISV in the manufacture of an agent for use
in the treatment or prophylaxis of asthma.
"Treatment" and "treating" as used herein refer
both to the alleviation of existing morbid conditions
and to the prophylactic prevention thereof by timely
administration of NISV before proinflammatory cytokine
levels have become dangerously elevated. The onset of
such conditions can often be foreseen, but there has
hitherto been no effective method of prophylaxis
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available.
The therapeutic agents according to the invention
may be administered by all conventional methods
including parenterally (e.g. intraperitoneally,
subcutaneously, intramuscularly, intradermally or
intravenously), topically (e.g. as a cream to the skin,
intra-articularly, mucosally (e.g. orally, nasally,
vaginally, rectally and via the intra-ocular route) or
by intrapulmonary delivery for example by means of
devices designed to deliver the agents directly into the
lungs and bronchial system such as inhaling devices and
nebulisers, and formulated according to conventional
methods of pharmacy optionally with one or more
pharmaceutically acceptable carriers or excipients, such
as for example those described in Remingtons
Pharmaceutical Sciences, ed. Gennaro, Mack Publishing
Company, Pennsylvania, USA (1990).
Such compositions are conveniently formulated in
unit dosage form eg. for mucosal, parenteral or oral
administration.
Although NISV are known per se, it has not
previously been proposed to divide such preparations
into unit dosages.
There~ore, in a further aspect, the present
invention provides a pharmaceutical composition
comprising empty NISV together with a pharmaceutically
acceptable carrier or excipient, conveniently in unit
dosage form.
In this context, "empty NISV" are NISV which have
no active agent entrapped or associated with them.
Actual treatment regimes or prophylactic regimes,
formulations and dosages will depend to a large extent
upon the individual patient and may be devised by the
medical practitioner based on the individual
circumstances.
The type of formulation will be appropriate to the
route of administration. For example, parenteral
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W O 97/04768 PCT/GB96/01861
administration of NISV by subcutaneous or intramuscular
injection may be with a sterile aqueous suspension of
~ NISV in PBS or water for injection, provided in
ampoules, vials or as measured doses in pre-filled
syringes or in the form of a lyophilisate for
reconstitution with PBS or water for injection prior to
administration.
The dosage of NISV for subcutaneous injection may
include from 2.5 to 50 mg eg. 2.5 to 25 mg of vesicles
formulated as described above for example in PBS or
water. ~m; ni stration regimes for the subcutaneous
route may be determined by the duration of action in
specific clinical situations. Frequency of
administration may range from daily injections to
injections weekly or fortnightly. Typical
administration regimes may for example comprise two
doses at fourteen day intervals, three doses at fourteen
day intervals, three doses at intervals of 0, 28 and 84
days, or three doses at seven day intervals.
Mucosal adminstration may for example follow these
regimes. ~he dosages for oral administration may be
between 2.5 to 50 mg of vesicles or considerably higher.
Suitable oral formulations include flavoured liquid
suspension of syrups, liquid/powder filled capsules.
For administration to the respiratory tract (nasally or
orally) metered spray inhalers or nebulisation of an
aqueous suspension of NISV may be used.
Although primarily of applicability to hllm~n.q, the
invention may also be used in veterinary medicine for
example to treat companion An i m~ 1 S such as cats and
dogs, and livestock, eg. poultry.
In general, the size of the vesicles is not
critical and the method is applicable to a wide size
range of vesicles, appropriate for ~mi ni stration by the
above-mentioned routes. A wide range of NISV sizes has
been described in the literature ranging for example in
the order of about 100 nm to several micrometers and can
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- 1 6
be used. We have however found that whilst the levels
of the proinflammatory cytokines and the cytokine
mediators of inflammation are affected by the vesicles
of the invention, the degree of modulation of cytokine
levels may be influenced by the size of the vesicles and
in particular that certain vesicle sizes may enhance the
reduction in cytokine levels which may be observed.
Thus we have found that effective down regulation of
pro-inflammatory cytokine production is particularly
pronounced above a threshold which our experiments have
shown to be within the range 150-215 nm. This effect
may vary depending on the system used ie. the nature of
the vesicle, the condition being treated and e~en the
animal species concerned. Appropriate vesicle sizes for
achieving this enhanced beneficial effect, may be
determined by appropriate tests. In vitro, murine and
human cells appear to show similar size thresholds for
enhancement of the beneficial effects of NISV. Thus,
for example, in the case of mice and humans our
experiments have shown that the beneficial enhanced
effect may be obtained with vesicles of greater than
about 200 nm. In particular larger vesicles, typically
of mean diameter greater than approximately 200 or 215
nm up to several micrometers (eg. 750-3000 nm) have been
shown to cause greater down-regulation of IL-5 and IL-l
as compared with smaller vesicles, for example those of
mean diameter approximately 160 nm. In vivo experiments
have demonstrated that vesicles up to several
micrometers are effective.
Thus, a preferred aspect of this invention
comprises all the methods, uses and compositions of the
invention wherein the vesicles are of mean diameter
greater than 200 nm, preferably greater than 215 nm and
more preferably greater than 250 nm.
This finding of the role of NISV size may be
important in all the cases of allergic and inflammatory
diseases associated with elevated levels of
. CA 02228298 l998-0l-29
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proin~lammatory cytokines and cytokine mediators of
inflammation.
If desired, the size distribution of vesicles
within a preparation may ~e modified for example to
reduce variability, exclude vesicles o~ certain size
ranges or obtain a homogenous preparation. This may be
achieved by extrusion through polycarbonate membranes
with pores of known diameter as described in Nayar et
al, Biochem. Biophys. Acta, 986, 200-206 (1989). It
will of course be appreciated that actual size of the
vesicles produced by this extrusion method may differ
~rom the stated pore diameter of the membrane used, and
it is there~ore desirable to ~urther characterise the
size of the vesicles once formed by other methods known
to those skilled in the art, such as photon correlation
spectroscopy (PCS) and electron microscopy (EM). Other
methods of preparing vesicles of relatively homogeneous
size include the use of a French press,
microfluidisation/ homogenisation and sonication as
described in Lasic, Liposomes: from physics to
applications, Elsevier, Amsterdam (1993). The size of
such preparations may be confirmed by the techniques
described above. If other preparative methods are
used, vesicles of desired size may be fractionated from
a more heterogeneous size population by a variety of
techniques known to those skilled in the art, including
for example size exclusion chromatography,
centrifugation etc.
The above discussion of cytokine pathways and
interactions is only a summary of the existing knowledge
about this highly complex and rapidly-advancing field.
It should be clearly appreciated that our invention is
not tied to any specific theory of cytokine activity but
is firmly based on experimental observations.
3s It will be appreciated that the activities of
certain cytokines vary according to cell type, and that
cytokines may have dif~erent properties according to the
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- 18
condition and its stage of development.
The method of the invention is applicable to the
production of therapeutic agents comprising NISV as the
sole therapeutic agent, and also to combinations
together with one or more other agents useful in the
treatment of the diseases or conditions concerned, for
example anti-inflammatory agents such as
corticosteroids, antihistamines and anti-cytokine
antibodies, therapeutic cytokines such as IL-2, IL-12
and IFN-y, and in the case of septic shock,
antibacterial agents. Such additional agents may be
administered simultaneously or sequentially with the
surfactant vesicles, preferably NISv and in the case of
simultaneous administration, the agents may be provided
in admixture with the vesicles, and/or entrapped
therein.
Thus viewed from a yet further aspect, the
invention provides a product containing NISV and at
least one other pharmaceutically active agent as a
combined preparation for simultaneous separate or
sequential use in therapy. The pharmaceutically active
agent will be selected according to the therapy, and
examples are given herein.
The NISV may also be administered in combination
with an agent(s) in order to counteract the unwanted
side effects of that agent(s) without removing the
therapeutic effect of the agent(s). For example, in the
treatment of cancer, the administration of NISV in a
combination therapy may be used to reduce the
undesirable side effects, such as cachexia, of
chemotherapeutic drugs.
Furthermore, in such combination therapies,
entrapping an agent(s) within NISV may, in addition to
providing the beneficial therapeutic effects of the
NISV, be used to improve the pharmacokinetic profile of
that agent(s), for example by providing a sustained
release vehicle for the agent(s) or by protecting the
CA 02228298 1998-01-29
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agent(s) from degradation or rapid clearance from the
system.
Methods by which other agents may be entrapped
within preformed NISV include the dehydration-
rehydration method (Kirby & Gregoriadis, Biotechnology,~, 979-984 (1984)) in which the agent present in the
aqueous phase is entrapped in pre-formed vesicles by
flash freezing ~ollowed by lyophilisation, and the
freeze-thaw technique (Pick, Arch. Biochem. Biophys.
212, 186-194 (1981)). In the latter technique, vesicles
are mixed with the agent concerned and repeatedly ~lash
~rozen in liquid nitrogen and e.g. warmed to
temperatures of the order of 60~C (ie. above the
transition temperature of the relevant surfactant). In
the case where NISV are prepared by homogenisation, the
agent may be entrapped during the homogenisation process
itself. In such a method the agent is dissolved in the
aqueous phase prior to homogenisation.
The invention will now be described by way of the
following non-limiting Examples, with reference to the
Figures which show:
Figure 1: The ability of NISV to reduce weight-loss
associated with T.Gondii-induced cachexia in mice.
Figure la: A bar chart showing the number of
T.Gondii cysts in brains o~ mice infected with T.Gondii
a~ter immunisations with PBS, NISV, soluble tachyzoite
antigen (STAg), STAg entrapped in NISV (NISV/STAg) or
STAg mixed with NISV (NISV+STAg).
Figure lb: Graphs showing the level of weight loss
after infection with T.Gondii in experimental mice
actively immunised with PBS, NISV, STAg or NISV/STAg.
Fi~ure 2: The ability of NISV to reduce weight loss
associated with FK-565 induced cachexia in mice.
Figure 2a: Graph showing weight loss in mice after
intraperitoneal injection with PBS, NISV, the
acyltripeptide FK-565 in PBS or FK-565 entrapped in
NISV.
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Figures 2b and 2c: Bar charts showing the levels of
IL-2 (Figure 2b) and IL-12 (Figure 2c) produced in ConA
stimulated splenocytes isolated from mice treated as per
Fig. 2a.
Fiaure 3: Reduction by NISV in levels of cytokines
produced by stimulated murine cells.
Fiaure 3a: Bar chart showing TNF-~ production by
the stimulation of the murine macrophage cell line J774
with LPS+IFN-y, IFNy, LPS, NISV+IFNy+LPS, NISV+IFN-y,
NISV+LPS or NISV.
Figure 3b: Bar chart showing TNF-~ production by
the stimulation of murine peritoneal macrophages with
PBS, LPS, LPS+NISV, LPS+IFN-y or LPS+NISV+IFN-y.
Figure 3c: Bar chart showing IL-6 production by the
stimulation of murine peritoneal macrophages with
LPS+NISV or LPS+PBS as compared to IL-6 produced by
unstimulated cells.
Fiaure 4: Reduction by NISV in levels of cytokines
produced by stimulated human cells.
Fiaures 4a and 4b: Graphs showing levels of IL-6
(Figure 4a) and TNF-~ (Figure 4b) in human peripheral
blood leucocytes treated with PBS, LPS or LPS+NISV.
Fiqure 5: Reduction by NISV in levels of cytokines
produced by stimulated human cells.
Fiqures 5a and 5b: Graphs showing levels of IL-6
(Figure 5a) and TNF-~ (Figure 5b) in human peripheral
blood leucocytes treated with PBS, LPS or LPS+NISV.
Fiaures 5c and 5d: Graphs showing the level of IL-
1~ (Figure 5c) and IL-1~ (Figure 5d) in human peripheral
blood leucocytes treated with PBS, LPS, NISV or
LPS+NISV.
F;aure 6: The effect of size of NISV on their
immunomodulatory effect.
F;qures 6a and 6b: IL-2 and IL-5 (Fig. 6a) and IL-5
and IFNy (Fig. 6b) produced by con-A stimulated lymph
node cells collected from mice treated with ovalbumin
entrapped in NISV prepared by extrusion though membranes
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of pore size 800 nm, 400 nm, 200 nm and 100 nm, and in
PBS.
Figures 7a and 7b: Bar charts showing levels of IL-
1~ (Figure 7a) and IL-1~ (Figure 7b) in cells from human
leucocyte pro-macrophage cell line U937 and PBLS from
healthy volunteers treated with PBS, LPS, NISV extruded
through 200 nm pore size membrane or non-extruded NISV;
Figures 8, 9 and 10: Suppresion of LPS induced
cytokine production by NISV in mice.
Figure 8 is a bar chart showing serum IL-6
production in mice in response to LPS administered 1, 4
or 14 days after subcutaneous injection of NISV. Figure
8 shows results from an NISV dose of 17 mg/kg, 3 hours
after LPS challenge (Figure 8a) or 6 hours after LPS
challenge (Figure 8b).
Figure 9 shows results from an NISV dose of 80
mg/kg 3 hours after LPS challenge (Figure 9a) or 6 hours
after LPS challenge (Figure 9b). (*: psO.05 v control;
**: psO.025 v control; ***: psO.0005 v control)
Figure 10 shows results from an NISV dose of 80
mg/kg after a second challenge with hPS at days 15, 18
or 28 days after the NISV dose. (*: psO.05 v control)
Figures 11 and 12: Reduction by NISV in levels of
TNF~ and IL-6 in LPS-stimulated human PBLs.
Figures lla and llb are graphs showing TNF~ levels
in LPS-stimulated (llb) or non-stimulated (lla) PBLs
extracted from a hllm~n volunteer before (dotted line)
and after (solid line) administration of NISV.
Figures 12a and 12b are graphs showing IL-6 levels
in LPS-stimulated (12b) or non-stimulated (12a) PBLs
extracted from a human volunteer before (dotted line)
and after (solid line) ~mi n; stration of NISV.
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EXAMPLES
~am~le 1
The modulation of cachexia
Cachexia is a serious condition characterised by
pronounced weight-loss caused by several underlying
conditions (e.g. chronic infections with microorganisms
such as viruses and parasites, tumours, congestive
cardiac failure) but mediated by proinflammatory
cytokines, in particular TNF-~ and IL-6. The induction
of this weight-loss is independent of the nutritional
status of the animal, which could be well fed, and is
characterised by loss of body mass. NISV were
investigated as an immunomodulator in two models for
prevention of cachexic weight-loss, one in which the
cachexia was induced by parasitism and the other in
which it was induced by a peptide drug.
A. Reduction in the level of weight-loss associated
wi th Toxoplasma ~ondii-induced cachexia in mice
treated with NISV
Materials and Methods
Vesicle preparation: Vesicles were prepared by the
methods previously described by Brewer and Alexander
(Immunology, 75, 570-575 (1992)). 1-Mono palmitoyl
glycerol (MPG), cholesterol (CHOh) and dicetyl phosphate
(DCP) (all Sigma, Poole, Dorset, U.K.) were mixed in a
15 ml pyrex test tube in the molar ratio 5:4:1 (MPG:
CHOL: DCP) to a total of 150 ~moles and then heated to
130~C in a dry-block (Grant) until melted. Empty
vesicles were formed when 5 ml aqueous buffer (PBS, pH
7.4) was added and the resulting suspension vortexed
vigorously for 1 minute and the suspension shaken at
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60~C for 2 hours. The vesicles were then ready for
storage or in vitro assay. Previous studies have shown
that vesicles prepared by this method yielded vesicles
of approximately 2 micron diameter.
Antigen ent~a~..a..t: Antigen entrapment into preformed
vesicles was achieved by the dehydration-rehydration
technique as described by Kirby and Gregoriadis,
(Biotechnology, 2, 979-984 (1984)). Briefly, 5 ml (150
~moles) of vesicle solution were mixed with 2 ml antigen
in PBS (5 mg/ml) in polypropylene centrifuge tubes
(Elkay Products Inc., Shrewsbury, MA, U.S.A.) and flash
frozen as a thin shell by swirling in liquid nitrogen.
Preparations were then lyophilised in a freeze drier at
0.1 torr overnight before rehydration in 0.5 ml
distilled water.
~n;m~1 8 and inoculationB: BALB/K mice were in-house
bred and inoculated when 8-10 weeks old. Groups of 5
mice were immunised subcutaneously with either 50 ~g
soluble tachyzoite antigen (STAg) (as prepared by
Roberts & Alexander, Parasitology, 104, 19-23 (1992)) in
PBS emulsified with 100 ~l FCA, 50 ~g STAg entrapped
within NISV, or 50 ~g STAg mixed with empty NISV.
Control groups were immunised with the same volume of
PBS or empty NISV. The inoculations were repeated after
2 weeks.
Mice were infected with 20 viable T. gondii cysts 2
weeks after the second immunisations. Four weeks later
cyst burdens were enllmerated mAnllAlly from brain
suspensions. The mean body weights of the mice were
measured for 32 days post infection.
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RE~ULTS
Active immunisation with STAg alone, STAg entrapped in
NISV (i.e. NISV/STAg) or STAg mixed with NISV (i.e. NISV
& STAg) resulted in a significant reduction in the
number of parasites encysted within the mice brains
(Figure la). There was no reduction in and no
significant difference between the number of cysts per
brain in the control groups treated with PBS or NISV
alone. NISV alone had little effect on the numbers of
cysts able to infect each mouse, i.e. they did not
provide any direct protection against the parasites or
directly affect the parasites themselves.
Figure lb shows the level of weight-loss post-infection
with the T. gondii cysts. Weight-loss (cachexia) was
severe in the control group receiving PBS. Mice in this
group lost approximately 12% of their body weight within
20 days post-infection. In contrast, the administration
of NISV in the other control group prevented any
significant metabolic weight loss in infected mice,
without altering their degree of parasitism. The
weights of mice treated with NISV alone were similar to
those exhibiting active, protective immunity after
inoculation with STAg, NISV/STAg or NISV-STAg.
The administration of NISV alone did not prevent
infection by Toxoplasma gondii parasites or effect
parasite viability as shown by the number of cysts in
the brain. However, NISV administration did prevent the
considerable weight-loss (cachexia) associated with this
condition. In contrast, mice in the PBS control group
exhibited radical losses of weight over 20 days as well
as high levels of parasitism.
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,
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B. Reduction in the level of weight-loss associated
with FK-565-induced cachexia in mice treated with
NISV
FK-565 is an experimental acidic acyltripeptide known to
have potent antitumour and antibacterial effects. It
enhances anti-tumour host defence activity by inhibiting
tumour growth. Repeated intraperitoneal injections of
FK-565 significantly activates the cytotoxicity of
murine peritoneal macrophages and natural killer (NK)
cells towards tumours and also augments their killing
potential. FK-565 exhibits further antitumour activity
by increasing the release of TNF-~, a potent anti-tumour
cytokine. However, the increased production of TNF-
~
and other proinflammatory cytokines by FK-565 is
associated with a significant degree of drug-induced
cachexia.
Materials and Methods
NISV were prepared as described by Brewer and Alexander
(Supra).
Drug entrapment: FK-565 was entrapped in preformed NISV
using the freeze-thaw method (Pick, Arch. Biochemistry,
Biophysics 212, 186-194 (1981)). The antigen vesicle
mixture was frozen in liquid nitrogen and then thawed to
60~C. This was repeated five times. The suspension was
shaken for a further 2 hours at 60~C. The level of
entrapment of FK-565 was assessed using a standard
ninhydrin assay.
Ar lm~ nd inoculations: Female BALB/c mice were in-
house bred and inoculated when 8-10 weeks old. Groups
of 5 mice were immunised intraperitoneally with either
100 ~l PBS, empty NISV (100 ~1; 5 mg), FK-565 in PBS
(200 ~l; 20 ~g in toto) or FK-565 entrapped in NISV (200
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- 2 6
~1; 6 ~g in toto in 10 mg vesicles). Mouse weights were
recorded prior to injection and at 1, 2 and 3 days after
injection of the above agents.
Cytok; n~ assay~: Spleens were collected ~rom the mice 3
days after injection and pooled in RPMI 1640 culture
medium supplemented with 2 mM L-glutamine, 100 units/ml
penicillin, 100 ~g/ml streptomycin, 0.05 mM ~-
mercaptoethanol and 10% (v/v) foetal calf serum (FCS)
(all Gibco, Paisley, U.K.). Splenocyte suspensions were
prepared by gently teasing the spleens apart with
forceps after which the suspensions were centrifuged at
200 x g for 10 minutes and resuspended in 0.5ml o~
medium. Viable cells were enumerated by Trypan Blue
exclusion test and cell suspensions adjusted to 5X106
cells/ml. 100 ~l/well aliquots of cell suspension,
containing 5x105 cells, were added to 96-well flat-
bottomed tissue culture plates (Costar, Cambridge, M.A.,
USA), followed by 100 ~l/well aliquots of Con A (5
~g/ml) in triplicate. Cultures were then incubated for
60 hours at 37~C in a 5~ CO2 atmosphere after which 150
~l aliquots of cell culture supernatants were removed
and stored at -70~C for cytokine assay.
Cytokines (IL-2 and IL-12) were detected by monospecific
ELISA. Flat-bottomed polystyrene plates (Dynatech,
Alexandria, VA, USA) were coated overnight at 4~C with
50 ~l/well anti-mouse cytokine monoclonal antibodies at
optimum concentrations (determined at 2 ~g/ml in each
case). (IL-2 reagents were obtained from Pharmingen,
San Diego, CA, USA, and IL-12 reagents from Wistar
Institute, USA). Plates were washed three times with
PBS/Tween (PBST) (pH 7.4, 0.05~ Tween 20) and blocked
with 200 ~l/well 10~ (v/v) FCS in PBS for 60 minutes at
37~C. Plates were washed three times in PBST, 100 ~l
samples of supernatants and standards (I~-2, 0-62.5
units/ml; IL-12, 0-500 pg/ml) added in duplicate to the
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wells and incubated for 2 hours at 37~C. After four
washes with PBST, 100 ,ul/well of biotinylated anti-mouse
cytokine monoclonal antibody (all l ~g/ml) was added and
the plates incubated for 45 minutes at 37 ~C. After
washing six times with PBST, 100 ,ul/well alkaline
phosphatase-streptavidin conjugate (Pharmingen), diluted
1/2000 in 10~ (v/v) FCS in PBS, was added and the plates
incubated for 30 minutes at 37~C. Plates were washed
eight times in PBST and 100 ,ul/well of para-nitrophenyl-
phosphate (pNPP) substrate (Sigma), prepared in glycine
buffer (0.1 M; pH 10.4), added. Plates were incubated
for 30 minutes at 37~C in darkness before the resulting
absorbances were read at 405 nm on a Titertek Multiskan
plate reader (Flow Laboratories, Irvine, Ayrshire,
U.K.). Cytokine concentrations in the cell cultures
were determined from the standard curve (regression
coefficient, r = 0.990 or better). Comparisons between
groups were made using a Student's T test.
RESUITS
Figure 2a shows the level of weight-loss after
intraperitoneal administration of FK-565. The control
groups which received either PBS or NISV exhibited
normal, unaltered body weights throughout. Mice treated
with FK-565 in PBS lost 16% of their body weight 3 days
post-administration of the drug. Mice which received
FK-565 entrapped in NISV showed a significant reduction
in weight-loss, with the mean body weight stabilising
after 1 day and rising 3 days after injection. The mice
lost approximately 596 mean body weight over 3 days.
IL-2 and IL-12 production in Con A stimulated
splenocytes isolated from mice after the various
treatments was compared (Figures 2b and 2c,
respectively). The administration of FK-565 entrapped
in NISV elicited a predomln~ntly Thl-type immune
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- 2 8
response as evidenced by the significant production of
IL-2 and IL-12. These cytokines were not produced in
levels significantly greater than controls after the
delivery of FK-565 in PBS which tends to produce
cytokines involved with a Th2 response and inflammation.
The administration of FK-565 entrapped in NISV did not
completely prevent the cachexic weight-loss attributable
to FK-565 but did considerably reduce it (from 16~ to 5
over 3 days). NISV, as is apparent from the data
presented below, prevented cachexia by direct down-
regulation in the production of the major
proinflammatory cytokines including TNF-~, IL-6 and IL-l
(~ and ~).
Example 2
Reduction of LPS-induced TNF-~ and IL-6 levels in in
vitro murine macrophage models treated with NISV
The results in Example 1 show that NISV has a
significant therapeutic ability to reduce whole-body
metabolic weight-loss caused by proinflammatory
cytokines. TNF-~ is a primary proinflammatory cytokine
associated with cachexic weight-loss. An experiment was
set up to confirm that this reduction was mediated by
NISV and the ability of NISV to reduce the levels of
TNF-~.
Materials and Methods
NISV were prepared by the method de5cribed in Example 1.
A. In vitro study using the murine macrophage cell
line J774.
The murine macrophage cell line, J774, harvested from
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BALB/c/NIH mice, were obtained as a gift from Professor
H. Harris/Dr R. Sutherland (Sir William Dunn School of
Pathology, Oxford, U.K.). The cells were maintained in
RPMI 1640 culture medium supplemented with 10~ (v/v)
FCS, 2 mM L-glutamine, 100 units/ml penicillin and 100
~g/ml streptomycin (all Gibco) at 37~C in a 5% CO2
atmosphere. Cells were washed extensively by
centrifugation at 1000 x g before activation with
various agents ln Dulbecco's modified Eagles medium
(DMEM) supplemented with 3 M D-glucose and 10% FCS.
100 ~1/well aliquots of cells, at a concentration of
approximately 1x106 cells/ml, were established in 96-well
flat-bottomed tissue culture plates (Costar). Seven
groups (5-6 replicates/group) of cells were treated with
10 ~l of the following preparations and incubated at 37OC
in a 5% C~2 atmosphere:
Group 1: LPS (40 ng/ml) + IFNy (10 units/ml)
Group 2: IFNy (10 units/ml)
Group 3: LPS (40 ng/ml)
Group 4: NISV (0.2 mg) + IFNy (10 units/ml) + LPS (40
ng/ml)
Group 5: NISV (0.2 mg) + IFN (10 units/ml)
Group 6: NISV (0.2 mg) + LPS (40 ng/ml)
Group 7: NISV (0.2 mg)
Cytokine assay: After 48 hours of stimulation,
supernatants were removed from the test culture wells
and tested for TNF-~ production using a monospecific
ELISA (Pharmingen). Flat-bottomed polystyrene plates
(Dynatech) were coated overnight at 37~C in a 5% CO2
atmosphere with 50 ~l/well rat anti-mouse TNF-~
~ monoclonal antibody. Plates were washed three times
with PBST and blocked with 200 ~l/well 10~ (v/v) FCS in
PBS for 60 minutes at 37~C. Plates were washed three
times with PBST, 100 ~l samples of supernatants and
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- 30 -
standards (20 ng/ml TNF-~), diluted in 10% (v/v) FCS in
PBS, added and incubated for 2 hours at 37~C. After
four washes with PBST, 100 ~l/well of biotinylated rat
anti-mouse TNF-~ monoclonal antibody (0.5 mg/ml in 10%
(v/v) FCS in PBS) was added and the plates incubated for
45 minutes at 37~C. Detection of TNF-~ levels using an
alkaline phosphatase-streptavidin conjugate and pNPP
substrate was exactly as described in the cytokine
assays in Example 1.
B. In vitro study using murine perito~e~l macrophages.
The above study was carried out in an established murine
cell culture (J774). In order to assess that the same
immunomodulatory effect was seen in cells freshly
removed from mice, which should arguably provide a more
accurate model of a true in vivo system, a parallel
study was performed using exudated murine peritoneal
macrophages.
Peritoneal macrophages were removed from BALB/c mice by
introduction of a nominal volume of RPMI culture medium
and subsequent removal via a fine-gauge needle. Cells
were washed as described in (A) in RPMI culture medium
supplemented with 3 M D-glucose and 10% FCS.
100 ~l/well aliquots of cells, at a concentration of
approximately 2.5x106 cells/ml, were incubated in 96-
well flat-bottomed tis~ue culture plates (Costar) for 4
hours at 37~C in a 5% C02 atmosphere. Non-adherent cells
were removed by aspirating the wells with 2 volumes of
RPMI culture medium supplemented with FCS. Eight groups
(3 replicates/group) of cells were treated with the
following preparations and incubated at 37OC in a 5% CO2
atmosphere:
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- 3 1
Group 1: Control (PBS, pH 7.4)
Group 2: LPS (200 ng/well)
Group 3: LPS (200 ng/well) + NISV (0.2 mg)
Group 4: LPS (200 ng/well) + IFN-y (10 units/well)
Group 5: LPS (200 ng/well) + NISV (0.2 mg) - IFN-y
(10 units/well)
Group 6: LPS (200 ng/well) + NISV (0.2 mg)
Group 7: LPS (200 ng/well) + control (PBS, pH 7.4)
Group 8: No stimulation (background)
Cytokine assay: After 24 hours stimulation, 100 ~l of
supernatants were removed from each test culture-well
and tested for:
i) TNF-~ production using a monospecific ELISA
(Pharmingen) exactly as described in (A) above
(Groups 1-5).
ii) IL-6 production using a monospecific ELISA
(Pharmingen) exactly as described in (A) above
(Groups 6-8).
RESULTS
Figure 3a shows the levels of TNF-~ release after
stimulation of J774 cells. Treatment of J774 murine
macrophages with NISV down-regulated the LPS-induced
production of the pivotal proinflammatory cytokine, TNF-
~. LPS treatment of the murine macrophages resulted in
levels of TNF-~ that are significantly higher than those
elicited after the treatment of J774 cells with the
other agents. Co-administration of NISV and LPS to J774
macrophages significantly reduced (p c 0.025) the
production of TNF-~, compared to the administration of
LPS alone. A similar reduction in TNF-~ release in J774
macrophages was observed after the co-administration of
NISV and IFN-y.
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Figure 3b shows the levels of TNF-~ release after LPS
stimulation of murine peritoneal macrophages (treatment
Groups 1-5). The administration of LPS alone to the
murine macrophages induced considerable release of TNF-
~
from the cells. The co-administration of LPS and IFN-y
caused a greater increase, although not significant, in
TNF-~ production than LPS alone. Co-administration of
NISV and LPS substantially reduced (by approximately
50~) the level of TNF-~ released. Similarly, the
reduction of TNF-~ release from the peritoneal
macrophages was even greater after the co-administration
of NISV and IFN-y (approximately 75%).
Figure 3c shows the levels of IL-6 release after LPS
stimulation of murine peritoneal macrophages (treatment
Groups 6-8). By co-administering NISV with LPS, the
level of IL-6 produced by murine peritoneal macrophages
was significantly reduced as compared to the
administration LPS in PBS. Unstimulated peritoneal
macrophages did not produce IL- 6.
This study shows that NISV can significantly reduce the
levels of TNF-~ in cultured murine macrophages after
stimulation with LPS, which mimics an inflammatory
event. The results from the peritoneal macrophages also
indicate that this effect is not specific to established
murine cell cultures and demonstrate the
immunomodulatory capacity of NISV to reduce the levels
of two pivotal proinflammatory cytokines, TNF-~ and
IL-6, after an inflammatory-type stimulus in cells
freshly removed from mice and maintained for 24 hours.
This data provides an important link between the
cellular events that result in prevention of
inflammatory weight-loss (cachexia), the whole-body
effect observed in Example 1. It indicates that the
immunomodulatory effects of NISV act to significantly
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WO 97/04768 PCT/GB96/01861
reduce both TNF-~ and IL-6. This reduction in
proinflammatory cytokines at a cellular level can result
~ in a benefit in inflammatory-mediated conditions, at the
physiological level. In vivo both the effects on TNF-~
~ 5 and IL-6 would occur in tandem and act to down-regulate
the inflammatory response to a greater extent than
either acting alone.
~xample 3
Reduction in the levels of LPS-induced TNF-~ and IL-6
and in an in vi tro human peripheral blood leucocvte
model treated with NIS~v~
This study was carried out to demonstrate that the
ability of NISV to down-regulate proinflammatory
cytokine production was not exclusive to murine systems
and that human cells responded equally as readily to the
anti-inflammatory effect of NISV.
Materials and Methods
NISV were prepared by the method described in Example l.
Peripheral blood mononuclear leucocytes (PBLs) were
prepared from heparinised/citrated venous blood from
healthy adult volunteers. Approximately 10 ml of whole
hllm~n venous blood was withdrawn into a heparinised
syringe and carefully layered onto 10 ml Ficoll/Paque,
room temperature-equilibrated in a sterile centrifuge
tube, with minimal perturbation of the interface between
the fluids. The blood/Ficoll gradient was centrifuged
at 1000 x g for 30 minutes at 20~C. The PBLs appeared
as a tight band of cells layered above the pelleted
erythrocytes and polymorphonuclear leucocytes and below
the straw-coloured pool of plasma and platelets. The
plasma and platelets were carefully removed and
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- 34 -
discarded leaving a very thin film of plasma above the
leucocytes. These were aspirated into a sterile 20 ml
Nunc tube containing 10 ml RPMI 1640 culture medium
supplemented with 10~ (v/v) FCS in PBS and stored at
4~C. The cells plus medium were re-centrifuged for 20
minutes at 25~C prior to use and the cell pellet
resuspended in 10 ml RPMI 1640 supplemented with FCS as
before. The cells were re-washed in 10 ml RPMI 1640
supplemented with FCS and re-suspended in 10 ml DMEM
supplemented with FCS and suitable antibiotics (1% w/v
tetracycline; 1% w/v streptomycin). The cells were then
ready ~or in vitro assay.
2 ml/well aliquots of human PBLs, at a concentration of
approximately 2X106 cells/ml, were established in 24-well
flat-bottomed tissue culture plates (Costar). Three
groups (3 replica'ces/group) of cells were treated with
the following preparations and incubated at 37~C in a 5
C~2 atmosphere:
Group 1: Control (PBS, pH 7.4)
Group 2: LPS (40 ng/ml)
Group 3: hPS (40 ng/ml) + NISV (1 mg)
The cells were maintained for 72 hours with aliquots of
the cell culture supernatan~s removed at 4, 24, 48 and
72 hours post-stimulation and stored at -70~C for
subsequent cytokine assay.
The supernatants from the cell cultures were assayed by
monospecific human ELISA for IL-6 (Genzyme) and TNF-o~
(Pharmingen) according to the manufacturers'
instructions. The ELISA plates were developed using a
suitable colorimetric solution and read at 405 nm on a
Titertek Multiskan plate reader (Flow Laboratories).
Cytokine concentrations in the cell culture supernatants
were determined from appropriate standard curves
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(regression coefficient r = 0.990 or better).
Preliminary statistical analyses were carried out. Any
~ signi~icance mentioned in the text refers to standard
deviation measurements (not shown).
R~.~UT TS
This study illustrates the response of human PBLs to in
vitro stimulus with LPS, which mimics an inflammatory
response. Figures 4a and 4b indicate that the
introduction of LPS to the cells resulted in the
production of the cytokines IL-6 and TNF-~,
respectively.
Figure 4a shows the release of IL-6. After stimulation
with LPS, the levels of IL-6 increased rapidly and
peaked at 2.5 ng/ml 24 hours after administration.
Stimulation of the PBLs with PBS resulted in negligible
levels of IL-6 release. The co-administration of NISV
with LPS completely prevented IL-6 release. Measurable
levels of IL-6 after NISV treatment were not
significantly different from those achieved after the
control administration of PBS.
Figure 4b shows the release of TNF-~. TNF-~ was
released very rapidly from human PBL after stimulation
with LPS and was present in high levels only 4 hours
after stimulation. Co-administration of NISV with LPS
reduced the level TNF-~ release at 4 hours post-
introduction, as compared with the group receiving LPS
alone, from 5.5 ng/ml to 3 ng/ml.
These results illustrate the considerable ability of
NISV to reduce the levels of the pivotal proinflammatory
cytokines IL-6 and TNF-~ released by human PBLs in vitro
after stimulation with LPS. Both IL-6 and TNF-c~ are
potential therapeutic targets for modulation, e.g. in
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the treatment of chronic diseases such as rheumatoid
arthritis as well as acute conditions such as SIRS. In
this study, NISV were observed to almost completely
prevent IL-6 release from stimulated PBL and greatly
reduce levels of TNF-~ release, as rapidly as 4 hours
after co-administration with LPS.
~ample 4
Reduction in the levels of LPS-induced TNF-~, IL-6 and
IL-1 in human PBLs with NISV.
Materials and Methods
NISV were prepared by the method described in Example 1.
PBLs were obtained from heparinised/citrated venous
blood from healthy adult volunteers and also from the
Scottish Blood Transfusion Service. PBLs were prepared
by density-dependent centrifugation using a
Ficoll/Plaque gradient medium as described in Example 3.
2 ml/well aliquots of human PBLs, at a concentration of
approximately 2X106 cells/ml, were established in 24-well
flat-bottomed tissue culture plates (Costar). Four
groups (3 replicates/group) of cells were treated with
the following preparations and incubated at 37~C in a 5%
C~2 atmosphere:
Group 1: Control (PBS, pH 7.4)
Group 2: LPS (40 ng/ml)
Group 3: NISV (1 mg)
Group 4: LPS (40 ng/ml) + NISV (1 mg)
The cells were maintained for 48 hours with aliquots of
the cell culture supernatants removed at 1.5, 4, 24 and
48 hours post-stimulation and stored at -70~C for
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subsequent cytokine assay.
The supernatants from the cell cultures were assayed by
monospecific human ELISA for IL-l (~ and ~) (Dynatech)
(all treatment groups), IL-6 (Genzyme) and TNF-
~(Pharmingen) (treatment groups 1, 2 & 4), according to
the manufacturers~ instructions. The ELISA plates were
developed using a suitable colorimetric solution and
read at 490nm on a Titertek Multiskan plate reader (Flow
Laboratories). Cytokine concentrations in the cell
culture supernatants were determined from appropriate
standard curves (regression coefficient r = 0.990 or
better). Preliminary statistical analyses were carried
out. Any significance mentioned in the text refers to
standard deviation measurements (not shown).
RESULTS
This study shows the ability of NISV to reduce the
levels of proinflammatory cytokines from stimulated
human PBL, in vitro. Figures 5a and 5b indicate that
the introduction of LPS to the cells resulted in the
production of the cytokines IL-6 and TNF-~,
respectively. Notably, the control PBLs in this
particular study happened to be derived from an
individual with an ongoing inflammatory response. As
such, the control PBLs consistently produced a
detectable level of the proinflammatory cytokines IL-6
and TNF~.
Figure 5a shows the release of IL-6. LPS-stimulated
PBLs produced peak levels of IL-6 after 24 hours of
culture. These levels were similar to those observed in
- Figure 4a. Control PBLs also produced measurable levels
of IL-6. The co-administration of NISV with LPS reduced
the level of IL-6 below control levels.
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Figure 5b shows the release of TNF-~. After stimulation
with LPS, the levels of TNF-~ increased rapidly and
peaked 4 hours post-administration. The co-
administration of NISV with LPS reduced the level of
TNF-~ by approximately 50%. Control PBLs also produced
measurable levels of TNF-~ in this system.
Figure 5c shows the release of IL-1~. NISV failed to
induce significant levels of IL-1~ in the absence of a
stimulatory signal (LPS). The administration of LPS
resulted in a considerable production of IL-lcx. The co-
administration of NISV with LPS substantially reduced
the release of IL-lc~ over 48 hours.
Figure 5d shows the release of IL-1~. NISV failed to
induce any significant levels of IL-1~ in the absence of
a stimulatory signal (LPS). The administration of LPS
resulted in a considerable production of IL-1~3. The co-
administration of NISV with LPS reduced the release of
IL-l,B over 48 hours.
This study represents a repeat investigation in which
the results from Example 3, showing the considerable
ability of NISV to reduce the pivotal proinflammatory
cytokines, were confirmed.
Interestingly, the vesicle preparation, at the
concentration used in this study, had no substantial
affect on the kinetics of release of these pro-
inflammatory cytokines (except in instances of completeprevention of release).
The control PBLS from this study represent a cell
population that are similar to those that would be
encountered in individuals afflicted by an ongoing
inflammatory response. As such, the ability of NISV to
reduce the "baseline" levels of one of the major
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proinflammatory cytokines, IL-6, can only be regarded as
beneficial and significant.
The results also demonstrate that NISV have the ability
~ 5 to reduce the overall levels of IL-1 elicited after LPS
stimulation of human PBLs. NISV have the ability to
significantly reduce the level of IL-1~ produced in this
system and, in addition, reduce the amounts of IL-1
elicited, both of which are important in the
inflammatory response.
~xam~le 5
Effect of size on the immunomodulatory effect of NISV
This study was designed to assess if the size of
vesicles influenced the level of therapeutic
immunomodulation achieved with NISV.
A. The effect of size of NISV on IL-5 levels.
Whether the size of the vesicles could influence the
therapeutic potential of NISV was determined from
immunogenicity studies using entrapped ovalbumin (OVA)
as an antigen. IL-5, a cytokine implicated in the onset
of asthma, was measured.
Materials and Methods
NISV were prepared by the method described in Example 1.
Antigen ent ~ _~t: OVA (grade V, Sigma) was entrapped
in preformed NISV using the freeze-thaw method (Pick,
1981). The antigen/vesicle mixture was frozen in liquid
nitrogen and then thawed to 60~C. This was repeated
five times. The suspension was shaken for a further 2
hours at 60~C and vesicle preparations of different
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sizes prepared by sequential extrusion through
decreasing pore size polycarbonate filters (Costar) at
60~C in a thermobarrel extruder (Lipex Biomembranes
Inc., Vancouver, Canada). The ~ree antigen was removed
by washing at 1000000 x g for 40 minutes at 4~C. The
protein concentration was measured by nitrogen assay
(Brewer et al., vaccine ~ (5), 1441-1444 (1995) ) .
Electron microscopy: The vesicles were examined by
electron microscopy as follows. A small sample of
vesicle suspension (approximately 10 ~1) was sandwiched
between clean copper plates (Balzers High Vacuum, Milton
Keynes, U.K.) and fast-frozen by plunging lnto liquid
propane at -190 ~C. Samples were then transferred to a
cold stage at -100~C in a diffusion pumped vacuum system
operating around 4x10-6 torr. The support plates were
fractured apart and the exposed surfaces shadowed
immediately with evaporated platinum/carbon at 45~
followed by a second strengthening coat of carbon
applied at 90~ to the exposed fracture faces. The
vesicle preparations were removed from the replica by
sequentlal washing in acetone/distilled water solution
of several decreasing acetone concentrations from pure
acetone. Finally, after several washes in distilled
water, the replicas were collected onto copper grids,
dried and examined under a transmission electron
microscope.
Anim~l 8 and inoculations: Female BALB/c mice were in-
house bred and inoculated when 8-10 weeks old. Groups
of 5 mice were inoculated in the footpad with 10 ,ul of
the following:
Group 1: 10 ~g OVA entrapped in NISV extruded through a
800 nm pore size membrane
Group 2: 10 ,ug OVA entrapped in NISV extruded through a
4 0 0 nm pore size membrane
Group 3: 10 ,ug OVA entrapped in NISV extruded through a
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200 nm pore size membrane
Group 4: 10 ~g OVA entrapped in NISV extruded through a
100 nm pore size membrane
Group 5: 10 ~g OVA in PBS (control)
Draining inguinal and popliteal lymph nodes were
collected from the mice 10-14 days after the treatments.
Inguinal and popliteal lymph nodes were aseptically
removed and cell suspensions prepared and enumerated as
described in Example 1. 100 ~l/well aliquots of cell
suspension, containing 5x105 cells, were added to 96-well
flat-bottomed tissue culture plates (Costar), followed
by 100 ~l/well ali~uots of Con A (5 ~g/ml) or OVA (2000
~g/ml) in triplicate. cultures were then incubated for
60 hours at 37~C in a 5% CO2 atmosphere after which 150
~1 aliquots of cell culture supernatants were removed
and stored at -70~C for cytokine assay.
Cytokines (IL-2, IL-5 and IFN-y) were detected by
monospecific ELISA. Flat-bottomed polystyrene plates
(Dynatech) were coated overnight at 4~C with 50 ~l/well
anti-mouse cytokine monoclonal antibodies at optimum
concentrations (determined at 2 ~g/ml in each case;
Pharmingen). Plates were washed three times with PBST
and bloc~ed with 200 ~l/well 10% (v/v) FCS in PBS for 60
minutes at 37~C. Plates were washed three times in
PBST, 100 ~l samples of supernatants and standards (IL-
2, 0-62.5 units/ml; IL-5, 0-1.6 ng/ml; IFN-y, 0-70
units/ml) added in duplicate to the wells and incubated
for 2 hours at 37~C. Detection of cytokine levels using
an alkaline phosphatase-streptavidin conjugate and pNPP
substrate was exactly as described in the cytokine
assays in Example 1.
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~7~UT~TS
Other experiments using photon correlation spectroscopy
(data not shown) confirmed that the actual size of
vesicles extruded through a polycarbonate filter of pore
diameter 200 nm is in the range 152-157 nm, and that of
vesicles extruded through a 400 nm pore diameter
membrane within the range 200-242 nm.
Figure 6a shows the levels of IL-2 and IL-5 production
in Con A stimulated lymph node cells. Figure 6b shows
the levels of IL-5 and IFN-y production in Con A and OVA
stimulated, respectively, lymph node cells. NISV
extruded through 400 nm membranes elicited the largest
amounts of IL-2 and IFN-y, whilst reducing the levels of
IL-5 to below (but not significantly) those achieved in
the control OVA group. NISV extruded through 800 nm
membranes produced levels of IL-5 that were similar to
those achieved with OVA alone. NISV extruded through
100 & 200 nm membranes produced significantly greater
levels of IL-5 than those elicited after the
administration of NISV extruded through 400 & 800 nm
membranes or the OVA control.
The size of the NISV used in a therapeutic application
may be important as the ability to act as an
immunomodulator appears to be influenced by this
parameter. In the system described in this Example NISV
of of mean diameter greater than 200 nm are particularly
well suited to generate a good therapeutic effect on the
down regulation of IL-5 and, also by implication IL-4
whose production is linked to that of IL-5.
B. The effect of size of NISV on IL-1 levels.
The size of NISV was studied in hl7m~n cell lines to
confirm that larger (i.e. > 200 nm) vesicles may also be
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more suitable for therapeutic indications in human
cells. Parallel studies were carried out in a cultured
human macrophage cell line and in human PBhS, freshly
derived from volunteers.
Materials and Method~
NISV were prepared by the method described in Example 1
and extruded through polycarbonate filters (Costar) as
described previously.
Human U937 cell line: Cells from the human leucocyte
pro-macrophage cell line U937 were maintained in RPMI
1640 culture medium supplemented with 10% (v/v) FCS, 2
mM L-glutamine, 100 units/ml penicillin and 100 ~g/ml
streptomycin (all Gibco) at 37~C in a 5~ CO2 atmosphere.
Cells were washed extensively by centrifugation at 1000
x g before activation with various agents in DMEM
supplemented with 10% FCS.
~llm~n PBLs: Human PBLs were prepared from healthy
volunteers as described in Example 3.
2 ml/well aliquots of either U937 or PBL cells, at a
concentration of approximately 2 X106 cells/ml, were
established in 24-well flat-bottomed tissue culture
plates (Costar). Four groups (3 replicates/group) of
each cell ~ype were treated with the following
preparations and incubated at 37~C in a 5% CO2
atmosphere:
Group 1: Control (PBS, pH 7.4)
Group 2: LPS (40 ng/ml)
Group 3: NISV extruded through a 200 nm pore size
membrane (1 mg) ("small")
Group 4: NISV non-extruded (approximately 1.6 ~m in
diameter) (1 mg) ("normal")
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The cells were maintained for 72 hours with aliquots of
the cell culture supernatants removed at 24, and 48
hours (PBLs) and 72 hours (U937 cells) post-stimulation
and stored at -70~C for subsequent cytokine assay.
The supernatants from both the U937 and PBL cell
cultures were assayed by monospecific human ELISA for
IL-lcx and IL-l,B, (Pharmigen) according to the
manufacturers instructions. The ELISA plates were
developed using a suitable colorimetric solution and
read at 490 nm on a Titertek Multiskan plate reader
(Flow Laboratories). Cytokine concentrations in the
cell culture supernatants were determined from
appropriate standard curves (regression coefficient r =
0.990 or better).
R~.'~UI.TS
Figures 7a and 7b show the levels of IL-lo~ and IL-l,B,
respectively. As expected, LPS stimulated the release
of IL-l from both types of cells between 24 and 72
hours, except that U937-releaSe of IL-lo~ appeared to be
refractory to LPS stimulation. Small, approximately 160
nm, NISV elicited the release of low levels of IL-lcx
from U937 cells and human PBLs. However, larger,
approximately 1.6 ~m, vesicles did not stimulate the
release of IL-lo~ to any detectable extent.
Small, approximately 160 ,um, NISV elicited significant
levels of IL-l,B in both U937 and human PBL cells. The
levels of this cytokine elicited by small NISV are
similar to those generated after the stimulation of
these cells with LPS. Larger, approximately 1.6 ,um,
NISV elicited levels of IL-1~ similar to those observed
in the control PBS group.
The results exhibited after the stimulation of human
U937 pro-macrophages and human PBLs with small and large
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NISV correlate with the results described for murine in
vivo systems. NISV greater than approx. 200 nm in mean
diameter nave a particularly good therapeutic profile
for immunomodulation in human cells.
Example 6
Sup~ression of LPS induced cytokine production by NISV,
administered by subcutaneous injection in mice
This study was carried out to demonstrate that the
ability of NISV to down regulate LPS-induced cytokine
production in vi tro was retained in a comparable in vivo
test system.
Materials and Methods
NISV were prepared from cholesterol, 1-monopalmitoyl
glycerol and dicetyl phosphate in a ratio 4:5:1 by
weight. These components were melted together at 135~C,
diluted with PBS to a final volume of 750 ml and a
concentration of 25 mg/ml at 65~C as in Example 1 then
homogenised at 8000 rpm for 30 minutes and sterilised by
autoclaving. NISV were administered to groups of BALB/c
mice (5 in each group) by subcutaneous injection at one
of two dose levels 2.5 mg (equivalent to approximately
83 mg/kg bodyweight) or 0.5 mg (equivalent to
approximately 17 mg/kg bodyweight). Three groups of
animals received each dose and a further two groups were
left untreated.
LPS was administered by interperitoneal injection (4 ~g
in 200 ~1) at different time points after NISV
injection.
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Treatment Group Day of LPS challenge(s)
(n=5) in relation to NISV dose
1st 2nd
1 - untreated control
2 - 0.5 mg NISV +l
3 - 0.5 mg NISV +4
4 - 0.5 mg NISV +14
5 - untreated control
6 - 2.5 mg NISV +1 +15
7 - 2.5 mg NISV +4 +18
8 - 2.5 mg NISV +14 +28
Animals allocated to 2.5 mg NISV received two LPS
challenges at 14 days intervals, whereas those allocated
to 0.5 mg NISV received one LPS challenge. The
respective control groups were similarly challenged.
Blood samples were taken for measurement of IL-6 levels
at 3 and 6 hours after each LPS challenge. The samples
were analysed using 'Quantikine' ELISA kits (R+D
Systems, Abingdon, Oxon) according to the manufacturer~s
instructions.
REsULTS
The results are presented in Figures 8, 9 and 10.
As expected, the control group showed elevation of serum
IL-6 levels post LPS challenge, the highest levels
occurring at the 3 hour post-challenge time point. Both
dose levels of NISV partially inhibited the increase in
IL- 6 levels.
Fig. 8 shows the effect of NISV at 17 mg/kg bodyweight.
The suppressive effect was greatest at 1 day post NISV
and lasted for at least 4 days (IL-6 levels at 14 days
post NISV were comparable to control levels).
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Fig. 9 shows the effect of NISV at 83 mg/kg bodyweight.
At this higher dose the onset of effect appeared slower
and was greatest in the group that received NISV 14 days
prior to LPS challenge.
Fig. 10 shows the response to the second LPS challenge
in the high dose NISV groups. The inhibition of IL-6
response to LPS appears maximal in the group challenged
15 days after NISV administration, but persists in part
for 28 days.
These results demonstrate that subcutaneous injection of
NISV inhibits the cytokine response to a noxious
stimulus (LPS) in vivo and that the duration of action
is dose dependent, suggesting a 'depot' effect when
administered by this route.
Example 7
Reduction in the levels of TNF~ and IL-6 in LPS
stimulated human PBLs
In a volunteer study a preliminary evaluation of
cytokine production ex vivo was undertaken using PBLs
stimulated with LPS.
Materials and Methods
Pre and post dose (+ 24 hr) venous blood samples were
taken from a volunteer who received a subcutaneous
injection of 25 mg NISV (prepared as in Example 6)
suspended in 1 ml of phosphate buffered saline.
Peripheral blood mononuclear leucocytes were separated
from the heparinised sample by density dependent
centrifugation using a Ficoll/Paque gradient and the
leucocyte layer further processed as described in
Example 3 to produce a cell suspension in Dulbecco's
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Minimal Essential Medium.
Aliquots from this suspension (containing approximately
2X106 cells/ml) were established in 24 well flat bottomed
tissue culture plates. Two sets of cells (3 replicates/
set) from each sample were treated as follows and
incubated at 37~C for 4 8 hours.
Set 1 Unstimulated cell culture
Set 2 LPS (40 ng/ml) stimulated cell culture
Aliquots of the cell culture supernatant were removed at
0, 1.5, 4, 24 and 48 hours of culture and stored at
-70~C until assayed for IL-6 and TNF~ using 'Quantik~ne'
kits (R&D Systems, Abingdon, Oxon). Cytokine
concentrations were determined from standard curves
based on results ~rom assay standards.
RESULTS
The results are shown in Figures 11 and 12.
Increases in both TNF~ and IL-6 levels occurred
irrespective of LPS stimulation in the pre-NISV dose
samples. However, in the volunteer, a demonstrable
difference was observed in comparing PBLs obtained prior
to and subsequent to NISV administration. A suppression
of TNF~ levels was seen in PBLs extracted at both 4 and
24 hours post NISV administration (see Figure 11) and
suppression of IL-6 levels was seen in PBL6 extracted at
24 hours post NISV administration (see Figure 12).
These preliminary data suggest that subcutaneous
injection of NISV suppresses the responsiveness of hllm~n
PBLs to LPS challenge.
