U.S. patent application number 10/357618 was filed with the patent office on 2004-06-03 for aggregates with increased deformability, comprising at least three amphipats, for improved transport through semi-permeable barriers and for the non-invasive drug application in vivo, especially through the skin.
Invention is credited to Cevc, Gregor, Vierl, Ulrich.
Application Number | 20040105881 10/357618 |
Document ID | / |
Family ID | 35265949 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040105881 |
Kind Code |
A1 |
Cevc, Gregor ; et
al. |
June 3, 2004 |
Aggregates with increased deformability, comprising at least three
amphipats, for improved transport through semi-permeable barriers
and for the non-invasive drug application in vivo, especially
through the skin
Abstract
The invention describes combinations of at least three
amphipatic substances forming aggregate suspensions in a polar
liquid. Judicious choice of system components, which differ at
least 2-times to 10-times in solubility, ensures said aggregates to
have extended, unusually adaptable surfaces. This is probably due
to simultaneous action on said aggregates of at least two more
soluble substances amongst said three system components, at least
one of which is an active ingredient and preferably a drug; the
third component, alternatively, can take the role of a drug. The
patent further deals with the use of said combinations in
pharmaceutical preparations capable of transporting drugs into the
body of warm blood creatures. This is made possible by the drug
loading capability of said aggregates with the highly flexible and
deformable coating, which renders the resulting drug carriers
highly adaptable. The patent finally reveals suitable methods and
favourable conditions for carrier manufacturing and
application.
Inventors: |
Cevc, Gregor; (Gauting,
DE) ; Vierl, Ulrich; (Munchen, DE) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
35265949 |
Appl. No.: |
10/357618 |
Filed: |
February 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60417847 |
Oct 11, 2002 |
|
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|
Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 31/196 20130101;
A61K 47/16 20130101; A61K 31/405 20130101; A61K 31/5415 20130101;
A61K 47/20 20130101; A61K 47/12 20130101; A61K 47/26 20130101; Y10S
514/886 20130101; A61K 47/34 20130101; A61K 9/127 20130101; A61K
47/10 20130101; A61K 9/1272 20130101; A61K 31/192 20130101; A61P
29/00 20180101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Claims
1. Preparation based on a combination of at least one first
(membrane forming component MFC), at least one second (membrane
destabilising component MDC), and at least one third (membrane
destabilising component MDC) amphipatic component suspended in a
suitable liquid medium in the form of corresponding mixed amphipat
aggregates with extended surface (ESAs) with one or a few mixed
amphipat coating(s), which are preferably bilayer like, wherein
said ESAs formed by a combination of all three said components have
surfaces in contact with said liquid medium that are at least 50%
more extended, on the average, than the typical surfaces of
aggregates comprising the said at least one second and at least one
third amphipatic component alone, at the same concentrations and,
in case, after adjustment for the physico-chemical effects of
resulting from the absence of said first amphipatic compound (MFC)
for application, administration or transport of an active
ingredient, which can be one of the three amphipatic components,
especially for biological, medical, immunological, or cosmetic
purposes, into and through the pores in semi-permeable barriers or
other constrictions, such as through the skin of warm blood
creatures or the like.
2. A combination of at least one first (membrane forming component
MFC), at least one second (membrane destabilising component MDC),
and at least one third (membrane destabilising component MDC)
amphipatic component suspended in a suitable liquid medium in the
form of mixed amphipat aggregates with extended surface (ESAs) with
one or a few mixed amphipat coating(s), which are preferably
bilayer like, wherein the said at least one first substance has a
tendency to self aggregate and is at least 10-times less soluble in
said liquid medium than said at least one second and said one third
substance, allowing the first to form extended surfaces, said at
least one second substance is at least 10-times more soluble than
said at least one first substance in said liquid medium and, on its
own, tends to form or supports the formation of surfaces that are
at least 2-times less extended than the surfaces containing the at
least one first substance alone, said at least one third substance
being also at least 10-times more soluble in said liquid medium
than the first substance and optionally forms self-aggregates with
aggregation number at least 10-times smaller than that of
self-aggregates of said first substance; and said extended surfaces
comprising said at least one first, at least one second and at last
one third substance, in equilibrium, have at least 50% greater
surface than the surfaces formed by the at least one second or one
third substance alone, at the same concentration and, in case,
after adjustment for the physico-chemical effects of the absence of
said first amphipatic compound (MFC) for a preparation for
application, administration or transport of at least one active
ingredient, which can be one of the three amphipatic components,
especially for medicinal or biological purposes, into and through
barriers and constrictions, such as the skin of warm blood
creatures or the like.
3. Extended-surface aggregates (ESAs) comprising at least one first
(membrane forming component, MFC), at least one second (membrane
destabilising component, MDC), and at least one third (membrane
destabilising component, MDC), all of which are amphipatic,
suspended in a suitable liquid medium, which permits said ESAs to
permeate barriers with the pores with at least 40% smaller radius
than the average ESAs radius, as measured after the ESAs have
permeated the barrier pores and assuming spherical ESAs
geometry.
4. Preparation based on a combination of at least one first
(membrane forming component MFC), at least one second (membrane
destabilising component MDC), and at least one third (membrane
destabilising component MDC) amphipatic component suspended in a
suitable liquid medium in the form of corresponding mixed amphipat
aggregates with an extended surface (ESAs) with one or a few,
preferably bilayer-like, mixed amphipat coating(s), wherein said
MFC alone forms extended-surface aggregates with aggregation number
of at least 5000, and preferably more than 10.000, and both MDCs
alone and the combination of both MDCs form smaller aggregates with
no substantially extended surface and aggregation number below
5000, and preferably below 1000 in contact with said suitable
liquid medium.
5. A combination according to claims 1 to 4, wherein the said
extended surfaces are in the form of membrane surfaces.
6. A combination according to any of preceding claims, wherein the
said at least one second substance increases the flexibility of
extended surfaces comprising said at least one first, at least one
second, and at least one third substance in comparison with the
surfaces formed merely by an at least one first substance or else
with the surfaces formed by at least one first and at least one
third substance.
7. A combination according any of preceding claims, wherein the
said at least one second and one third substance together increase
the permeability of extended surfaces containing the said at least
one first, at least one second, and at least one third substance,
in comparison with the surfaces formed merely by the at least one
first substance or else with the surfaces formed by at least one
first and at least one third substance.
8. A combination according to any of preceding claims, wherein the
said at least one second substance or the said at least one third
substance increases the ability to tolerate high curvature, as
assessed by relative stability of said extended surface comprising
said one first, said one second and said one third substance
against enforced higher curvature during passing through a
constriction with maximum diameter at least 1.4 times smaller than
the average diameter of an extended surface formed by an at least
one first substance alone.
9. A combination according to any of preceding claims, wherein the
at least one first substance and the at least one second substance
or the at least one third substance differ in solubility on the
average at least 10-fold.
10. A combination according to any of preceding claims, wherein the
at least one second substance and the at least one third substance
differ in solubility on the average at least 2-fold.
11. A combination according to any of preceding claims, wherein the
at least one second substance or the at least one third substance
have the hydrophilicity-lipophilicity ratio between 10 and 20.
12. A combination according to any of preceding claims, wherein the
concentration of said at least one second substance used in the
combination with said one first and said one third substance is
below 80% of the concentration that would be needed to render the
aggregates comprising only said one first and said one second
substance as adaptable to ambient stress as the selected
combination of all at least three substances, whereby the said one
second and said one third substance can exchange roles.
13. A combination according to any of preceding claims, wherein the
concentration of said at least one second substance or of said at
least one third substance, as the case may be, amounts to at least
0.1% of the relative concentration as defined in claim 8.
14. A combination according to any of preceding claims, wherein the
concentration of said at least one second or of said at least one
third substance amounts to 1-80% of the relative concentration as
defined in claim 8.
15. The combination according to any of preceding claim, wherein
relative concentration of said at least one third substance used in
combination with said one first and said one second substance is
above 0.1% of maximum possible concentration of the said at least
one third substance in the system, a) as defined in terms of the
solubility of said third substance in the system or in said at
least three-component aggregates, or else b) as determined by the
negative action of said at least one third substance on the
stability of said at least three-component aggregates, whereby the
said one third and one second substance can also exchange
roles.
16. The combination according to any of preceding claims, wherein
relative concentration of said at least one third substance used in
combination with said one first and with said one second substance
is between 1% and 99% of maximum possible concentration of said at
least one third substance, a) as defined in terms of the solubility
of said third substance in the system or in said at least
three-component aggregates, b) or else as determined by the
detrimental effect of said at least one third substance on the
stability of said at least three-component aggregates, whereby the
said one third and one second substance can also exchange
roles.
17. The combination according to any of preceding claims, wherein
relative concentration of said at least one third substance used in
combination with the said one first and the said one second
substance is between 10% and 95% of the maximum possible
concentration of said at least one third substance, as defined in
terms of the third substance solubility in the system or in said
aggregates, or else as determined by the detrimental effect of said
at least one third substance on the stability of said at least
three-component aggregates, whereby the said one third and one
second substance can also exchange roles.
18. The combination according to any of preceding claims, wherein
relative concentration of said at least one third substance used in
combination with the said one first and the said one second
substance is between 25% and 90% of the maximum possible
concentration of said at least one third substance, as defined in
terms of the third substance solubility in the system or in said
aggregates, or else as determined by the detrimental effect of said
at least one third substance on the stability of said at least
three-component aggregates, whereby the said one third and one
second substance can also exchange roles.
19. The combination according to any of preceding claims, wherein
the total dry mass of all at least three amphipatic substances,
which together form highly adaptable aggregates with an extended
surface, is between 0.01 weight-% and 50 weight-%.
20. The combination according to any of preceding claims, wherein
total dry mass of all at least three substances, which together
form highly adaptable aggregates with an extended surface, is
between 0.5 weight-% and 30 weight-%.
21. The combination according to any of preceding claims, wherein
total dry mass of all at least three substances, which together
form highly adaptable aggregates with an extended surface, is
between 1 weight-% and 15 weight-%.
22. A combination according to any of preceding claims, wherein the
extended surfaces with a high adaptability, which contain said at
least three substances, have an average curvature corresponding to
an average radius between 15 nm and 5000 nm.
23. A combination according to any of preceding claims, wherein the
extended surfaces with a high adaptability, which contain said at
least three substances, have an average curvature corresponding to
an average radius between 30 nm and 1000 nm.
24. A combination according to any of preceding claims, wherein the
extended surfaces with a high adaptability, which contain said at
least three substances, have an average curvature corresponding to
an average radius between 40 nm and 300 nm.
25. A combination according to any of preceding claims, wherein the
extended surfaces with a high adaptability, which contain said at
least three substances, have an average curvature corresponding to
an average radius between 50 nm and 150 nm.
26. The combination of substances according to any of preceding
claims, wherein the concentration and the composition of the
electrolyte in which the extended surfaces with at least one first,
at least one second, and at least one third substance are
suspended, and which comprises mono and/or oligovalent ions, is
chosen to have ionic strength between I=0.001 and I=1.
27. The combination of substances according to any of preceding
claims, wherein the concentration and the composition of the
electrolyte, in which the extended surfaces with at least one
first, at least one second, and at least one third substance are
suspended, and which comprise mono and/or oligovalent ions, is
chosen to have pH value a) in the vicinity of the logarithm of the
apparent ionisation constant (pKa) of said at least one second
substance, if the latter is mono-ionizable, or in the vicinity of
such pKa value that maximises the solubility of said at least one
second substance, if the latter has several ionizable groups, or
else b) in the vicinity of pH optimum for the most rapidly decaying
or the otherwise most sensitive amongst the said at least three
substances, if the said at least one second substance is not
ionizable.
28. The combination of substances according to any of preceding
claims, wherein the pH value of the polar medium in which the ESAs
comprising at least one first, at least one second, and at least
one third substance are suspended is between pH=pKa-3 and
pH=pKa+3.
29. The combination according to any of preceding claims, wherein
the at least one first substance being less soluble in the liquid
medium, and/or being the surface-building substance in the system,
is a lipid, whereas the at least one second substance being more
soluble in the liquid medium and/or increasing the tolerable
surface curvature or adaptability of said extended surface, is a
membrane destabilising amphipat, which is typically a surfactant,
and said at least one third substance is either a biologically
active amphipatic ingredient, which has a capability of its own to
increase the tolerable surface curvature, or adaptability of said
extended surface, or else is a different surfactant different from
the said at least second substance.
30. The combination according to any of preceding claims, wherein
the molecules are arranged in the form of minute fluid droplets
suspended or dispersed in a liquid medium and surrounded by a
coating of one or several layers of the at least one first
substance, which is capable of self-aggregation, and of at least
one second substance and of at least one third substance, which are
both amphipatic, such that a) the former substance and the latter
two substances differ in solubility in a suitable liquid medium at
least 10-fold, or such that b) the average radius of
homo-aggregates of the more soluble amongst the at least one second
and third substance or of hetero-aggregates of the at least one
first, the at least one second and the at least one third substance
is smaller than the average radius of homo-aggregates of said at
least one first substance, which is the least soluble amongst the
three.
31. Combination according to any of preceding claims, wherein the
at least one first substance is a polar or a non-polar,
surface-forming lipid.
32. Combination according to any of preceding claims, wherein the
at least one first substance in extended surfaces is capable of
forming bilayer membranes and preferably forms bilayers on its
own.
33. Combination according to any of preceding claims, wherein the
solubility of the at least one first substance in a polar liquid
medium is between 10.sup.-12 M and 10.sup.-7M.
34. Combination according to any of preceding claims, wherein the
at least one first substance forming extended surfaces is selected
from the group comprising lipids, lipoids from a biological source,
corresponding synthetic lipids, or modifications thereof.
35. Combination according to any of preceding claims, wherein said
at least one first substance forming extended surfaces is selected
from the group comprising glycerides, glycolipids,
glycerophospholipids, isoprenoidlipids, sphingolipids, steroids,
sterines or sterols, sulphur-containing lipids, lipids containing
at least one carbohydrate residue, or other polar fatty
derivatives.
36. Combination according to any of preceding claims, wherein said
at least one first substance forming extended surfaces is selected
from the group comprising phosphatidylcholines,
phosphatidylethanolamines, phosphatidylglycerols,
phosphatidylinositols, phosphatidic acids, phosphatidylserines,
sphingomyelins, sphingophospholipids, glycosphingolipids,
cerebrosides, ceramidpolyhexosides, sulphatides,
sphingoplasmalogenes, or gangliosides.
37. Combination according to any of preceding claims, wherein said
extended surface-forming substance is selected from the group
comprising lipids with one or two, not necessarily identical, fatty
chains, especially with acyl-, alkanoyl-, alkyl-, alkylene-,
alkenoyl-, alkoxy, or chains with omega-cyclohexyl-,
cyclo-propane-, iso- or anteiso-branched segments, or the
corresponding chains mixtures.
38. Combination according to any of preceding claims, wherein said
substance that forms extended surfaces is selected from the group
comprising lipids with n-decyl, n-dodecyl (lauryl), n-tetradecyl
(myristyl), n-hexadecyl (cetyl), n-octadecyl (stearyl), n-eicosyl
(arachinyl), n-docosyl (behenyl) or n-tetracosyl (lignoceryl),
9-cis-dodecenyl (lauroleyl), 9-cis-tetradecenyl (myristoleyl),
9-cis-hexadecenyl (palmitoleinyl), 9-cis-octadecenyl
(petroselinyl), 6-trans-octadecenyl (petroselaidinylj,
9-cis-octadecenyl (oleyl), 9-trans-octadecenyl (elaidinyl),
9-cis-eicosenyl (gadoleinyl), 9-cis-docosenyl (cetoleinyl) or
n-9-cis-tetracosoyl (nervonyl), n-decyloxy, n-dodecyloxy
(lauryloxy), n-tetradecyloxy (myristyloxy), n-hexadecyloxy
(cetyloxy), n-octadecyloxy (stearyloxy), n-eicosyloxy
(arachinyloxy), n-docosoyloxy (behenyloxy) or n-tetracosoyloxy
(lignoceryloxy), 9-cis-dodecenyloxy (lauroleyloxy),
9-cis-tetradecenyloxy (myristoleyloxy), 9-cis-hexadecenyloxy
(palmitoleinyloxy), 6-cis-octadecenyloxy, (petroselinyloxy),
6-trans-octadecenyloxy (petroselaidinyloxy), 9-cis-octadecenyloxy
(oleyloxy), 9-trans-octadecenyloxy (elaidinyloxy), and
9-cis-eicosenyl (gadoleinyloxy), 9-cis-docosenyl (cetoleinyloxy) or
n-9-cis-tetracosoyl (nervonyloxy), n-decanoyloxy, n-dodecanoyloxy
(lauroyloxy), n-tetradecanoyloxy (myristoyloxy), n-hexadecanoyloxy
(palmitoyloxy) I n-octadecanoyloxy (stearoyloxy), n-eicosanoyloxy
(arachinoyloxy), n-n-docosoanyloxy (behenoyloxy) and
n-tetracosanoyloxy (lignoceroyloxy), 9-cis-dodecenyloxy
(lauroleoyloxy), 9-cis-tetradecenoyloxy (myristoleoyloxy),
9-cis-hexadecenoyloxy (palmitoleinoyloxy), 6-cis-octadecenoyloxy
(petroselinoyloxy), 6-trans-octadecenoyloxy (petroselaidinoyloxy),
9-cis-octadecenoyloxy (oleoyloxy),
9-trans-octadecenoyloxyelaidinoyloxy), and 9-cis-eicosenoyloxy
(gadoleinoyloxy), 9-cis-docosenoyloxy (cetoleinoyloxy) and
9-cis-tetracosenoyloxy (nervonoyloxy) or the corresponding
sphingosine derivative chains.
39. Combination according to any of preceding claims, wherein said
at least one second substance is a surfactant.
40. Combination according to any of preceding claims, wherein said
surfactant is selected from the group comprising nonionic,
zwitterionic, anionic and cationic surfactants.
41. Combination according to any of preceding claims, wherein said
surfactant has the solubility in a polar liquid in which the
extended surfaces are prepared between 10.sup.-6 M and 10.sup.-2
M.
42. Combination according to any of preceding claims, wherein said
surfactant is selected from the group comprising long-chain fatty
acids or long chain fatty alcohols, long chain fatty ammonium
salts, such as alkyl- or alkenoyl-trimethyl-, -dimethyl- and
-methyl-ammonium salts, alkyl- or alkenoyl-sulphate salts, or
monovalent salts of cholate, deoxycholate, glycocholate,
glycodeoxycholate, taurodeoxycholate, taurocholate, acyl- or
alkenoyl-dimethyl-aminoxides, long fatty chain, for example
alkanoyl, dimethyl-aminoxides and especially dodecyl
dimethyl-aminoxide, long fatty chain, for example alkyl-N--,
methylglucamides and alkanoyl-N-methylglucamides, long fatty
chain-N,N-dimethylglycines, for example
N-alkyl-N,N-dimethylglycines, 3-(long fatty
chain-dimethylammonio)-alkanesulphonates, for example
3-(acyldimethylammonio)-alkanesulphonates, long fatty chain
derivatives of sulphosuccinate salts, long fatty
chain-sulphobetaines, for example N-acyl-sulphobetaines, long fatty
chain betaines, polyethylen-glycol-acyl- phenyl ethers,
polyethylene-long fatty chain-ethers such as polyethylene-acyl
ethers, polyethyleneglycol-iso long fatty chain ethers, such as
polyethyleneglycol-isoacyl ethers, polyethyleneglycol-sorbitane-l-
ong fatty chain esters, for example
polyethyleneglycol-sorbitane-acyl esters and especially
polyethylenglykol-monolaurate (e.g. Tween 20),
polyethylenglykol-sorbitan-monooleate (e.g. Tween 80),
polyhydroxyethylene-long fatty chain ethers, for example
polyhydroxyethylene-acyl ethers (Brij series), or the corresponding
polyhydroxyethylene-acyl esters (Myrj series) and polyethoxylated
castor oil 40 (Cremophor EL), sorbitane-mono long fatty chain, for
example alkylate (Arlacel or Span series), long fatty chain
--N-methylglucamides, such as acyl-N-methylglucamides or
alkanoyl-N-methylglucamides, long fatty chain sulphates, for
example alkyl-sulphates and their salts; long fatty chain
thioglucosides, such as alkylthioglucosides, long fatty chain
derivatives of various carbohydrates, such as pentoses, hexoses and
disaccharides, especially alkyl-glucosides and maltosides; further
lysolipids, such as long fatty chains derivatives of common
phospholipids, especially lyso-glycerophosphatidylcholine
(lysolecithin), lyso-glycerophosphatidylethanolamine
(lysokephalin), lyso-glycerophosphatidic acid,
lyso-glycerophosphorylglycerol, lyso-glycerophosphorylserine,
corresponding short-chain phospholipids, and membrane destabilising
oligo or polypeptides.
43. Combination according to any of preceding claims, wherein the
at least one second substance is charged if the at least one third
substance is uncharged, and the at least one second substance is
uncharged if the at least one third substance is charged, similar
preferred combinations also being possible for the said at least
one first and one second or for the said at least one first and one
third substance.
44. Combination according to any of preceding claims, wherein the
surface, formed by the at least one first, one second and one third
substance, at least one of which is charged, contains between 1%
and 75% of the charged component.
45. Combination according to any of preceding claims, wherein the
surface, formed by the at least one first, one second and one third
substance, at least one of which is charged, contains between 5%
and 50% of the charged component.
46. Combination according to any of preceding claims, wherein the
surface, formed by the at least one first, one second and one third
substance, at least one of which is charged, contains between 10%
and 30% of the charged component.
47. Combination according to any of previous claims, wherein the
surface-supporting at least one first substance is a
phosphatidylcholine, a phosphatidylethanolamine-N-mono- or
N-di-methyl, phosphatidic acid or its methyl ester,
phosphatidylserine and/or phosphatidylglycerol and the at least one
second substance which on its own forms small aggregates is a
lysophospholipid, especially a lysophosphatidic acid,
lysomethylphosphatidic acid, lysophosphatidylglycerol,
lysophosphatidylcholine, a partially N-methylated
lysophosphatidylethanol- amine, a monovalent salt of cholate,
deoxycholate, glycocholate, glycodeoxycholate, taurocholate, or a
sufficiently polar sterol derivative, a laurate, myristate,
palmitate, oleate, palmitoleate, elaidate or other long-chain fatty
acid salt and/or a Tween-, a Myrj-, or a Brij-surfactant, or a
Triton, a long-chain fatty sulphonate, -sulphobetaine,
--N-glucamide or -sorbitane (Arlacel or Span) surfactant.
48. Combination according to any of preceding claims, wherein the
at least one third substance, if not a surfactant different from
the at least one second substance, but otherwise selected from
similar surfactant classes, is a biologically active amphipat whibh
can destabilise lipid membranes.
49. Combination according to any of preceding claims, wherein the
solubility of at least one third or of one second substance in a
polar liquid is between 5.times.1 0-6 M and 1 M.
50. Combination according to any of preceding claims, wherein the
at least one third amphipat or the at least one second amphipat
adsorbs to the surface of lipid bilayer membrane but is well
miscible with or soluble in the polar liquid in which the said
extended surfaces are formed.
51. Combination according to any of previous claims, wherein the at
least one third or one second substance is a drug.
52. Combination according to any of preceding claims, wherein the
amphipatic compound with biological activity, which can act as a
drug, is a substituted ammonium compound of the formula 9in which
a) Ra represents a hydrophobic group, and Rb, Rc, and Rd,
independently of one another, each represents hydrogen,
C1-C4-alkyl, 2-hydroxyethyl, allyl or
cycle-C3-C6-alkyl-C1-C3-alkyl, or two of the radicals Rb, Rc and Rd
together represent C4- or C5-alkylene interrupted by --HN--,
--N(C1-C4-alkyl)-, --N(2-hydroxyethyl)- or by oxygen, or; b) Ra and
Rb are two hydrophobic groups or together represent a hydrophobic
group, and Rc and Rd, independently of one another, each represents
hydrogen, C1-C4-alkyl, allyl or cyclo-C3-C6-alkyl-C1-C3-alkyl, or
c) Ra, Rb and Rc together represent a hydrophobic group, and Rd
represents hydrogen or C1-C4-alkyl, and A.sup.- represents the
anion of a pharmaceutically acceptable acid, as a carboxylic acid
salt of the formula Ra-COO.sup.-Y.sup.+ (2) in which Ra represents
a hydrophobic group, and Y+represents the cation of a
pharmaceutically acceptable base, as an alpha-amino acid compound
of the formula 10in which Ra represents a hydrophobic group, and Rb
and Rc, independently of one another, each represents hydrogen or
C1-C4-alkyl, as a phosphoric acid monoester of the formula 11in
which Ra represents a hydrophobic group and Y+represents the cation
of a pharmaceutically acceptable base, or as an acid addition salt
of a compound having a hydrophobic group Ra and an imidazoline,
imidazolidine or hydrazino group as hydrophilic group.
53. Combination according to any of preceding claims, wherein the
said at least one third or one second amphipatic substance with
biological activity, which can act as a drug, is a substituted
ammonium compound of the formula 1 in which a) the hydrophobic
group can be an aliphatic hydrocarbon radical that can be
interrupted by an oxygen or sulphur atom, may contain the groups
--CO(.dbd.O)--, --O--C(.dbd.O)--, --C(.dbd.O)--NH--,
--O--C(.dbd.O)--NH-- or hydroxy, and can be substituted by from 1
to 3 monocyclic, aliphatic or aromatic hydrocarbon radicals, by a
bi- or tri-cyclic, aromatic or partially saturated hydrocarbon
radical, by a monocyclic, aromatic, partially saturated or
saturated heterocycle or by a bi- or tri-cyclic, aromatic,
partially saturated or benzo-fused heterocycle, or can be a
mono-cyclic, aliphatic or aromatic hydrocarbon radical or a
bicyclic, aliphatic or benzo-fused hydrocarbon radical, and the
hydrophilic group is a group of the formula 12in which Rb, Rc, and
Rd, independently of one another, each represents hydrogen,
C1-C4-hydrogen, C1-C4-alkyl or 2-hydroxyethyl, or in which two of
the radicals Rb, Rc and Rd together represent piperidino,
piperazinyl, 1-methylpiperazinyl, 1-(2-hydroxyethyl-piperazinyl or
morpholino, and the other radical represents hydrogen, or, b) the
hydrophobic groups Ra and Rb can be two aliphatic hydrocarbon
radicals which can be substituted by one or two monocyclic,
aliphatic or aromatic hydrocarbon radicals or by substituted,
monocyclic, aromatic, partially saturated or saturated heterocycle,
or Ra and Rb together represent a monocyclic, aromatic, saturated,
partially saturated or benzo-fused heterocycle, and the hydrophilic
group is a group of the formula 13in which Rc and Rd, independently
of one another each represents hydrogen or C1-C4-alkyl, or c) the
hydrophobic group is formed by Ra, Rb and Rc together and
represents an aromatic, partially saturated or benzo-fused
heterocycle and the hydrophilic group is a group of the formula
14in which Rd represents hydrogen or C1-C4-alkyl, preferably
methyl, and k is the anion of a pharmaceutically acceptable acid,
or a carboxylic acid salt of the formula 2 in which the hydrophobic
group Ra can be an aliphatic hydrocarbon radical, which can be
substituted by a monocyclic, aromatic hydrocarbon radical, or by a
bi- or tri-cyclic, aromatic or partially saturated hydrocarbon
radical, by a monocyclic, aromatic or partially saturated
heterocycle or by a bi- or tri-cyclic, aromatic, partially
saturated or benzo-fused heterocycle or by a steroid radical, or Ra
can be a monocyclic, aromatic hydrocarbon radical, a bi- or
tri-cyclic, aromatic or partially saturated hydrocarbon radical, a
monocyclic, aromatic or partially saturated heterocycle or a bi- or
tri-cyclic, aromatic, partially saturated or benzo-fused
heterocycle, and Y+is the cation of a pharmaceutically acceptable
base.
54. Combination according to any of preceding claims, wherein the
said at least one third or one second amphipatic substance, which
acts as a drug, is a substituted ammonium compound or the
corresponding amino compound that can be converted into the
ammonium compound by salt formation, such as acetylcholine
chloride, methacholine chloride, carbachol, muscarine, pilocarpine,
arecoline, phyostigmine, neostigmine, pyridostigmine bromide,
serotonin, histamine, tryptamine, bufotenine, psilocybin, morphine,
hydromorphone, oxymorphone, levorphanol, codeine, hydrocodone,
oxycodone, nalorphine, naloxone, naltrexon, buprenophine,
butorphanol, nalbiphine, pholcodine, pentazocine, ketamine,
metazocine, pentazocine, cyclazocine, pethidine, cetobemidon,
alphaphrodine, ethoheptazine, prodilidine, profadol, methadone,
normethadone, isomethadone, dipipanone, phenadoxone, dimephethanol,
dextromoramide, D-propoxyphene,
1-benzyl-2-dimethylaminomethyl-1-propanoyloxytetralin, tramadol,
dimethylthiambutene, diampromide, phenampromide, propiram,
tilidine, metopholine, etonitazene, ergotamine, dihydroergotamine,
dihydroergocryptine, methysergide, lisuride, dimetotiazin,
dizotifen, oxetoron, cyproheptadine, procaine, chloroprocaine,
hydroxyprocaine, propoxycaine I oxy-buprocaine, propoxymetacaine,
piridocaine, leucinocaine, butacaine p tetracaine,
hydroxytetracaine, cornecaine, edan, piperocaine, cyclomethycaine,
parethoxysaine, stadacain, cinchocaine, lidocaine, pyrrocaine,
granocaine, butanilicaine, tolycaine, mepivacaine, bupivacaine,
prilocaine, carticaine, dipiperidon, propicocaine, dyclonine,
pramocaine, fomocaine, quinisocaine, profenamine, promethazine,
periciazine, perimethazine, chlorpromazine, perphenazine,
prochlorperazine, triflumpromazine, trifluoperazine, fluphenazine,
thioridazine, mesoridazine, piperacetazine, acetophenazine,
ethymemazine, dimethacrine, opipramol, clomipramine, imipramine,
desimipramine, trimipramine, chloroprothixene, thiothixene,
amitriptyline, nortriptyline, doxepin, thiepin, protriptyline,
prothipendyl, femoxetin, citalopram, zimelidine, trebenzomin,
viloxazine, nomifensine, femoxetin, tranylcygromine, pargyline,
etryptamine, flurazepam, mescaline,
Nalpha,Nalpha-dimethyl-tryptamine, bufotenine, psilocin,
psilocylein, scopolamine, atropine, benzatropine, trihexyphenidyl,
cycrimine, pridinol, biperidine, procyclidine, caramiphene,
phenglutarimide, orphenadrine, chlor-phenoxamine, metixen,
doxapram, amphetamine, methamphetamine, propylhexedrine,
prolintane, fencamfamine, methylphenidol, pipradrol, phenmetrazine,
diethylpropion, meclofenoxat, naftidrofuryl, dexamphetamine,
phentermin, chlorphentermine, fenfluramine, amfepramone,
phenmetrazine, phendimetrazine, tubocumarin, alcuronium chloride,
gallamin triethiodide, hexacarbacholine bromide, pancuronium
bromide, suxamethonium chloride, decamethonium bromide, scopolamine
butyl bromide, bevonium methyl sulphate, valethamate bromide,
methanteline bromide, camylofine, hexahydroadiphenine, adiphenine,
fencarbamide, benzyclamine, ditaxol, chloroquine, tamoxifen,
ethamoxytriphetol, phenbenzamine, tripelenamin, chlorpyramine,
mepyramine, metaphenilene, metapyrilene, chloropyrilene,
histpyrroclin, bamipin, thenalidine, clemizole, meth-dilazine,
isothipendyl, oxomenazine, diphenhydramine, medrylamine,
chlorophenoxamine, silachlorophenoxamin, carbinoxamine,
diphenpyraline, clemastine, ametho-benzepine, pheniramine,
chlorophenamine, bromo-pheniramine, triprolidine, cycliramine,
phenindamine, dimetindene, cyproheptadine, ketotifen, epinephrine
(adrenaline), norepinephrine (noradrenaline), dopamine, nordefrin,
ethylnorepinephrine, isoprenaline, iso--ethorine, metaproterenol,
orciprenaline, metaraminol, phenylephrine, hydroxyamphetamine,
methoxyphenamine, methoxamine, albuterol, ephedrine, norephedrine,
fenfluramine, phenylpropanolamine, pholedrine, tyramine,
dichloroisoprenaline, norfenefrine, octopamine, etilefrin,
acebutolol, atenolol, meto-prolol, toliprolol, alprenolol,
oxprenolol, bunitrolol, bupranolol, talinolol, phenbutolol,
bufetolol, varbian (R,S- or S-form), propanolol, indenolol,
pindolol, mepindolol, nadolol, bunolol, sofalol, nifenalol,
cabetalol, bufenalol, reserpine, rescinnamine, syringopine,
chlorotetracycline, oxytetracycline, tetracycline,
demethylchlorotetracycline, metacycline, doxycycline, minocycline,
rolitetracycline, quinine, conquinidine, quinidine, cinchonine,
pamaquine, primaquine, pentaquine, chloroquine, santoquine,
hydroxychloroquine, amodiaquine, mepacrin, biguanid-1,3,5-triazin,
proguanil, bromoguanil, chloroproguanil, nitroguanil,
cycloguanilembonate, pyrimethamine, tri-methoprim, lucanthone,
hycanthone, miracil A or B, amantadine, cyclooctylamine,
rimantadin, prednisolone diethylaminoacetate.
55. Combination according to any of preceding claims, wherein the
said at least one third or one second amphipatic substance takes
the role of a drug as the substituted ammonium compound or as the
corresponding amino compound that can be converted into the
ammonium compound by salt formation, and is a compound selected
from the group of the acid addition salts of antidepressants of the
formula in which R1 represents lower alkyl, for example methyl, A
represents the group N-.nu. R1, oxygen or sulphur, and R2
represents hydrogen or cyano; acid addition salts of
antidepressants of the formula 15in which R1 represents lower
alkylamino-lower alkyl, for example 3-methylamino-n-propyl,
di-lower alkyl-amino-lower alkyl, for example
3-dimethylamino-n-propyl or
3-(4-(2-hydroxyethyl)-piperazin-1-yl)-n-propyl and A represents
ethylene or vinylene, or acid addition salts of amphetamine,
methamphetamine, benzphetamine, propyl-hexedrine, prolintan,
fencamfin, methylphenidate, pipradrol, phenmetrazine, adiphenine,
epinephrine, norepinephrine, dopamine, nordefrin,
ethyl-norepinephrine, isoprenaline, isoethorine, meta-proterenol,
orciprenaline, metaraminol, phenylephrine, hydroxyamphetamine,
methoxyphenamine, ephedrine, norephedrine, pholedrine, tyramine,
norfenefrin, octopamine, acebutolol, atenolol, toliprolol,
alprenolol, oxprenolol, bunitrolol, bupranolol, talinolol,
phenbutolol, bufetolol, varbian (R,S-form and S-form), reserpine,
rescinnamine, syringopine or prednisolone diethylaminoacetate.
56. Combina tion according to any of preceding claims, wherein the
said at least one third or second amphipatic substance takes the
role of a drug as the substituted ammonium compound of the formula
1 or as the corresponding amino compound that can be converted into
the ammonium compound by salt formation,
1-(2R-2-hydroxy-3-methylaminopropyl)dibenzo[b-
,e]bicyclo[2.2.2]octadiene, and the 2R,S-isomeric mixture,
maprotiline, benzoctamine,
3-methyldibenzo[2,3:6,7]-oxepino[4,5-diazepine hydrochloride,
7-cyano-3-methyl-2,3,4,5-tetrahydro-1H-dibenzo[2,3:6,7]-th-
iepino[4,5-d]azepine methanesulphonate,
3,10-dimethyl-1,2,3,4,5,10-hexahyd-
rodibenzo[b,f]azepino[4,5]azepine maleate, clomipramine, opipramol,
desipramine, imipramine or imipramine N-oxide, ephedrine,
norephedrine,
1-iso-propylamino-3-[4-(2-methylthioethoxy)-phenoxy]-propan-2-ol,
1-isopropylamino-3-(2-pyrrol-1-ylphenoxy)-propan-2-ol, oxprenolol,
prenalterol, adiphenine, prednisolone diethylaminoacetate, or
reserpine.
57. Combination according to any of preceding claims, wherein the
said at least one third or second amphipatic substance takes the
role of a drug as the as the carboxylic acid salt or the carboxylic
acid compound that can be converted into the carboxylic acid salt
by salt formation, methylprednisolone sodium succinate,
prednisolone sodium succinate, 3,20-dioxo-5'-pregnane, hydroxydione
succinate sodium, 11,20-dioxo-3alpha-hydroxy-5alpha-pregnane,
alphadolon, a cholic acid or deoxycholic acid salt, alclofenac,
ibufenac, ibuprofen, clindanac, fenclorac, ketoprofen, fenoprofen,
indoprofen, fenclofenac, diclofenac, flurbiprofen, pirprofen,
naproxan, benoxaprofen, carprofen, cicloprofen, mefenamic acid,
flufenamic acid, tolfenamic acid, meclofenamic acid, milflumic
acid, clonixin, flunixin, indometacin, oxmetacin, intrazol,
acemetazin, cinmetacin, zomepirac, tolmetin, colpirac, tiaprofenic
acid, benzadac, PGE2 (dinoprostone), PGF2alpha (dinoprost), 15
(S)-15-methyl-PGE2, 15 (S)-15-methyl-PGF2alpha (carboprost), (+)15
(Xi)-15-methyl-13,14-dihydro-11-deoxy-PGE1 (deprostil), 15
(S)-15-methyl-11-deoxy-PGE, (doxaprost), 16,16-dimethyl-PGE2,
17-phenyl-18,19,20-trinor-PGF2alpha,
16-phenoxy-17,18,19,20-tetranor-PGF2- , or
N-methylsulphonyl-16-phenoxy-17,18,19,20-tetranor-PGF2 alpha
(sulproston), nalixidic acid, cinoxacin, oxolinic acid, pironidic
acid, pipenidic acid, penicillin G or V, phenethicillin,
propicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin,
flucloxacillin, cyclacillin, epicillin, mecillinam, methicillin,
azlocillin, sulbenicillin, ticarcillin, mezlocillin, piperacillin,
carindacillin, azidocillin, ciclazillin, cefaclor, cefuroxime,
cefazlur, cephacetrile, cefazolin, cephalexin, cefadroxil,
cephaloglycin, cefoxitin, cephaloridine, cephsulodin, cefotiam,
ceftazidine, cefonicid, cefotaxime, cefinenoxime, ceftizoxime,
cephalothin, cephradine, cefamandol, cephanone, cephapirin,
cefroxadin, cefatrizine, cefazedonep ceftrixon, ceforanid,
moxalactam, clavulanic acid, nocardicine A, sulbactam, aztreonam,
thienamycin, chlorambucil or methotrexate.
58. Combination according to any of preceding claims, wherein the
said at least one third or second amphipatic substance, which takes
to role of a drug, acts as an adrenocorticostatic, a
.beta.-adrenolytic, an androgen an antiandrogen, an antiparasitic,
an anabolic, an anaesthetic, an analgesic, an analeptic, an
antiallergic, an antiarrhythmic, an antiarterosclerotic, an
antiasthmatic, a bronchospasmolytic, an antibiotic, an
antidrepressive, an antipsychotic, an antidiabetic, an antidot, an
antiemetic, an antiepileptic, an antifibrinolytic, an
anticonvulsive, an anticholinergic, an enzyme, a coenzyme or
corresponding inhibitor, an antihistaminic, an antihypertonic, a
biological inhibitor of drug activity, an antihypotonic, an
anticoagulant, an antimycotic, an antimyasthenic, an agent against
Morbus Parkinson or Morbus Alzheimer, an antiphlogistic, an
antipyretic, an antirheumatic, an antiseptic, a respiratory
analeptic or a respiratory stimulant, a broncholytic, a
cardiotonic, a chemotherapeutic, a coronary dilatator, a
cytostatic, a diuretic, a ganglium-blocker, a glucocorticoid, an
antiflew agent, a haemostatic, a hypnotic, an immunoglobuline or
its fragment, an immunologically active substance, a bioactive
carbohydrate, a bioactive carbohydrate derivative, a contraceptive,
an anti-migraine agent, a mineralo-corticoid, a
morphine-antagonist, a muscle relaxant, a narcotic, a
neurotherapeutic, a neuroleptic, a neurotransmitter or its
antagonist, a small peptide, a small peptide derivative, an
ophthalmic, a sympaticomimetic or a sympathicolytic, a
para-sympaticomimetic or a para-sympathicolytic, a psoriasis drug,
a neurodermitis drug, a mydriatic, a psychostimulant, a rhinologic,
a sleep-inducing agent or its antagonist, a sedating agent, a
spasmolytic, tuberculostatic, an urologic agent, a vasoconstrictor
or vasodilatator, a virustatic, a wound-healing substance, or a
combination of aforesaid agents.
59. Combination according to any of preceding claims, wherein the
drug content is between 0.1 rel.% and 60 rel.% compared to the
total mass of all three said substances that form said extended
surfaces.
60. Combination according to any of preceding claims, wherein said
at least one third or second substance is a low molecular weight
immunomodulator.
61. Combination according to any of preceding claims, wherein said
at least one third or second substance is a bio-catalyst.
62. Combination according to any of preceding claims, wherein said
at least one third or second substance is a low molecular weight
agonist or antagonist of some biological substance action.
63. Combination according to any of preceding claims, wherein said
at least one third or second substance is a co-enzyme.
64. Combination according to any of preceding claims, wherein said
at least one third or second substance is a hormone.
65. Combination according to any of preceding claims, wherein said
at least one third or second substance is a low to intermediate
weight polypeptide with membrane destabilising properties.
66. Combination according to any of preceding claims, wherein said
at last one second substance is a cyclooxygenase or lipoxygenase
inhibitor and at least one third substance is a non-ionic
surfactant with solubility in 1-10 .mu.M range that preferably
belongs to the class of sorbitane-polyoxyethylene-alkyl or
-alkylene esters or else is a polyoxyethylene-alkyl or -alkylene
ether.
67. The use of a combination of substances according to any of
preceding claims, in drug carriers, drug depots, or for other kind
of medicinal or biological application by providing the extended
surfaces in the form of membranes formed by the at least one first
substance, the at least one second and the at least one third
substance, which together surround miniature droplets, wherein the
substance with biological activity, being a drug, is mainly
associated with said droplet surface or else is mainly incorporated
into the droplet and then carried by the droplet to the place where
the drug is intended to act.
68. The use of a combination of substances according to any of
preceding claims for the manufacture of a preparation for the
transport of an active ingredient, which can be one of the three
amphipatic components, especially for biological, medical,
immunological, or cosmetic purposes, into and through the skin of
warm blood creatures.
69. A method of preparing a combination according to any of
preceding claims in the form of a formulation of a biologically,
cosmetically and/or pharmaceutically active agent, comprising the
steps of selecting the at least one first and the at least one
second substance which together form extended surfaces, when in
contact with said medium, such that said extended surfaces formed
by the at least one first and the at least one second substance are
more adaptable than the at least one first substance alone and the
surfaces formed by the at least one second substance alone form
small aggregates; alternatively selecting the at least one first
and the at least one third substance which together form extended
surfaces, when in contact with said medium, such that said extended
surfaces formed by the at least one first and the at least one
third substance are more adaptable than the at least one first
substance alone and the surfaces formed by the at least one third
substance alone form small aggregates, if this substance
self-aggregates; and generating said surface-forming combination
from at least one first, at least one second, and at least one
third substance, such that the surface of resulting at least three
component combination is even more adaptable than the surface
prepared from at least one first and one second substance alone or
of the surfaces formed by the at least one first and one third
substance alone, bringing the combination of at least two or all
three said substances into suspension by means of controlled
mechanical fragmentation, in the presence of or before being mixed
with the at least one third substance, such that said third
substance is incorporated at least partly in said extended surface
formed by controlled mechanical fragmentation to obtain final
preparation.
70. The method according to any of preceding claims, wherein said
means of controlled mechanical fragmentation includes on
filtration, pressure change or mechanical homogenisation, shaking,
stirring, or mixing.
71. The method according to any of preceding claims, wherein the
liquid medium suspension characteristics correspond to any one of
claims 1 to 65.
72. The method according to any of preceding claims, wherein said
active agent is selected from the group comprising anti-diabetic
agents, growth factors, immunomodulators, enzymes, recognition
molecules, adrenocorticostatics, adrenolytics, androgens,
antiandrogens, antiparasitics, anabolics, anaesthetics, analgesics,
analeptics, antiallergics, antiarrhythmics, antiarteroscierotics,
antiasthmatics, bronchospasmolytics, antibiotics, antidrepressiva,
antipsychotics, antidots, antiemetics, antiepileptics,
antifibrinolytics, anticonvulsiva, anticholinergics, enzyme,
coenzymes or corresponding inhibitors, antihistaminics,
antihypertonics, biological inhibitors of drug activity,
antihypotonics, anticoagulants, antimycotics, antimyasthenics,
agents against Morbus Parkinson or Morbus Alzheimer,
antiphlogistics, antipyretics, antirheumatics, antiseptics,
respiratory analeptics or respiratory stimulants, broncholytics,
cardiotonics, chemotherapeutics, coronary dilatators, cytostatics,
diuretics, ganglium-blockers, glucocorticoids, antiflew agents,
haemostatics, hypnotics, immunologically active substances,
contraceptives, anti-migraine agents, mineralo-corticoids,
morphine-antagonists, muscle relaxants, narcotics,
neurotherapeutics, neuroleptics, neurotransmitters or their
antagonists, peptides, peptide derivatives, opthalmics,
sympaticomimetics or sympathicolytics, para-sympaticomimetics or
para-sympathicolytics, anti-psoriasis drugs, neurodermitis drugs,
mydriatics, psychostimulants, rhinologics, sleep-inducing agents or
their antagonists, sedating agents, spasmolytics, tuberculostatics,
urologics, vasoconstrictors or vasodilatators, virustatics,
wound-healing substances, or a combination of aforesaid agents.
73. The method according to any of preceding claims, wherein said
at least three amphiphilic substances are either used as such, or
dissolved in a physiologically compatible polar fluid, comprising
water or water-miscible fluids, or in a solvation-mediating agent,
together with a polar solution.
74. The method according to any of preceding claims, wherein the
said polar solution contains at least one surfactant or
surfactant-like amphipat, which destabilises bilayer membrane, and
at least one more membrane destabilising, biologically active
ingredient or an additional surfactant.
75. The method according to any of preceding claims, wherein the
formation of said surfaces is induced by substance addition into a
fluid phase, evaporation from a reverse phase, by injection or
dialysis, or with the aid of mechanical stress.
76. The method according to any of preceding claims, wherein the
formation of said surfaces is induced by filtration, the filtering
material having pores diameters between 0.01 .mu.m and 0.8 .mu.m,
the preferred choice of pore diameter being dependent on the
desired final aggregate dimensions.
77. The method according to any of preceding claims, wherein
several filters are used sequentially or in parallel.
78. The method according to any of preceding claims, wherein said
agents and carriers are made to associate, at least partly, after
formation of said extended surfaces.
79. The method according to any of preceding claims, wherein said
extended surfaces, with which the agent molecules are allowed to
associate, are prepared just before the application of the
formulation, if convenient from a suitable concentrate or a
lyophylisate.
80. A container comprising the pharmaceutical composition based a
combination of substances according to any preceding claim.
81. A package comprising at least one container comprising the
pharmaceutical composition based on a combination of substances
according to any preceding claims.
82. A method for generating a therapeutic effect on a warm blood
creature by applying a pharmaceutical composition based on a
combination of substances according to any of previous claims onto
or into such living creature body.
83. The method according to any of preceding claims, wherein
different administration volumes are selected to control the
applied medicament dose and the outcome of therapeutic
application.
84. The method according to any of preceding claims, wherein a
suspension of drug-free aggregates is loaded with the drug to be
associated therewith during the day prior to an administration,
preferably 360 min, more preferably 60 min and even more preferably
30 min before administering the resulting formulation in or on the
body.
85. The method of any of preceding claims, characterised in that at
least one dose of the pharmaceutical composition with therapeutic
activity is administered.
86. The method according to any of preceding claims, wherein the
flux of penetrants that carry a drug through the various pores in a
well-defined barrier is determined as a function of a suitable
driving force or a pressure acting across the barrier and the data
are then conveniently described by a characteristic curve which, in
turn, is employed to optimise the formulation or application
further.
87. The method according to any of preceding claims, wherein the
characteristic, e.g. penetrability vs. pressure, curve is analysed
in terms of eq. (*) or alike.
88. The combination of any of preceding claims, wherein the
adaptability of extended surface aggregates comprising all three
said amphipatic components exceeds by at least 20% or by at least
twice the standard deviation of a typical measurement, whichever is
smaller, the adaptability of the extended surface aggregates
comprising the at least one first and the at least one second
amphipatic component, used at the corresponding concentrations, or
the adaptability of the extended surface comprising the at least
one first and the at least one third amphipatic component, used at
the corresponding concentrations, whichever is smaller.
89. The combination of any of preceding claims, wherein the
adaptability of extended surface aggregates comprising all three
said amphipatic components exceeds by at least 30% the adaptability
of the extended surface aggregates comprising the at least one
first and the at least one second amphipatic component, used at the
corresponding concentrations, or the adaptability of the extended
surface comprising the at least one first and the at least one
third amphipatic component, used at the corresponding
concentrations, whichever is smaller.
90. The combination of any of preceding claims, wherein the total
concentration of said at least one second and said at least one
third compound in the ESAs comprising all three said amphipatic
components is equal to or less than the concentration of said at
least one second compound in the ESAs comprising at least one first
and at least one second compound and the corresponding
concentration of the at least one first compound.
91. The combination of any of preceding claims, wherein the total
concentration of said at least one second and said at least one
third compound in the ESAs comprising all three said amphipatic
components is equal to or less than the concentration of said at
least one third compound in the ESAs comprising at least one first
and at least one second compound at the corresponding concentration
of the at least one first compound.
92. The combination of any of preceding claims, wherein the
adaptability is expressed as the inverse value of the p* value
corresponding to a predefined fraction of P.sub.max-value, which is
often selected around 60% and preferably is 57% of P.sub.max-value.
Description
[0001] The present application claims the benefit of U.S.
provisional application No. 60/417,847 filed on Oct. 11, 2002,
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to aggregates with extended surface
(extended-surface aggregates, ESAs) with increased deformability
and improved barrier penetration capability, said ESAs being
suspendable in a suitable liquid medium and comprising at least
three amphipats (amphipatic components) and being capable to
improve the transport of actives through semi-permeable barriers,
such as the skin, especially for the non-invasive drug application
in vivo by means of barrier penetration by such aggregates. The
three amphipats include at least one membrane forming compound
(MFC), which can form the membrane of said ESAs, and at least two
membrane destabilising compounds (MDC.sub.1 and MDC.sub.2)
differentiated by their capability of forming smaller aggregates
(with no extended surfaces) by either themselves or else in
combination with each other and/or characterized by their
relatively high solubility in said suitable liquid medium. The ESAs
are loaded with at least one biologically active compound, which
can be one of the at least three amphipats. The invention relates
also to preparations comprising extended surface aggregates (ESAs),
that can penetrate barriers even when the typical ESAs radius (when
an ESA is considered to be spherical) is at least 40% (and
preferably at least 50% or even more) greater than the average
radius of a pore in the barrier before and after the ESAs have
penetrated the barrier.
BACKGROUND INFORMATION
[0003] Administration of active ingredients frequently is limited
by natural barriers, such as the skin, which prevent adequate
absorption of the active molecules due to the low barrier
permeability for such ingredients.
[0004] Availability and use of preparations that can overcome this
barrier impermeability problem and allow non-invasive active
ingredient administration would be advantageous in many cases. In
humans and animals, for example, a percutaneous administration of
such preparations would protect the active ingredients against
decomposition in the gastrointestinal tract and possibly would
result in a modified, therapeutically attractive distribution of
the agent in the body; such non-invasive administration could also
affect the pharmacokinetics of the active ingredient and permit
less frequent and/or simpler disease treatment (G. Cevc. Exp. Opin.
Invest. Drugs (1997) 6: 1887-1937.). In the case of plants,
improved penetration through or into the cuticle could lower the
concentration of active ingredient that is required for the desired
effect and, in addition, could significantly decrease contamination
of the environment (Price, C. E. (1981) in: The Plant Cuticle (D.
F. Cutler, K. L. Alvin, C. E. Price, Publisher), Academic, New
York, pp. 237-252).
[0005] Many methods for increasing the skin permeability have been
discussed (see, for example, G. Cevc, 1997, op. cit.). Most
prominent are jet injection (for a classical review see Siddiqui
& Chien Crit. Rev. Ther. Drug. Carrier Syst. (1987) 3:
195-208), the use of electrical (Bumette & Ongpipattanakul J.
Pharm. Sci. (1987) 76: 765-773) or accoustic (Vyas et al., J
Microencapsul (1995) 12: 149-54) skin perturbation or else the use
of chemical additives, such as certain solvents or surfactants.
Such chemicals generally act as the skin permeation enhancers by
increasing the partitioning and/or diffusivity of the active
ingredient in the skin lipids.
[0006] Most often used permeation enhancers are non-ionic short or
long-chain alcohols and uncharged surfactants etc., anionic
materials (particularly fatty acids), cationic long-chain amines,
sulfoxides, as well as various amino derivatives, and amphoteric
glycinates and betaines. None of these, however, solves the problem
of active ingredient transport through the skin or mucous barrier
to general satisfaction.
[0007] An overview of the measures, which have been used for the
purpose of increasing active ingredient penetration through plant
cuticles, is summarised in the work of Price (1981, op. cit.).
[0008] Epidermal use of one or several amphipatic substances in the
form of a suspension or an O/W or W/O emulsion, has also brought
about too little improvement. An extensive review written by G.
Cevc (1997, op. cit.) explains why liposomes, at best, can modify
drug retention time or stability on the skin and or improve
transcutaneous drug transport by partly occluding the skin surface.
Japanese patent application JP 61/271204 A2 (86/27 1204) provides
an example for stabilizing effect of liposomes on the skin, relying
on hydroquinone glucosidal as stabilizing material.
[0009] The use of lipid vesicles loaded with an active ingredient
combined with a gel-forming agent in the form of "transdermal
patches" was proposed in WO 87/1938 A1. However, the ability of the
active ingredient to permeate the skin was not appreciably
increased. Massive use of permeation--promoting polyethylene glycol
and of fatty acids, together with lipid vesicles, was required by
Gesztes and Mezei (1988, Anesth. Analg. 67, 1079-1081) to attain
only a moderate local analgesia with lidocaine-containing
formulations applied for several hours under occlusion on the
skin.
[0010] U.S. Pat. No. 6,193,996 describes a pressure sensitive skin
adhesive that uses skin permeation enhancers. European Patent
applications EPA 102 324 and EPA 0 088 046, and U.S. Pat. No.
4,619,794, all by H. Hauser, describe methods for preparing
unilamellar vesicles, using a single membrane destabilising
component. The vesicles may be used as carriers for different
drugs. However, such vesicles are not used on the skin or for
transport through semi-permeable barriers. European Patent
application EPA 0 152 379 by Muntwyler and Hauser similarly
describes the preparation of unilamellar vesicles. However, these
vesicles often need to be separated from the residual multilamellar
liposomes, facilitated by the presence of charged drugs, for final
use of the former for treating human or animal body. The authors
also point the potential need to neutralize the drug during vesicle
preparation to obtain the desired unilamellar liposomes. Further,
such vesicles are not used for transport of drugs through a
semi-permeable barrier.
[0011] European patent EP 0 475 160, corresponding U.S. Pat. No.
6,165,500 and Canadian patent 2,067,754, all with the title
"Preparation for the application of agents in mini-droplets",
describe special preparations related to the suspensions described
in this application. These documents report the use of different
agents associated with minuscule droplets or, in particular, with
the vesicles consisting of one or a few membrane-like amphiphile
assemblies for overcoming semi-permeable barriers including the
skin. These references describe preparations having a single
membrane destabilising component. WO 98/17255 and AU 724218,
likewise, describe vesicles for the transport of a variety of drugs
through the skin.
[0012] In two relatively early reports on dermal liposomal
tetracaine (Gesztes A, Mezei M. "Topical anesthesia of the skin by
liposome-encapsulated tetracaine." Anesth. Analg. (1988),
67:1079-1081) and lidocaine (Foldvari M, Gesztes A, Mezei M.
"Dermal drug delivery by liposome encapsulation: clinical and
electron microscopic studies." J Microencapsul (1990), 7:479-489),
Mezei's group reported anaesthetic performance of such locally used
drugs and corresponding autoradiography data. Drug was found in the
epidermis and in dermis of humans and guinea pigs when the skin was
treated under an impermeable (occlusive) coating with the
liposome-encapsulated anaesthetics. The formulations always
contained multilamellar soybean phosphatidylcholine vesicles.
However, the reports demonstrate no liposome-mediated drug
transport through the skin. (Foldvari M. "In vitro cutaneous and
percutaneous delivery and in vivo efficacy of tetracaine from
liposomal and conventional vehicles." Pharm Res (1994)
11:1593-1598) and with an additional oily ingredient (Foldvari M.
"Effect of vehicle on topical liposomal drug delivery: petrolatum
bases." J Microencapsul (1996), 13:589-600). This conclusion is
supported by the fact that the reported maximum transported drug
dose (5.3%) was more than 20-times higher than the reported
transported lipid dose (0.2%) (Foldvari, 1994). Further, Foldvari's
formulations evidently were not optimised for adaptability but
rather for best drug retention/release.
[0013] P. Gonzalez, M. E. Planas, L. Rodriguez, S. Sanchez, and G.
Cevc in an article on "Noninvasive, percutaneous induction of
topical analgesia by a new type of drug carriers and prolongation
of the local pain-insensitivity by analgesic liposomes" (Anesth.
Analg. (1992), 95: 615-621) report the results of investigations
with surfactant-containing formulations, typically loaded with
lidocaine (2%, as a free base) in a mixed lipid 4-8% suspension
(w/v). Lipid aggregates were prepared from a 4/1 mol/mol
phosphatidylcholine/sodium cholate mixture, starting with an
ethanolic lipid solution (7-3 w-% EtOH in the final product) for
easier manufacturing. However, all the tested suspensions were
reported by Planas et al. to be unstable. Further, Planas et al.
failed to disclose how a stable drug formulation could be prepared,
which would be suitable for transdermal drug delivery. Peters and
Moll (1995) ("Pharmacodynamics of a liposomal preparation for local
anaesthesia". Arzneimittelforschung (1995), 45:1253-6, describe
permeation of a topically applied drug through the skin. The
permeation is enhanced by ethanol, is based on diffusion, and is
achieved under occlusion.
[0014] Carafa and colleagues describe the use of surfactant-based,
phospholipid-free vesicles (Carafa et al., 2002 ("Lidocaine-loaded
non-ionic surfactant vesicles: characterisation and in vitro
permeation studies." Int J Pharm (2002), 231:21-32). However, such
vesicles do not simultaneously include both a MFC and a MDC, and
are unsatisfactory.
SUMMARY OF THE INVENTION
[0015] Applicants have discovered that incorporation of a
surfactant into a bilayer membrane that is built from another less
soluble amphipat, such as a phospholipid, can increase the
flexibility of the resulting complex membrane. This promotes the
capability of complex aggregates in the form of droplets covered by
the bi-component membranes to cross pores in a semi-permeable
barrier that otherwise would prevent comparably large aggregates
from crossing. Further, the use of aggregates with highly
deformable membrane coating can mediate agent transport into and/or
across mammalian skin. This can be achieved by selecting a
surfactant, which is a membrane destabilising component (=MDC), and
a less soluble amphipat, which is the membrane forming component
(=MFC), so as to maximize the mixed membrane flexibility and the
mixed aggregate stability. Further the surfactant can be selected
to increase bilayer membrane adaptability. Patent applications by
applicant, especially WO 92/03122 and WO 98/172550 describe basic
requirements for the use of lipid/surfactant mixtures for
transbarrier transport.
[0016] It is an objective of the invention to provide preparations
that can transport active ingredients through a barrier in the form
of vesicles or other extended surface aggregates (ESAs) comprising
said actives, said preparations having improved permeation
capability through semi-permeable barriers.
[0017] It is an aspect of the invention to provide preparations
comprising extended surface aggregates (ESAs) which permit the ESAs
to permeate barriers, with the radius of the ESAs (when considered
as spherical) being at least 40% (and preferably at least 50% or
even more) greater than the average pore radius of the barrier,
after the ESAs have permeated the barrier pores.
[0018] It is a further aspect of the invention to provide a
preparation based on a combination of at least one first (membrane
forming component MFC), at least one second (membrane destabilising
component MDC), and at least one third (membrane destabilising
component MDC) amphipatic component suspended in a suitable liquid
medium in the form of corresponding mixed amphipat extended surface
aggregates (ESAs) with one or a few bilayer-like, mixed amphipat
coating(s), wherein said ESAs formed by a combination of all three
said components have surfaces in contact with said liquid medium,
that are at least 50% more extended, on the average, than the
typical surfaces of aggregates comprising the said at least one
second and at least one third amphipatic component alone, at the
same concentrations and, in case, after adjustment for
physico-chemical effects of the absence of said first amphipatic
compound (MFC).
[0019] A further aspect of the invention is to provide suspensions
of extended surface aggregates in a liquid medium comprising: at
least one first membrane forming component (MFC); at least one
second membrane destabilising component (MDC); at least one third
membrane destabilising component (MDC), the third component
typically being a drug, such that said complex extended surface
aggregates (ESAs) can penetrate intact mammalian skin and thus
increase drug concentration in the skin and/or increase the reach
of drug distribution below the skin, in comparison with the result
of the same drug application in a solution on the skin. In a
special version of said suspensions, said extended surface
aggregates are membrane-enclosed, liquid-filled vesicles, said
first component is a membrane-forming lipid, and said second and
third components are membrane-destabilising components.
[0020] Another aspect of the invention provides a combination of at
least one first (membrane forming, component MFC), at least one
second (membrane destabilising component MDC), and at least one
third (membrane destabilising component MDC) amphipatic component
suspended in a suitable liquid medium in the form of mixed amphipat
extended surface aggregates (ESAs) with one or a few bilayer-like,
mixed amphipat coating(s), wherein the
[0021] said at least one first substance has a tendency to self
aggregate and is at least 10-times less soluble in said liquid
medium than said at least one second and said one third substance,
allowing the first to form extended surfaces,
[0022] said at least one second substance is at least 10-times more
soluble than said at least one first substance in said liquid
medium and, on its own, tends to form or supports the formation of
surfaces, that are at least 2-times less extended than the surfaces
containing the at least one first substance alone,
[0023] said at least one third substance being also at least
10-times more soluble in said liquid medium than the first
substance and optionally forms self-aggregates with aggregation
numbers at least 10-times smaller than that of self-aggregates of
said first substance; and
[0024] said extended surfaces comprising said at least one first,
at least one second and at last one third substance, in
equilibrium, have at least 50% more extended surfaces than the
surfaces formed by the at least one second or one third substance
alone, at the same concentration and, in case, after adjustment for
physico-chemical effects of the absence of said first amphipatic
compound (MFC).
[0025] Yet another aspect of the invention is a preparation based
on a combination of at least one first (membrane forming component
MFC), at least one second (membrane destabilising component MDC),
and at least one third (membrane destabilising component MDC)
amphipatic component suspended in a suitable liquid medium in the
form of corresponding mixed aggregates with an extended surface
(ESAs) with one or a few, preferably bilayer-like, mixed amphipat
coating(s), wherein said MFC alone forms extended-surface
aggregates with aggregation number of at least 5000, and preferably
more than 10.000, and both MDCs alone and the combination of both
MDCs form smaller aggregates with no really extended surface and
aggregation number below 5000, and preferably below 1000 in contact
with said suitable liquid medium.
[0026] All compositions according to the present invention
comprising three amphipatic compounds which together form extended
surface aggregates either have a defined solubilization point, or
do comprise more than 0.1 mol % of the solubilizing amount of those
components which at higher concentrations would solubilize the
extended surface aggregates.
[0027] All embodiments of the invention are useful in preparations
for the application, administration or transport of at least one
active ingredient which can be amongst said three substances,
especially for medicinal or biological purposes, into and through
barriers and constrictions, such as the skin of warm blood
creatures or the like.
[0028] Preferably the adaptability of extended surface comprising
all three said amphipatic components to ambient stress exceeds by
at least 20% or by at least twice the standard deviation of a
typical measurement (whichever is smaller) the adaptability of the
extended surface comprising the at least one first and the at least
one second amphipatic component used at the corresponding
concentrations or the adaptability of the extended surface
comprising the at least one first and the at least one third
amphipatic component at corresponding concentrations, whichever is
smaller.
[0029] The adaptability can be expressed as the inverse value of
the p* value. This specific p* value is typically higher than 50%,
often is around 60% and preferably is 57% of P.sub.max-value.
[0030] Further objectives and advantages of the instant invention
will become apparent from the following description of preferred
embodiments, which include a best mode preparation.
[0031] In the present description, the general terms employed
hereinbefore and hereinafter have the following meanings.
[0032] The term "aggregate" denotes a group of more than just a few
amphipats of similar or different kind. A small aggregate, as used
in the context of this invention, has an aggregation number
n.sub.a>3, that is, contains at least 3 molecules, but does not
exceed n.sub.a<5000 or more preferably n.sub.a<1000, that is,
contains no more than 5000 or 1000 molecules. The "extended surface
aggregate (ESA)", "an aggregate with extended surface", a "vesicle"
or an "extended surface" as used in the context of this invention,
all have aggregation number.gtoreq.5000, that is, contain a minimum
of 5000 molecules, and most often are characterized by an even
higher aggregation number, that is, contain an even higher number
of molecules. Preferred ESAs have aggregation numbers of
n.sub.a>10000 and even more preferably n.sub.a>50000. For a
preparation containing aggregates, the reference will always be
made to the average aggregation number or to the average number of
molecules per aggregate, except if indicated otherwise. The term
"aggregation number" equals the number of molecules which together
form an aggregate. Corresponding methods of n.sub.a determination
are well known in the art.
[0033] When a lipid aggregate is water filled and surrounded with
at least one membrane it is called a lipid vesicle. The membrane as
defined in this description is a mixture of at least three
amphipats (MFC+MDC.sub.1+MDC.sub.2) preferably in the form of a
bilayer; a membrane destabilising component hereby is potentially a
MFC-MDC combination (i.e. a mixed amphipat associate).
[0034] The aggregates of the invention are coated with one half,
one, or several bilayers. These may also be called mixed amphipat
coating(s), and correspond to a lipid monolayer, bilayer or
oligo-layers, respectively.
[0035] For a solid aggregate with the surface comprising only one
layer of molecules (a monolayer), the aggregate surface
S.sub.aggegate is given by the product of aggregate number and the
exposed single molecule surface S.sub.molecule:
S.sub.aggregate=n.sub.aS.sub.molecule
[0036] S.sub.molecule can either be measured directly, e.g. in a
Langmuir trough or with diffractometric or reflectometric method,
or else can be calculated with any suitable computer model (e.g.
HyperChem).
[0037] An aggregate with a bilayer coating has only half as large
surface area:
S.sub.aggregate(bilayer, n.sub.a)=0.5 S.sub.aggregate(monolayer,
n.sub.a).
[0038] "Aggregate radius" n.sub.a for a spherical aggregate is
proportional to the square root of the aggregate surface:
r.sub.aggregate=(S.sub.aggregate/4/4.sup.{circle over
(6)}).sup.0.5
[0039] other aggregate geometries requiring appropriate formula
adaptation.
[0040] A "barrier" in the context of this invention is (as in, for
example, EP 0 475 160 and WO 98/17255) a body with
through-extending narrow pores, such narrow pores having a radius
which is at least 25% smaller than the radius of the ESAs
(considered as spherical) before said ESAs permeate through such
pores.
[0041] The term "narrow" used in connection with a pore implies
that the pore radius is significantly, typically at least 25%,
smaller than the radius of the entity tested with regard to its
ability to cross the pore. The necessary difference typically
should be greater for the narrower pores. Using 25% limit is
therefore quite suitable for >150 nm diameter whereas >100%
difference requirement is more appropriate for the smaller systems,
e.g. with <50 nm diameter. For diameters around 20 nm, aggregate
diameter difference of at least 200% is often required.
[0042] The term "semipermeable" used in connection with a barrier
implies that a solution can cross transbarrier openings whereas a
suspension of non-adaptable aggregates (large enough for the above
definition of "narrow" pores to apply) cannot. Conventional lipid
vesicles (liposomes) made from any common phosphatidylcholine in
the gel lamellar phase or else from any biological
phosphatidylcholine/cholesterol 1/1 mol/mol mixture or else
comparably large oil droplets, all having the specified relative
diameter, are three examples for such non-adaptable aggregates.
[0043] The term "stable" means that the tested aggregates do not
change their diameter spontaneously or under the transport related
mechanical stress (e.g. during passage through a semipermeable
barrier) unacceptably, which most often means only to a
pharmaceutically acceptable degree. A 20-40% change is normally
considered acceptable; the halving or doubling of aggregate
diameter is borderline and a greater change in diameter is
typically unacceptable. Alternatively and very conveniently, the
change in aggregate diameter resulting from pore crossing under
pressure is used to assess system stability; the same criteria are
then applied as for "narrow" pores, mutatis mutandis. To obtain the
correct value for aggregate diameter change, a correction for
flux/vortex effects may be necessary. These procedures are
described in greater detail in the publication of the applicant in
Cevc G., Schtzlein A., Richardsen H. (2002) Ultradeformable Lipid
Vesicles Can Penetrate the Skin and other Semi-Permeable Barriers
Intact. Evidence from Double Label CLSM Experiments and Direct Size
Measurements. Biochim. Biophys. Acta 1564:21-30.
[0044] The term "barrier transport resistance" describes the
resistance of a given barrier to the transport of a given fluid
with or without suspended aggregates. Mathematically speaking, this
resistance is given by the ratio of transport driving pressure and
of transport rate (=flow): resistance=delta p/j.sub.a. In more
qualitative terms, used in some of the examples in this document,
barrier resistance is identified with the total fluid volume that
can be filtered through a given barrier by certain pressure within
given time. Alternatively the pressure needed to achieve certain
flux can be used to describe functionally barrier resistance.
[0045] Barrier transport resistance generally decreases linearly
with the number and total area of pores in the given transport
obstacle. For relatively small pores the resistance value can also
depend on average pore diameter, mainly due to friction/viscosity
effects. In addition to this, barrier transport resistance is
sensitive to transported fluid/suspension characteristics and thus
strongly depends on the suspended particle adaptability and
sometimes concentration. In first approximation, this later
sensitivity is due to elastic and viscous loss during
transport.
[0046] The term aggregate "adaptability" which governs the
"tolerable surface curvature" is defined as the ability of a given
aggregate to change easily, and essentially reversibly, its
properties, such as shape, elongation ratio, and surface to volume
ratio. Essential for this invention is the adjustment of aggregate
shape and properties to the anisotropic stress caused by pore
crossing. Sufficient adaptability implies that an aggregate is able
to sustain different unidirectional forces or stress, such as
pressure, without significant fragmentation, which defines a
"stable" aggregate. If an aggregate passes through a barrier
fulfilling this condition the terms "adaptability" and (shape)
"deformability" plus "permeability" are essentially equivalent.
[0047] Non-destructing passage of ultradeformable, mixed lipid
aggregates through narrow pores in a semi-permeable barrier is thus
diagnostic of high aggregate adaptability. If pore radius is two
times smaller than the average aggregate radius the aggregate must
change its shape and surface-to-volume ratio at least 100% to pass
without fragmentation through the barrier. An easy and reversible
change in aggregate shape inevitably implies high aggregate
deformability and requires large surface-to-volume ratio
adaptation. A change in surface-to-volume ratio per se implies: a)
high volume compressibility, e.g. in the case of compact droplets
containing material other than, and immiscible with, the suspending
fluid; b) high aggregate membrane permeability, e.g. in the case of
vesicles that are free to exchange fluid between inner and outer
vesicle volume.
[0048] Measuring capability of given aggregate suspension to cross
a semi-permeable barrier with narrow pores thus offers simple means
for functionally testing aggregate adaptability, as is described in
Practical Examples. This capability for suspensions of sufficiently
stable aggregates is inversely proportional to the effective
barrier transport resistance and, in the first approximation, to
vesicle adaptability a.sub.v.eta.a.sub.a (subscripts v and a
denoting vesicle and aggregate, respectively). If no other
adaptability value is available, the inverse value of barrier
transport resistance or 1/p* value, which are defined further in
the text, can be used to characterise adaptability of aggregates in
a suspension.
[0049] The adaptability of a vesicle-like aggregate depends on
reversible vesicle membrane permeability and deformability. Lipid
bilayer permeability can be assessed by the well-established
methods, such as the osmotic swelling method that is described in
many scientific papers and in Phospholipids Handbook, edited by G.
Cevc for Marcel Dekker Publishers (New York, 1993). Less directly
and quantitatively, but still telling, vesicle bilayer permeability
can be checked by comparing the average aggregate diameter before
and after pore crossing: vesicle bursting and fragmentation is
indicative of aggregate membrane impermeability. In case of lipid
vesicles, the latter is identical to lipid bilayer impermeability.
Open membrane deformability is governed by lipid bilayer
flexibility. This quantity is proportional to bilayer bending
elasticity and is hence determined by the elastic membrane bending
modulus=the elastic curvature modulus of a bilayer=B. The latter
parameter can be measured with several methods known in the art,
including pipette aspiration measurements, vesicle shape or
fluctuation analysis, bilayer deformation under stress in an atomic
force microscope, etc. Bilayer curvature elastic energy density of
a vesicle with radius r.sub.ves is given by B/2r.sub.ves.sup.2,
which shows that most elastic/flexible bilayers, with smallest
B-values, are most deformable. For phosphatidylcholine bilayers in
the fluid lamellar phase B-value is typically of the order
10.sup.-19 J. This value is at least one order of magnitude higher
than the corresponding value determined for a suitable MDC-MFC or
MDC.sub.1-MDC.sub.2-MFC mixture, which is B.about.5 10.sup.-17 J.
This explains why the described three-component amphipat mixtures
form very flexible bilayers and highly deformable vesicles.
[0050] It is important to realise that any system property that
tends to lower aggregate shape adaptability also lowers the
likelihood for aggregate motion through the pores with a radius
smaller than the average aggregate radius. Incorporation of large
incompressible bodies (e.g. oil droplets) into or between the
shape-deformable aggregates therefore lowers, if not blocks,
trans-barrier transport. Incompressibility of aggregate core has
similarly negative effect. Aggregates in the form of (lipid)
vesicles suspended in and filled with nearly incompressible water
must therefore expel some water from vesicle interior during
aggregate deformation to attain high/maximum adaptability.
Introduction of membrane stiffening agents (including cholesterol
and other sterols, little polar long chain lipids, etc., as
quasi-MFC) into bilayers also lowers the adaptability of the
resulting mixed aggregates. Vesicle-like aggregates with many
bilayer coatings (=membranes) are also relatively non-adaptable
(i.e. have lower a.sub.a value, as defined further in the text) and
must be pushed with a higher force (i.e. have a higher p* value, as
defined further in the text) through narrow pores than the
aggregates with just a few or only one such coating(s). The reasons
for this are obvious: in the simplest approximation, aggregate
adaptability is inversely proportional to the number of bilayers
enshrining liquid core of an aggregate. Further system changes that
negatively impact on aggregate adaptability can be analysed in
similar fashion.
[0051] If a vesicle can pass through a narrow pore without
irreversibly adjusting its diameter to the pore diameter within 50%
or even 100% uncertainty range, the vesicle bilayer membrane under
terms of this document is declared to be permeable as well as
flexible. To assess lipid aggregate adaptability it is therefore
useful to employ another aspect of the invention, by using the
following method:
[0052] 1) measure the flux j.sub.a of aggregate suspension through
a semi-permeable barrier (e.g. gravimetrically) for different
transport-driving trans-barrier pressures delta p;
[0053] 2) calculate the pressure dependence of barrier
penetrability P for the given suspension by dividing each measured
flux value with the corresponding driving pressure value: P (delta
p)=j.sub.a (delta p)/delta p;
[0054] 3) monitor the ratio of final and starting vesicle diameter
2r.sub.ves (delta p)/2r.sub.ves,0 (e.g. with the dynamic light
scattering), wherein 2r.sub.ves (delta p)/ is the vesicle diameter
after semi-permeable barrier passage driven by delta p and
2r.sub.ves,0 is the starting vesicle diameter, and if necessary
making corrections for the flow-rate effects;
[0055] 4) align both data sets P (delta p) vs. r.sub.ves (delta
p)/r.sub.ves,0, to determine the co-existence range for high
aggregate adaptability and stability; it is also useful, but not
absolutely essential, to parameterise experimental penetrability
data within the framework of Maxwell-approximation in terms of the
necessary pressure value p* and of maximum penetrability value
P.sub.max, which are defined graphically in the following
illustrative schemes.
[0056] FIGS. 1 to 4 illustrate schematically the physical and
molecular principles underlying the abovementioned approach and the
mathematical model used to analyse the corresponding experimental
data.
[0057] It is plausible to sum-up all the contributions to a moving
aggregate energy (deformation energy/ies, thermal energy, the
shearing work, etc.) into a single, total energy. The equilibrium
population density of aggregate's energetic levels then may be
taken to correspond to Maxwell's distribution. All aggregates with
a total energy greater than the activation energy, E f E.sub.A, are
finally concluded to penetrate the barrier. The pore-crossing
probability for such aggregates is then given by: 1 P ( e ) = 1 -
erf ( 1 e ) + 4 e exp [ - 1 e ] ,
[0058] e being dimensionless aggregate energy in units of the
activation energy EA.
[0059] It is therefore plausible to write barrier penetrability to
a given suspension as a function of transport driving pressure
(=driving pressure difference) p (=delta p) as: 2 P ( p ) = p max {
1 - erf ( p * p ) + 4 p * p exp [ - p * p ] } (* )
[0060] P.sub.max is the maximum possible penetrability of a given
barrier. (For the aggregates with zero transport resistance this
penetrability is identical to the penetrability of the suspending
medium flux.) p* is an adjustable parameter that describes the
pressure sensitivity, and thus the transport resistance, of the
tested system. (For barriers with a fixed pore radius this
sensitivity is a function of aggregate properties solely. For
non-interacting particles the sensitivity is dominated by aggregate
adaptability, allowing to make the assumption: a.sub.a proportional
to 1/p*.)
[0061] In a presently preferred embodiment of the invention, the
experimental approach to quantitative aggregate adaptability
determination is to identify vesicle adaptability value with the
inverse pressure difference needed to attain certain predefined,
practically relevant fraction of maximum achievable flux-pressure
ratio with the vesicle suspension; using 50-60% maximum
penetrability criterion (P.sub.max) gives reasonable results.
Specifically, all p* values given in this document correspond to
57% of P.sub.max-value. Adaptability value, up to an uninteresting
constant, is then given by the inverse value of the p* value that
corresponds to 57% of the P.sub.max-value.
[0062] By making a few more reasonable suppositions one can use the
experimentally determined p*-value to calculate the activation
energy E.sub.A for transbarrier transport of adaptable vesicular
aggregates. The dominant energetic contribution to the work of
bilayer deformation--bilayer elastic energy; bilayer
permeabilisation energy, as the case may be--can then be deduced
from E.sub.A-value. Finally, bilayer elastic energy can be
translated into bilayer curvature elastic energy density, which
depends on the elastic curvature modulus of bilayer, B, as is
explained earlier in the text. Bilayer permeabilisation energy
independently can be related to the work needed to break a bilayer
membrane, and thus to bilayer lysis tension, assuming that elastic
energy is much smaller than membrane permeabilisation energy. For
simple lipid vesicles this has been done by the group of B. Frisken
(cf. Biophys. J. 74: 2996-3002 (1998) and Langmuir 16: 928-933
(2000)), amongst others. Such detailed analysis is not necessary
for optimising aggregate suspensions for transbarrier transport,
however, and therefore is not used in the present application.
[0063] The "liquid suspending medium" or "liquid medium" or
"suitable liquid medium" is well known and is defined in EP 0 475
160 and in WO 98/17255.
[0064] An "amphipat" (or an amphipatic component) is any substance
capable of forming an ESA or of modifying the adaptability of an
ESA, when brought into contact with the liquid suspending
medium.
[0065] For the broadest definition, the amphipats are divided into
two subgroups, the "membrane forming compounds" (MFCs) or "surface
building" or "extended surface-forming or "surface-supporting
substance", which are capably of forming extended surface
aggregates (ESAs), and "the membrane destabilising compounds"
(MDCs). The latter typically render the ESAs formed by the MFCs
more adaptable.
[0066] In some aspects the three amphipatic compounds, one MFC and
two MDCs forming the ESAs are then defined that the MFC alone forms
ESAs, the one MDC alone forms small aggregates, the other MDC alone
optionally forms small aggregates and the combination of both MDCs
forms small aggregates, in contact with said liquid suspending
medium. The ESAs and the small aggregates being defined in terms of
aggregation numbers as stated above.
[0067] In some aspects the three amphipatic compounds, one MFC and
two MDCs forming the ESAs are then characterised by their
solubility in the liquid suspending medium. The MFCs are then
defined to be less soluble than the MDCs at least by a factor of 2.
In more preferred embodiments the MFCs are then defined to be less
soluble than the MDCs at least by a factor of 10 and in preferred
embodiments the solubilities of the two MDCs differ at least by a
factor of 2. Alternatively or simultaneously the MFCs are defined
to be less soluble than the MDCs at least by a factor of 10, one
MDC forms aggregates with surfaces that are at least 2 times less
extended than the surfaces of aggregates formed by the MFC and the
other MDC forms aggregates with aggregation numbers at least 10
times smaller than the aggregation numbers of aggregates formed by
the MFC. Yet another possibility is to define MDC as molecules,
which are typically characterised by hydrophilicity-lipophilicity
ratio (HLB) between 10 and 20, even better between 12 and 18 and
most preferred between 13 and 17.
[0068] In some aspects the MFC and MDCs are defined to form in the
combination of one MFC and two different MDCs extended surface
aggregates with surfaces that are at least 50% more extended,
extended meaning larger, on the average than surfaces of aggregates
comprising only the two different MDCs alone, at the same
concentrations and, in case, after adjustment for physico-chemical
effects of the absence of said MFC.
[0069] For some aspects a selection or all definitions at once
apply.
[0070] The amphipats within the meaning of the present invention
comprise the membrane forming substances and the "edge-active
(surface active)" substances also known from EP 0 475 160 and WO
98/17255, but within the limitations defined in the attached
claims.
[0071] The term "drug" means a biologically or therapeutically
active ingredient, e.g. a medicament. Unless indicated otherwise,
the generic names proposed by the world Health Organisation (WHC))
(Recommended International Non-proprietary Names), such as can be
found e.g. in the Merck Index, are used for the drugs, which are
specified in greater detailed further in the text.
[0072] The term "low" used in connection with molecular weight of a
polypeptide means molar mass below 1500 and the term "intermediate"
in similar context implies molar mass between 1500 and 5000.
[0073] The term "lower" used in connection with organic radicals,
for example lower alkyl, lower alkylene, lower alkoxy, lower
alkanoyl, etc., means that such organic radicals, unless expressly
defined otherwise, contain up to and including 7, preferably up to
and including 4, carbon atoms.
[0074] The term "long" used in connection with a fatty residue
attached to a lipid, a surfactant or a drug implies the presence of
10 to 24 carbon atoms in alkyl, alkenyl, alkoxy, alkenyloxy or
acyloxy chains, which individually or together, as the case may be,
bear the class name of "fatty chains". Implicitly included in this
term, but not further specified in detail, are "fatty chains" with
at least one branched or a cyclic, but unpolar or little polar,
segment.
[0075] The use of square brackets in the text relates to molar
concentrations of the substance put between the brackets, except if
indicated otherwise.
[0076] The terms "surface active" and "edge active" relates to the
ability of a certain third compound to change the surface tension
and/or interface tension in systems comprising at least two
compounds forming a surface of interface.
[0077] In this specification the terms "compound", "substance" and
"component" generally indicate a single chemical species, which
needs, however, not to be totally uniform.
DESCRIPTION OF FIGURES
[0078] FIG. 1: Schematic representation of aggregate shape
deformation during pore crossing.
[0079] FIG. 2: Energy level associated with different states of
aggregate deformation that result from an enforced aggregate
passing through a narrow pore in a semi-permeable barrier.
[0080] FIG. 3: Penetrability of a semi-permeable porous barrier to
the suspension of vesicles smaller the average pore diameter in the
barrier as a function of transbarrier pressure which drives the
suspension through the barrier.
[0081] FIG. 4: Molecular redistribution in an aggregate-enshrining
lipid bilayer during aggregate deformation and pore crossing, which
lowers the activation energy for transbarrier transport.
[0082] FIG. 5: Schematic illustration of the role played by
membrane destabilising component(s) on lipid bilayer adaptability.
The effect of relative concentration of the second membrane
destabilising component is shown in inset.
[0083] FIG. 6 shows the effect of changing molar ratio of the
second (Tween80=) and the third (surfactant; SDS) amphipatic system
component, relatively to the first amphipatic system component
(phospholipid; SPC), on the resistance of resulting mixed lipid
suspension to the filtration through a barrier with 0.2 micrometer
pore-diameter (left panel). The starting and final vesicle diameter
was significantly greater than the average pore diameter.
[0084] FIG. 7 exemplifies the effect of a charged biosurfactant,
sodium cholate, in mixtures with another surfactant (Tween 80)
containing phospholipid bilayers on the ability of the resulting
lipid vesicle suspensions to penetrate through a semipermeable
barrier under influence of transbarrier hydrostatic pressure.
Pressure dependence barrier penetrability to three different
suspensions of mixed bilayer vesicles, pushed through narrow pores,
as a function of the second surfactant concentration.
[0085] FIG. 8 illustrates penetrability of the suspensions prepared
as described in examples 143 and 144. The curves were calculated
within the framework of Maxwell's energy distribution model, by
using formula (*).
Detailed description of the Invention
[0086] The invention describes suspensions of complex ESAs with at
least three amphipatic components, one of which is membrane forming
and at least two of which are membrane destabilising, which can be
suspended in a suitable, e.g. pharmaceutically acceptable, polar
liquid medium and loaded with at least one biologically active
compound, which can correspond to one of the amphipats. An
essential characteristic of such, relatively large, aggregates is
the ability to penetrate pores in semi-permeable barriers even when
the pore radius is significantly, i.e. at least 25% and often is
more than 40% or even better more than 50% and most preferably is
more than 70% smaller than the average aggregate radius before
barrier crossing. Another important characteristic of aggregates
introduced in this document is the relatively low concentration of
one of the two membrane destabilising components, which is below
the concentration needed to achieve high aggregate shape
deformability when this component is used for the purpose on its
own. High aggregate deformability is a prerequisite for reaching
practically useful-i.e. sufficiently high-suspension flux through a
barrier, such that approaches in order of magnitude the flux of
suspending medium. The other necessary condition is sufficient
aggregate stability, which ensures that the average aggregate
radius after barrier crossing is still at least 40%, more often is
at least 50% and most typically is at least 100% larger than the
pore radius. High deformability and sufficient stability of
aggregates that can cross semipermeable barriers are sub-summarised
in the term aggregate adaptability, which is parameterised as
a.sub.a. Highly adaptable complex aggregates excel in their ability
to transport active ingredients through semi-permeable barriers,
such as mammalian skin.
[0087] The present invention specially relates to the selection of
one membrane destabilizing amphipatic component of the system such
that can boost the deformability of mixed aggregates supported by
judicious choice of the other system components to the effect of
improving barrier penetration by such aggregates. The invention
also teaches how to select the right total amphipat concentration
of and, in case, amphipat ionisation in mixed aggregate
suspensions. The invention further relates to the preparation and
application of resulting suspensions in pharmaceutical
formulations, with a focus on epicutaneous application on, or less
frequently in, the warm blood creatures.
[0088] We discovered unexpectedly that incorporation of an
additional, suitable amphipatic membrane destabilising component
(MDC.sub.2) in aforementioned bi-component (MFC+MDC,) aggregates
can increase the resulting three-component
(MFC+MDC.sub.1+MDC.sub.2) aggregate adaptability
a.sub.a(MFC+MDC.sub.1+MDC.sub.2)>a.sub.a(MFC+MDC.sub.1) and thus
augments the shape deformability of resulting aggregates. This
lowers the pressure p* needed to drive substantial suspension flux
through a barrier:
p*(MFC+MDC.sub.1+MDC.sub.2)>[p*(MFC+MDC.sub.1). The capability
of said at least three-component aggregates to move through a
semi-permeable barrier is therefore increased. This finding is
surprising taken that the droplets covered by a bi-component
bilayer membrane already have a rather high barrier crossing
ability compared to the droplets enshrined by a simple lipid
bilayer: a.sub.a(MFC+MDC.sub.1)>&- gt;a.sub.a(MFC).
[0089] Apparently, the third aggregate component, which acts as a
second membrane destabilising component, can increase or support
transport-permitting aggregate adaptability beyond normal
expectation:
a.sub.a(MFC+MDC.sub.1+MDC.sub.2)>a.sub.a(MFC+MDC.sub.1) and
a.sub.a(MFC+MDC.sub.1+MDC.sub.2)>a.sub.a(MFC+MDC.sub.2. This is
illustrated in inset to FIG. 5.
[0090] The three-component bilayer membrane comprising a lipid
(MFC), a suitable first surfactant/amphipatic drug (MDC.sub.1) and
a suitable second surfactant/amphipatic drug (MDC.sub.2) may also
require a lower driving pressure to achieve transbarrier transport:
p*(MFC+MDC.sub.1+MDC.sub.2)<p *(MFC+MDC.sub.2). Additionally or
alternatively, a lower total amount of bilayer destabilising second
amphipat may suffice for obtaining sufficiently adaptable
aggregates, such that can cross a semipermeable barrier. The role
of both membrane destabilising compounds is potentially, but not
necessarily quantitatively, interchangeable (cf. FIG. 5).
[0091] Specifically, we found that relative concentration of said
third component, which acts as membrane destabilising amphipat in
the at least quaternary suspension (liquid suspending
medium+MFC+MDC.sub.1+MDC.sub.2 preferably
water+lipid+drug+surfactant) containing aggregates with a high
adaptability, can be kept below the necessary MDC.sub.2, preferably
the surfactant, concentration in a ternary suspension (liquid
suspending medium+MFC+MDC, preferably water+lipid+surfactant)
containing aggregates of similar adaptability:
a.sub.a(MFC+MDC.sub.1+MDC.sub.2).apprxeq.a.sub.a- (MFC+MDC.sub.2)
and [MDC.sub.2].sub.three-component<[MDC.sub.2].sub.bi--
component or
a.sub.a(MFC+MDC.sub.1+MDC.sub.2).apprxeq.a.sub.a(MFC+MDC.sub.- 1)
and [MDC].sub.three-component<[MDC.sub.1].sub.bi-component,
values in square brackets denoting molar membrane component
concentrations. Practical Examples provide several illustrations
for this. In our opinion, this phenomenon reflects a synergy
between the action of two bilayer components, e.g. between both
membrane destabilising constituents (preferably amphipatic drug(s),
surfactant(s); MDC.sub.1, MDC.sub.2). The dependence of
adaptability curve on the magnitude of coupling parameter m,
documented in inset to FIG. 5, supports such notion. We furthermore
suggest that the interacting two membrane destabilizing components
together make said three-component lipid bilayers more permeable
and/or more flexible than the two-component bilayer membranes in
which one of these MDC is lacking. This means that:
a.sub.a([MFC]+[MDC.sub.1]+[MDC.sub-
.2])>a.sub.a([MFC]+[MDC.sub.1]) and
a.sub.a([MFC']+[MDC.sub.1']+[MDC.su-
b.2'])>a.sub.a([MFC']+[MDC.sub.2']), similar concentration
symbols meaning identical membrane component concentration. The
corresponding p* values typically exhibit the inverse behaviour of
a.sub.a values.
[0092] Preferably, the aggregate adaptability fulfils the condition
a.sub.a([MFC]+[MDC.sub.1]+[MDC.sub.2])>a.sub.a([MFC]+[MDC.sub.1],)
and/or
a.sub.a([MFC]+[MDC.sub.1]+[MDC.sub.2]>a.sub.a([MFC]+[MDC.sub.2]-
), wherein the combined molar concentration of both membrane
destabilizing compounds [MDC.sub.1]+[MDC.sub.2] in aggregates
comprising three amphipats (MFC+MDC.sub.1+MDC.sub.2) is equal or
less than the molar concentration of [MDC.sub.1] in the aggregates
that comprise only two amphipats (MFC+MDC.sub.1) and/or is less
than the molar concentration of [MDC.sub.2]in the aggregates
comprising only two amphipats (MFC+MDC.sub.2), at the same molar
concentration of MFC, or the aggregate adaptability fulfils the
condition a.sub.a([MFC]+[MDC.sub.1]+[MDC.sub.2])-
.apprxeq.a.sub.a([MFC]+[MDC.sub.1]) and/or
a.sub.a([MFC]+[MDC.sub.1]+[MDC.-
sub.2]).apprxeq.a.sub.a([MFC]+[MDC.sub.2]), wherein the combined
molar concentration [MDC.sub.1]+[MDC.sub.2] in aggregates
comprising three amphipats (MFC+MDC.sub.1+MDC.sub.2) is less than
the molar concentration of [MDC.sub.1] in the aggregates comprising
only two amphipats (MFC+MDC.sub.1) and/or is less than the molar
concentration of [MDC.sub.2] in the aggregates comprising only two
amphipats (MFC+MDC.sub.2), at the same molar concentration of MFC.
The corresponding p* values typically exhibit the inverse behaviour
of a.sub.a values.
[0093] Therefore, a second membrane destabilizing compound can be
used to form aggregates comprising three amphipates (MFC+2
different MDCs) and thus achieve aggregate adaptability a.sub.a
which is higher than that of an aggregate comprising only two
amphipats (MFC+MDC). Accordingly MDC.sub.1 can be used to increase
the adaptability a.sub.a of an aggregate comprising MFC and
MDC.sub.2, and MDC.sub.2 can be used to increase the adaptability
a.sub.a of an aggregate comprising MFC and MDC.sub.1 by forming an
aggregate comprising three amphipats (MFC+2 different MDCs).
Likewise the second membrane destabilizing compound can be used to
decrease the amount of the first membrane destabilizing compound
which would be necessary to achieve a certain adaptability a.sub.a
when used alone in an aggregate comprising two amphipats.
Accordingly MDC.sub.1 can be used to form an aggregate comprising
three amphipats (MFC+MDC.sub.1 and MDC.sub.2) to lessen the amount
of MDC.sub.2 necessary when used alone in an aggregate comprising
MFC and MDC.sub.2 to achieve a certain adaptability a.sub.a and/or
MDC.sub.2 can be used to form an aggregate comprising three
amphipats (MFC+MDC.sub.1 and MDC.sub.2) to lessen the amount of
MDC.sub.1 necessary when used alone in an aggregate comprising MFC
and MDC.sub.1 to achieve a certain adaptability. Preferably the
second membrane destabilizing compound MDC.sub.1 or MDC.sub.2 is
used to form an aggregate comprising three amphipats
(MFC+MDC.sub.1+MDC.sub.2) whereby the total molar amount of
destabilizing compound necessary to achieve a certain adaptability
of an aggregate comprising two amphipats, one membrane forming
compound and the respective other membrane destabilizing compound
(MFC+MDC.sub.1) or (MFC+MDC.sub.2), is reduced, so
[MDC.sub.1]+[MDC.sub.2] in amphipats comprising
[MFC]+[MDC.sub.1]+[MDC.sub.2] is less than [MDC.sub.1] in amphipats
comprising [MFC]+[MDC.sub.1] and/or [MDC.sub.2] in amphipats
comprising [MFC]+[MDC.sub.2].
[0094] We note that the characteristics listed in previous
paragraph favourably affect the transport of said pluri-component
mixed lipid vesicles through the skin. Simultaneous presence of at
least two bilayer destabilising amphipats in aggregate suspension
based on the lipid that forms stable bilayers is therefore
beneficial for application of corresponding pharmaceutical
formulations on semi-permeable barriers, such as the skin.
[0095] We thus unveil a fairly general, previously unknown
phenomenon with great practical and commercial potential. An
example is the transport of drugs across various biological
barriers mediated by the three-component aggregates (typically
vesicles comprising two membrane-destabilising amphipats) in said
at least quaternary mixture. The requirement for this is the
capability of complex aggregates to cross pores with a radius at
least 25% smaller than the average aggregate radius before passage
through the pores. The pores can also be part of the pathway
through the skin, which makes said at least quaternary mixtures
suitable for transdermal drug delivery. Quaternary mixtures
containing at least one polar, but poorly soluble lipid (which on
its own forms extended aggregates) and at least two relatively
highly soluble amphipats (surfactants/drugs, which tend to
destabilise the aforementioned lipid bilayer), consequently can
improve drug transport into the body of warm blood creatures.
[0096] Most drugs are amphipatic. Many such molecules, especially
in the ionised form, are also edge active and are thus attracted to
the hydrophilic-hydrophobic boundaries. Some drugs may
self-aggregate or at least tend to adsorb to an air-water or
lipid-water interface; this is mainly due to hydrophobic, ionic, or
H-bond interactions between drugs and lipid (aggregates), which can
lead to the creation of weak drug-lipid associates. The solubility
and/or amphipaty of such associates typically are greater than that
of the involved lipid or drug alone. This is the reason why
amphipatic drugs under certain conditions can destabilise or even
permeate and solubilise lipid bilayer membranes. Such drugs then
act as membrane destabilising components (MDC) in the sense of the
present invention, but this is not necessarily the case under all
conditions. Typically, sufficiently high drug solubility and
sufficiently high drug partition coefficient in or binding constant
to a bilayer membrane are both required for the effect. The
specific, suitable value for these two parameters depends on choice
of other system characteristics (pH, salt and its concentration,
lipid concentration, water activity, etc.). The rule of thumb is
that the highest membrane-concentration of the most water-soluble
drug form normally will work best, stability considerations
permitting.
[0097] To solve the above mentioned problems, this invention
describes preparations based on a combination of at least one
first, at least one second, and at least one third amphipatic
component suspended in a suitable liquid medium in the form of
corresponding mixed amphipat aggregates with one or a few
bilayer-like, mixed amphipat coating(s), in which the combination
of all three said components form extended surfaces in contact with
said liquid medium that are at least 50% more extended, on the
average, than the typical surface of the aggregates comprising the
said at least one second and at least one third amphipatic
component alone and the adaptability of extended surface aggregates
comprising all three said amphipatic components to ambient stress
exceeds by at least 20% or by at least twice the standard deviation
of a typical measurement, whichever is smaller, the adaptability of
the aggregates with extended surface that comprises the at least
one first and the at least one second amphipatic component used at
the corresponding concentrations or the adaptability of the
extended surface comprising the at least one first and the at least
one third amphipatic component at corresponding concentrations,
whichever is smaller, for the application, administration or
transport of an active ingredient, which can be one of the three
amphipatic components, especially for biological, medical,
immunological, or cosmetic purposes, into and through the pores in
semi-permeable barriers or other constrictions, such as through the
skin of warm blood creatures or the like.
[0098] In an alternative definition of the described problems
solution, a combination of at least one first, at least one second,
and at least one third amphipatic component suspended in a suitable
liquid medium in the form of mixed amphipat aggregates with one or
a few bilayer-like, mixed amphipat coating(s), and thus with an
extended surface, is used, in which the said at least one first
amphipatic component, on the one hand, and said at least one second
and one third amphipatic components, on the other hand, have at
least 2-times different solubilities in said liquid medium, and
said at least one first substance has a tendency to self aggregate
and is at least 10-times less soluble in said liquid medium than
said at least one second and said one third substance, allowing the
first to form extended surfaces; furthermore, said at least one
second substance is at least 10-times more soluble in said liquid
medium and, on its own, tends to form or supports the formation of
surfaces that are at least 2-times less extended than the surfaces
containing the at least one first substance alone and said at least
one third substance is also at least 10-times more soluble in said
liquid medium than the first substance and may, but needs not, form
self-aggregates with aggregation numbers at least 10-times smaller
than that of self-aggregates of said first substance, and said
extended surfaces comprising said at least one first, at least one
second and at last one third substance, in equilibrium, have at
least 50% greater extended surfaces than the surfaces formed by the
at least one second or one third substance alone and/or both
together, and preferably the aggregates with an extended surface
comprising all three said amphipatic components have adaptability
to ambient stress that exceeds by at least 20% or by at least twice
the standard deviation of a typical measurement, whichever is
smaller, provided that the adaptability of the extended surface
comprising the at least one first and the at least one second
amphipatic component used at the corresponding concentrations or
the adaptability of the extended surface comprising the at least
one first and the at least one third amphipatic component at the
corresponding concentrations, whichever is smaller, all of which
serves the purpose of application, administration or transport of
at least one active ingredient, which can be amongst said three
substances, especially for medicinal or biological purposes, into
and through barriers and constrictions, such as the skin of warm
blood creatures or the like.
[0099] A favourable problem solution relies on use of said extended
surfaces n the form of membrane surfaces.
[0100] Suitable combinations also fulfil the requirements as stated
in previous paragraphs, simultaneously ensuring that the said at
least one second substance increases the flexibility of extended
surfaces comprising said at least one first, at least one second,
and at least one third substance in comparison with the surfaces
formed merely by an at least one first substance or else with the
surfaces formed by at least one first and at least one third
substance.
[0101] Further suitable combinations fulfil the requirements by
ensuring that the said at least one second and one third substance
together increase the permeability of extended surfaces containing
the said at least one first, at least one second, and at least one
third substance, in comparison with the surfaces formed merely by
the at least one first substance or else with the surfaces formed
by at least one first and at least one third substance.
[0102] Combinations, which also fulfil said requirements contain
said at least one second substance such that increases the ability
to tolerate high curvature, as assessed by relative stability of
said aggregates with an extended surface comprising said one first,
said one second and said one third substance against enforced
higher curvature during passing through a constriction with maximum
diameter at least 1.4 times smaller than the average diameter of an
extended surface formed by an at least one first substance
alone.
[0103] When expressed in terms of relative solubilities of
different components, combinations as taught by this application
preferably comprise at least one first substance and the at least
one second substance that differ in solubility in the suspending
medium on the average at least 10-fold. Alternatively, the at least
one second substance and the at least one third substance differ in
solubility in the suspending medium on the average at least
2-fold.
[0104] It is furthermore recommendable to use combinations, as
described in previous paragraphs, characterised by the fact that
the concentration of said at least one second substance used in the
combination with said one first and said one third substance is
below 80% of the concentration that would be needed to render the
aggregates comprising only said one first and said one second
substance as adaptable to ambient stress as the selected
combination of all at least three substances. In preferred
combinations according to the description in penultimate paragraph,
the concentration of said at least one second substance amounts to
at least 0.1% of the relative stated concentration. In further
preferred combination, the concentration of said at least one
second substance amounts to 1-80% of the relative stated
concentration.
[0105] It is also possible to define the combination suitable for
solving the problems described in this application by selecting
relative concentration of said at least one third substance, used
in combination with said one first and said one second substance,
to be above 0.1% of maximum possible concentration of the said at
least one third substance in the system, a) as defined in terms of
the solubility of said third substance in the system or in said at
least three-component aggregates, or else b) as determined by the
negative action of said at least one third substance on the
stability of said at least three-component aggregates. This means
that more than 0.1% of saturating concentration of said third
substance in the at least three component aggregates is preferably
used or else, that the 0.1% limit pertains to maximum possible
concentration of said third substance which results in at least
three-component aggregates to fail to fulfil the necessary
aggregate stability criterion defined previously in the text.
[0106] Furthermore, it is possible to define a suitable combination
by requesting relative concentration of said at least one third
substance used in combination with said one first and with said one
second substance to be between 1% and 99%, more favourably to be
between 10% and 95%, and most preferably to be between 25% and 90%
of maximum possible concentration of said at least one third
substance, a) as defined in terms of the solubility of said third
substance in the system or in said at least three-component
aggregates, or else b) as determined by the detrimental effect of
said at least one third substance on the stability of said at least
three-component aggregates, the qualitative meaning of these
definitions being described in previous paragraph.
[0107] Problem solving amphipat combination preferably contains
between 0.01 weight-% and 50 weight-% dry mass as total mass of all
at least three amphipatic substances, which together form highly
adaptable aggregates with an extended surface. In more preferred
formulations, this mass is selected to be between 0.1 weight-% and
40 weight-%, even more preferably between 0.5 weight-% and 30
weight-% and most preferably between 1 weight-% and 15
weight-%.
[0108] Amphipat combinations designed according to this application
form extended surfaces with a high adaptability, containing said at
least three substances, preferably with an average surface
curvature corresponding to an average radius between 15 nm and 5000
nm. A particularly favoured choice are the systems with extended
highly adaptable surfaces, which contain said at least three
substances, with an average curvature corresponding to an average
radius between 30 nm and 1000 nm, more preferably between 40 nm and
300 nm and most preferably between 50 nm and 150 nm.
[0109] Electrolyte composition and concentration affects the
desirable properties of said amphipat combinations. It is therefore
preferred to select these characteristics of the electrolyte in
which the extended surfaces with at least one first, at least one
second, and at least one third substance are suspended, and which
comprises mono and/or oligovalent ions, so as to attain ionic
strength between I=0.001 and I=1. A more preferred choice yields
ionic strength between I=0.02 and I=0.5 which even more preferably
is selected to be between I=0.1 and I=0.3.
[0110] Proton concentration in the selected electrolyte is an
important parameter in case of ionizable systems. pH value of the
suspending electrolyte therefore preferably should be chosen: a) in
the vicinity of the logarithm of the apparent ionisation constant
(pKa) of said at least one second substance, if the latter is
mono-ionizable, or in the vicinity of such pKa value that maximises
the solubility of said at least one second substance, if the latter
has several ionizable groups, or else b) in the vicinity of pH
optimum for the most rapidly decaying or the otherwise most
sensitive amongst the said at least three substances, if the said
at least one second substance is not ionizable. More specifically,
the pH value of the polar medium in which the extended surfaces
comprising at least one first, at least one second, and at least
one third substance are suspended should be between pH=pKa-3 and
pH=pKa+3, the final pH selection being also affected by said
stability considerations. When a narrower choice is desirable,
fixing electrolyte a) between pH=pKa-1.5 and pH=pKa+2, if said at
least one third substance is more soluble at high pH, and b)
between pH=pKa-2 and pH=pKa+1.5, if said at least one third
substance is more soluble at low pH, is recommendable, the final pH
selection again being subject to stability considerations.
[0111] A preferred solution to outlined problems is the use of said
combinations characterised in that the at least one first
substance, which is less soluble in the liquid medium and/or is the
surface-building substance in the system, is a lipid, in that the
at least one second substance, which is more soluble in the liquid
medium and/or increases the tolerable surface curvature or
adaptability of said extended surface, is a membrane destabilising
amphipat and typically a surfactant, and in that said at least one
third substance is either a biologically active amphipatic
ingredient, which has a capability of its own to increase the
tolerable surface curvature or adaptability of said extended
surface, or else is a different surfactant different from the said
at least second substance. The second and third substance may be
interchanged.
[0112] Some preferred amphipat combinations that can conveniently
be used to solved the outlined problems are favourably arranged in
the form of minute fluid droplets suspended or dispersed in a
liquid, and surrounded by a coating of one or several layers of the
at least one first substance, which is capable of self-aggregation,
and of at least one second substance and of at least one third
substance, which are both amphipatic, provided that a) the former
substance and the latter two substances differ in solubility in a
suitable liquid suspending medium at least 10-fold, or else
provided that b) the average radius of homo-aggregates of the more
soluble amongst the at least one second and third substance or of
hetero-aggregates of the at least one first, the at least one
second and the at least one third substance is smaller than the
average radius of homo-aggregates of said at least-one first
substance, which is the least soluble amongst the three.
[0113] A preferred and practically very useful choice for the at
least one first substance, as defined herein, is a polar or a
non-polar, surface-forming lipid. This lipid is most often capable
of forming bilayer membranes and preferably forms bilayers on its
own. When looked upon from the solubility point of view, such
surface-forming lipid can be dissolved in the liquid suspending
medium e.g. suspension supporting polar medium preferably in a
concentration range between 10.sup.-12 M and 10.sup.-7 M.
[0114] For biological applications it is commendable to select the
at least one first substance forming extended surfaces as described
in this document from the group of lipids, lipoids from a
biological source, corresponding synthetic lipids and biochemical
or chemical modifications, i.e. derivatives, thereof.
[0115] Particularly preferred and attractive in the sense of
previous paragraph is the group comprising glycerides, glycolipids,
glycerophospholipids, isoprenoidlipids, sphingolipids, steroids,
sterines or sterols, sulphur-containing lipids, lipids containing
at least one carbohydrate residue, or other polar fatty
derivatives, which are therefore all suitable candidates for said
at least one first substance that forms said extended surfaces.
More specifically, the selection is made amongst
phosphatidylcholines, phosphatidyl-ethanolamines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids,
phosphatidylserines, sphingomyelins, sphingophospholipids,
glycosphingolipids, cerebrosides, ceramidpolyhexosides,
sulphatides, sphingoplasmalogenes, or gangliosides.
[0116] Said extended surface-forming substance, which solves the
problems outlined in the application, is preferably selected from
the group of lipoids or lipids with one or two, often different,
fatty chains, especially with acyl-, alkanoyl-, alkyl-, alkylene-,
alkenoyl-, alkoxy, or chains with omega-cyclohexyl-,
cyclo-propane-, iso- or anteiso-branched segments, or any other
practically useful aliphatic chain. There is some preference to use
lipids with n-decyl, n-dodecyl (lauryl), n-tetradecyl (myristyl),
n-hexadecyl (cetyl), n-octadecyl (stearyl), n-eicosyl (arachinyl),
n-docosyl (behenyl) or n-tetracosyl (lignoceryl), 9-cis-dodecenyl
(lauroleyl), 9-cis-tetradecenyl (myristoleyl), 9-cis-hexadecenyl
(palmitoleinyl), 9-cis-octadecenyl (petroselinyl),
6-trans-octadecenyl (petroselaidinylj, 9-cis-octadecenyl (oleyl),
9-trans-octadecenyl (elaidinyl), 9-cis-eicosenyl (gadoleinyl),
9-cis-docosenyl (cetoleinyl) or n-9-cis-tetracosoyl (nervonyl),
n-decyloxy, n-dodecyloxy (lauryloxy), n-tetradecyloxy
(myristyloxy), n-hexadecyloxy (cetyloxy), n-octadecyloxy
(stearyloxy), n-eicosyloxy (arachinyloxy), n-docosoyloxy
(behenyloxy) or n-tetracosoyloxy (lignoceryloxy),
9-cis-dodecenyloxy (lauroleyloxy), 9-cis-tetradecenyloxy
(myristoleyloxy), 9-cis-hexadecenyloxy (palmitoleinyloxy),
6-cis-octadecenyloxy, (petroselinyloxy), 6-trans-octadecenyloxy
(petroselaidinyloxy), 9-cis-octadecenyloxy (oleyloxy),
9-trans-octadecenyloxy (elaidinyloxy), and 9-cis-eicosenyl
(gadoleinyloxy), 9-cis-docosenyl (cetoleinyloxy) or
n-9-cis-tetracosoyl (nervonyloxy), n-decanoyloxy, n-dodecanoyloxy
(lauroyloxy), n-tetradecanoyloxy (myristoyloxy), n-hexadecanoyloxy
(palmitoyloxy) I n-octadecanoyloxy (stearoyloxy), n-eicosanoyloxy
(arachinoyloxy), n-n-docosoanyloxy (behenoyloxy) and
n-tetracosanoyloxy (lignoceroyloxy), 9-cis-dodecenyloxy
(lauroleoyloxy), 9-cis-tetradecenoyloxy (myristoleoyloxy),
9-cis-hexadecenoyloxy (palmitoleinoyloxy), 6-cis-octadecenoyloxy
(petroselinoyloxy), 6-trans-octadecenoyloxy (petroselaidinoyloxy),
9-cis-octadecenoyloxy (oleoyloxy),
9-trans-octadecenoyloxyelaidinoyloxy), and 9-cis-eicosenoyloxy
(gadoleinoyloxy), 9-cis-docosenoyloxy (cetoleinoyloxy) and
9-cis-tetracosenoyloxy (nervonoyloxy) or the corresponding
sphingosine derivative chains.
[0117] A preferred suitable solution to the problems outlined
herein are amphipat combinations in which said at least one second
substance is a surface active substance such as a
surfactant/detergent. The latter is preferably selected from the
group comprising nonionic, zwitterionic, anionic and cationic
surfactants. It is preferred to use a surfactant with the
solubility in a liquid suspending medium such as a polar liquid, in
which the extended surfaces are prepared, in the
range5.times.10.sup.-7 M to 10.sup.-2 M.
[0118] A long list of surfactants that qualify for the use in said
quaternary suspensions, and a number of definitions, is given in EP
0 475 160 and U.S. Pat. No. 6,165,500, which are herewith
explicitly included by reference. Valuable source of information on
the substances which qualify in this sense are also different
specialist handbooks, such as Handbook of Industrial Surfactants.
The following list therefore only offers a selection, which is by
no means complete or exclusive, of several surfactant classes that
are particularly common or useful in conjunction with the present
patent application. This includes ionized long-chain fatty acids or
long chain fatty alcohols, long chain fatty ammonium salts, such as
alkyl- or alkenoyl-trimethyl-, -dimethyl- and -methyl-ammonium
salts, alkyl- or alkenoyl-sulphate salts, long fatty chain
dimethyl-aminoxides, such as alkyl- or
alkenoyl-dimethyl-aminoxides- , long fatty chain, for example
alkanoyl, dimethyl-aminoxides and especially dodecyl
dimethyl-aminoxide, long fatty chain, for example
alkyl-N-methylglucamides and alkanoyl-N-methylglucamides, such as
MEGA-8, MEGA-9 and MEGA-10, N-long fatty
chain-N,N-dimethylglycines, for example
N-alkyl-N,N-dimethylglycines, 3-(long fatty
chain-dimethylammonio)-alkane- sulphonates, for example
3-(acyldimethylammonio)-alkanesulphonates, long fatty chain
derivatives of sulphosuccinate salts, such as bis(2-ethylalkyl)
sulphosuccinate salts, long fatty chain-sulphobetaines, for example
acyl-sulphobetaines, long fatty chain betaines, such as EMPIGEN BB
or ZWITTERGENT-3-16, -3-14, -3-12, -3-10, or -3-8, or
polyethylen-glycol-acylphenyl ethers, especially
nonaethylen-glycol-octyl- phenyl ether, polyethylene-long fatty
chain-ethers, especially polyethylene-acyl ethers, such as
nonaethylen-decyl ether, nonaethylen-dodecyl ether or
octaethylene-dodecyl ether, polyethyleneglycol-isoacyl ethers, such
as octaethyleneglycol-isotridecyl ether,
polyethyleneglycol-sorbitane-long fatty chain esters, for example
polyethyleneglycol-sorbitane-acyl esters and especially
polyethylenglykol-monolaurate (e.g. Tween 20),
polyethylenglykol-sorbitan- -monooleate (e.g. Tween 80),
polyethylenglykol-sorbitan-monolauroleylate,
polyethylenglykol-sorbitan-monopetroselinate,
polyethylenglykol-sorbitan-- monoelaidate,
polyethylenglykol-sorbitan-myristoleylate,
polyethylenglykol-sorbitan-palmitoleinylate,
polyethylenglykol-sorbitan-p- etroselinylate,
polyhydroxyethylene-long fatty chain ethers, for example
polyhydroxyethylene-acyl ethers, such as polyhydroxyethylene-lauryl
ethers, polyhydroxyethylene-myristoyl ethers,
polyhydroxyethylene-cetylst- earyl, polyhydroxyethylene-palmityl
ethers, polyhydroxyethylene-oleoyl ethers,
polyhydroxyethylene-palmitoleoyl ethers, polyhydroxyethylene-lino-
leyl, polyhydroxyethylen-4, or 6, or 8, or 10, or 12-lauryl,
miristoyl, palmitoyl, palmitoleyl, oleoyl or linoeyl ethers (Brij
series), or in the corresponding esters,
polyhydroxyethylen-laurate-, -myristate, -palmitate, -stearate or
-oleate, especially polyhydroxyethylen-8-stearat- e (Myrj 45) and
polyhydroxyethylen-8-oleate or -8-laurate, polyethoxylated castor
oil 40 (Cremophor EL), sorbitane-mono long fatty chain, for example
alkylate (Arlacel or Span series), especially as
sorbitane-monolaurate (Arlacel 20, Span 20) or -monooleaate a long
fatty chain, for example acyl-N-methylglucamide,
alkanoyl-N-methylglucamide, especially decanoyl-N-methylglucamide,
dodecanoyl-N-methylglucamide or octadecenoyl-N-methylglucamide;
also suitable are long fatty chain sulphates, for example
alkyl-sulphates, alkyl sulphate salts, such as lauryl-sulphate
(SDS), oleoyl-sulphate; long fatty chain thioglucosides, such as
alkylthioglucosides and especially heptyl-, octyl- nonyl- and
decyl-beta-D-thioglucopyranoside; long fatty chain derivatives of
various carbohydrates, such as pentoses, hexoses and disaccharides,
especially alkyl-glucosides and maltosides, such as hexyl-,
heptyl-, octyl-, nonyl- and decyl-beta-D-glucopyranoside or
-D-maltopyranoside; further salts, especially sodium or potasium
salts, of cholate, deoxycholate, glycocholate, glycodeoxycholate,
taurodeoxycholate, taurocholate; corresponding fatty acid salts,
especially oleate, elaidate, linoleate, laurate, or myristate;
furthermore lysophospholipids, such as
n-octadecylene-glycerophosphatidic acid,
octadecylene-phosphorylglycerol, octadecylene-phosphorylserine, or
-phosphatidylcholine, n-long fatty chain-glycero-phosphatidic
acids, such as n-acyl-glycero-phosphatidic acids, especially lauryl
glycero-phosphatidic acids, oleoyl-glycero-phosphatidic acid,
n-long fatty chain-phosphorylglycerol, such as
n-acyl-phosphorylglycerol, especially lauryl-, myristoyl-, oleoyl-
or palmitoeloyl-phosphorylglycerol, n-long fatty
chain-phosphorylserine, such as n-acyl-phosphorylserine, especially
lauryl-, myristoyl-, oleoyl- or palmitoeloyl-phosphorylserine,
n-tetradecyl-glycero-phosphatidic acid,
n-tetradecyl-phosphorylglycerol, n-tetradecyl-phosphorylserine,
corresponding -, elaidoyl-, vaccenyl-lysophospholipids,
corresponding short-chain phospholipids, as well as all surface
active and thus membrane destabilising polypeptides.
[0119] For the solution of problems addressed by the application,
charge-charge or charge-polar headgroup interactions amongst the
involved amphipats may be important. If so, the following
consideration can be made: if the at least one second substance is
charged the at least one third substance can be is uncharged and if
the at least one second substance is uncharged the at least one
third substance ideally should be charged; similar preference of
combinations is also possible for the said at least one first and
one second or for the said at least one first and one third
substance, respectively. When at least one charged amphipat is used
to prepare aggregates with at least three different components, the
extended aggregate surface, formed by the at least one first, one
second and one third substance, at least one of which is charged,
is preferably chosen to contain between 1% and 75% of the charged
component. An even more favourable choice is to use combinations of
at least one first, one second and one third substance, at least
one of which is charged, that contain between 5% and 50% of the
charged component and most preferably between 10% and 30% of the
charged component.
[0120] In some cases it is preferred to use combinations according
to claims of this application such that contain a
phosphatidylcholine, a phosphatidylethanolamine-N-mono- or
N-di-methyl, phosphatidic acid or its methyl ester,
phosphatidylserine and/or phosphatidylglycerol as the
surface-supporting at least one first substance and a
lysophospholipid, especially a lysophosphatidic acid,
lysomethylphosphatidic acid, lysophosphatidylglycerol,
lysophosphatidylcholine, a partially N-methylated
lysophosphatidylethanolamine, or else a monovalent salt of cholate,
deoxycholate, glycocholate, glycodeoxycholate, or a sufficiently
polar sterol-derivative, or a suitable salt form of laurate,
myristate, palmitate, oleate, palmitoleate, elaidate or some other
pharmaceutically acceptable long-chain fatty acid salt and/or a
Tween-, a Myrj-, or a Brij-surfactant with said aliphatic chains,
or a Triton, a long-chain fatty sulphonate, -sulphobetaine,
--N-glucamide or -sorbitane (Arlacel or Span) surfactant, any of
which can take the role of the at least one second or of at least
one third substance, as the case may be, such second/third
substance on its own forming less extended surfaces than the at
least one first substance on its own.
[0121] Preferred combinations that conveniently solve the outlined
problems may alternatively contain a biologically active amphipat,
which can destabilise lipid membranes, as the least one second or
one third substance, as the case may be, unless a surfactant
different from the at least one second substance or one third
substance, but otherwise selected from similar surfactant classes,
is selected for the purpose.
[0122] As a useful rule of the thumb, which can be applied to
select a suitable at least one third or second substance, is
preferably to select the solubility of such substance in the liquid
suspending medium, such as a polar liquid, to be between 10.sup.-6
M and 1 M.
[0123] For some embodiments it is preferred to seek such molecule
taking the role of at least one third or second amphipat that
adsorbs to the surface of lipid bilayers but is also well miscible
with or reasonably soluble in the polar liquid in which said
extended lipid bilayer surfaces are formed.
[0124] It is furthermore preferred, and practically useful, to use
such drug or drug form that can take the role of as the at least
one third or second substance, as the case may be, especially when
this role is not taken by the at least one first and/or the at
least one second or third substance, respectively. If so, such
ionisation or salt form of the drug is chosen that serve the
purpose best. To the effect, the bulk pH, electrolyte composition
and concentration value, and in case of need also co-solvents
including different short chain alcohols or other short chain polar
amphipats are selected appropriately.
[0125] Drugs suitable for solving the problems sketched in this
work can belong to the class of substituted ammonium compounds of
the formula 1
[0126] in which a) Ra represents a hydrophobic group, and Rb, Rc,
and Rd, independently of one another, each represents hydrogen,
C1-C4-alkyl, 2-hydroxyethyl, allyl or
cycle-C3-C6-alkyl-Cl--C3-alkyl, or two of the radicals Rb, Rc and
Rd together represent C4- or C5-alkylene optionally interrupted by
--HN--, --N(C1-C4-alkyl)-, --N(2-hydroxyethyl)- or by oxygen, or;
b) Ra and Rb are two hydrophobic groups or together represent a
hydrophobic group, and Rc and Rd, independently of one another,
each represents hydrogen, C1-C4-alkyl, allyl or
cyclo-C3-C6-alkyl-C1-C3-alkyl, or c) Ra, Rb and Rc together
represent a hydrophobic group, and Rd, represents hydrogen or
C1-C4-alkyl, and A.sup.- represents the anion of a pharmaceutically
acceptable acid, as a carboxylic acid salt of the formula
Ra-COO.sup.-Y.sup.+ (2)
[0127] Ra representing a hydrophobic group and Y+representing the
cation of a pharmaceutically acceptable base,
[0128] as an alpha-amino acid compound of the formula 2
[0129] In the above formula 3, Ra represents a hydrophobic group
and Rb and Rc, independently of one another, each represents
hydrogen or C1-C4-alkyl, as a phosphoric acid monoester of the
formula 3
[0130] wherein Ra represents a hydrophobic group and Y+represents
the cation of a pharmaceutically acceptable base, or as an acid
addition salt of a compound having a hydrophobic group Ra and an
imidazoline, imidasolidine or hydrasino group as hydrophilic
group.
[0131] In a substituted ammonium compound of the formula 1 that can
be used as a medicament, in case a) the hydrophobic group Ra is an
aliphatic hydrocarbon radical that can be interrupted by an oxygen
or sulphur atom, may contain the groups --CO(.dbd.O)--,
--O--C(.dbd.O)--, --C(.dbd.O)--NH--, --O--C(.dbd.O)--NH-- or
hydroxy, and can be substituted by from 1 to 3 optionally
substituted, mono-cyclic, aliphatic or aromatic hydrocarbon
radicals, by an optionally substituted, bi- or tri-cyclic, aromatic
or partially saturated hydrocarbon radical, by an optionally
substituted, monocyclic, aromatic, partially saturated or saturated
heterocycle or by an optionally substituted, bi- or tri-cyclic,
aromatic, partially saturated or benzo-fused heterocycle.
[0132] The hydrophobic group Ra can also be an optionally
substituted, monocyclic, aliphatic or aromatic hydrocarbon radical
or a bicyclic, aliphatic or benzo-fused hydrocarbon radical. The
hydrophilic group is, for example, a group of the formula 4
[0133] wherein Rb, Rc, and Rd, independently of one another, each
represents hydrogen, C.sub.1-C.sub.4-alkyl, for example methyl,
ethyl, isopropyl or n-propyl, or 2-hydroxyethyl, or in which two of
the radicals Rb, Rc, and Rd together represent piperidino,
piperazinyl, 1-methylpiperazinyl, 1-(2-hydroxyethyl)-piperazinyl or
morpholino, and the other radical represents hydrogen.
[0134] In a substituted ammonium compound of the formula 1 that can
be used as a medicament, in case b) Ra and Rb are two hydrophobic
groups, for example two aliphatic hydrocarbon radicals, which can
be substituted by one or two optionally substituted, monocyclic,
aliphatic or aromatic hydrocarban radicals or by an optionally
substituted, monocyclic, aromatic, partially saturated or saturated
heterocycle, or Ra and Rb together represent an optionally
substituted, monocyclic, aromatic, saturated, partially saturated
or benzo-fused heterocycle. The hydrophilic group is a group of the
formula 5
[0135] in which Rc and Rd, independently of one another each
represents hydrogen or C1-C4-alkyl, preferably methyl.
[0136] In a substituted ammonium compound of the formula 1, which
can be used as a medicament, in case c) Ra, Rb, and Rc form the
hydrophobic group and together represent an optionally substituted,
aromatic, partially saturated or benzo-fused heterocycle. The
hydrophilic group is a group of the formula 6
[0137] in which Rd represents hydrogen or C1-C4-alkyl, preferably
methyl.
[0138] A.sup.- is the anion of a pharmaceutically acceptable acid,
for example a mineral acid, for example the chloride, hydrogen
sulphate or dihydrogen phosphate ion, the bromide or iodide ion, or
the anion of an organic acid, for example a lower alkanecarboxylic
acid, for example the acetate ion, of an unsaturated carboxylic
acid, for example the fumarate or maleate ion, of a hydroxy acid,
for example the lactate, tartrate or citrate ion, or of an aromatic
acid, for example the salicylate ion.
[0139] In a carboxylic acid salt of the formula 2, which can be
used as a medicament, the hydrophobic group Ra is an aliphatic
hydrocarbon radical that can be substituted by an optionally
substituted, monocyclic, aromatic hydrocarbon radical or by an
optionally substituted, bi- or tri-cyclic, aromatic or partially
saturated hydrocarbon radical, by an optionally substituted,
monocyclic, aromatic or partially saturated heterocycle or by an
optionally substituted, bi- or tri-cyclic, aromatic, partially
saturated or benzo-fused heterocycle or by a steroid radical, or Ra
is an optionally substituted, monocyclic, aromatic hydrocarbon
radical, an optionally substituted, bi- or tri-cyclic, aromatic or
partially saturated hydrocarbon radical, an optionally substituted,
monocyclic, aromatic or partially saturated heterocycle or an
saturated or benzo-fused heterocycle.
[0140] The cation Y.sup.+ of a pharmaceutically acceptable base is,
for example, an alkali metal ion, for example a lithium, sodium or
potassium ion, an alkaline earth metal ion, for example a magnesium
or calcium ion, or an ammonium or mono-, di- or
tri-C1-C4-alkylammonium ion, for example a trimethyl-, ethyl-,
diethyl- or triethyl-ammonium ion, a
2-hydroxyethyl-tri-C1-C4-alkylammonium ion, for example cholinyl,
or the cation of a basic amino acid, for example lysine or
arginine.
[0141] Carboxylic acid salts of the formula 2 having biological
activity or carboxylic acids that can be converted into them by
salt formation are, for example, salts of glucocorticoids that are
esterified in the 21-position by a dicarboxylic acid, for example
methylprednisolone sodium succinate, prednisolone sodium succinate;
short-term narcotics of the 3,20-dioxo-5.beta.-pregnane type that
can be esterified by succinic acid, for example hydroxydione
succinate sodium or 11,20-dioxo-3alpha-hydroxy-5- alpha-pregnane,
for example alphaxolone, or the 21-compound, for example
alphadolone; salts of choleritics, for example cholic acid salts or
deoxycholic acid salts; analgesics, for example salts of
substituted phenylacetic acids or 2-phenylpropionic acids, for
example alclofenac, ibufenac, ibuprofen, clindanac, fenclorac,
ketoprofen, fenoprofen, indoprofen, fenclofenac, diclofenac,
flurbiprofen, pirprofen, naproxen, benoxaprofen, carprofen or
cicloprofen; analgesically active anthranilic acid derivatives, for
example of the formula optionally substituted, bi- or tri-cyclic,
aromatic, 7
[0142] in which R1, R2 and R3 independently of one another, each
represents hydrogen, methyl, chlorine or trifluoromethyl, for
example mefenamic acid, flufenamic acid, tolfenamic acid or
meclofenamic acid; analgesically active anilino-substituted
nicotinic acid derivatives, for example miflumic acid, clonixin or
flunixin; analgesically active heteroarylacetic acids or
2-heteroarylpropionic acids having a 2-indol-3-yl or pyrrol-2-yl
radical, for example indomethacin, oxmetacin, intrazol, acemetazin,
cinmetacin, zomepirac, tolmetin, colpirac or tiaprofenic acid;
analgesically active indenylacetic acids, for example sulindac;
analgesically active heteroaryloxyacetic acids, for example
benzadac, prostanoic acids that stimulate the smooth musculature,
for example PGE2 (dinoprostone), PGF2alpha (dinoprost), 15
(S)-15-methyl-PGE2, 15 (S)-15-methyl-PG F2alpha, (carboprost),
(!)15 (Xi)-15-methyl-13,14-dihydro-11-deoxy-PGE1 (deprostil),
15(S)-15-methyl-11-deoxy-PGE 1 (doxaprost), 16,16-dimethyl-PGE2,
17-phenyl-18,19,20-trinor-PGF2alpha,
16-phenoxy-17,18,19,20-tetranor-PGF2- alpha, for example
cloprostenol or fluprostenol, or
N-methylsulphonyl-15-phenoxy-17,18,19,20-tetranor-PGF2alpha
(sulproston); bacteriostatics, for example salts of nalixidic acid
derivatives, for example salts of nalixidic acid, cinoxacin,
oxolinic acid, pironidic acid or pipenidic acid, penicillanic acid
and cephalosporanic acid derivatives having antibiotic activity
with 6'- or 7.beta.-acylamino groups, which are present in
fermentatively, semi-synthetically or totally synthetically
obtainable 6'-acylamino-penicillanic acid or
7.beta.-acylaminocephalosporanic acid derivatives or
7'-acylaminocephalosporanic acid derivatives modified in the
3-position, for example penicillanic acid derivatives that have
become known under the names penicillin G or V, phenethicillin,
propicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin,
flucloxacillin, cyclacillin, epicillin, mecillinam, methicillin,
azlocillin, sulbenicillin, ticarcillin, meziocillin, piperacillin,
carindacillin, azidocillin or ciclazillin, or cephalosporin
derivatives that have become known under the names cefaclor,
cefuroxime, cefazlur, cephacetrile, cefazolin, cephalexin,
cefadroxil, cephaloglycin, cefoxitin, cephaloridine, cephsulodin,
cefotiam, ceftazidine, cefonicid, cefotaxime, cefinenoxime,
ceftizoxime, cephalothin, cephradine, cefamandol, cephanone,
cephapirin, cefroxadin, cefatrizine, cefazedone, ceftrixon or
ceforanid, and other R-lactam antibiotics, for example moxalactam,
clavulanic acid, nocardicine A, sulbactam, aztreonam or
thienamycin; or antineoplastics having a
4-[bis-(2-chloroethyl)-amino-phenyl]-butyric acid structure, for
example chlorambucil, or antineoplastics having two carboxy grows,
for example methotrexate.
[0143] Compounds of the formula 3 having a biological activity are,
for example, neurotransmitters in which the hydrophobic group is
methyl substituted by hydroxyphenyl, for example L-tyrosine,
L-dopa, alpha-methyldopa or metirosine; thyroid hormones having
iodine-substituted phenyl radicals, for example levo-thyrosine,
diiodotyrosine or liothyronine; or anti-neoplastics having an amino
acid structure, for example melphalen.
[0144] In a compound of the formula 4 having biological activity
the non-polar, hydrophobic group Ra is a glucocorticoid radical and
A+is sodium, for example betamethasone disodium phosphate,
dexamethasone disodium phosphate, cortisone phosphate,
hydrocortisone phosphate, prednisolone disodium phosphate or
paramethasone-21-disodium phosphate, Salt-type compounds having a
hydrophobic group and an imidazoline, imidazolidine or hydrazino
group as hydrophilic group are, for example, salts of
anti-depressantly active hydrazine derivatives, for example
iproniazid, nialamide, isocarboxazid, pheneizine, pheniprazine,
mebanazine or fenoxypropazine; a-sympathomimetics having an
imidazoline structure, for example naphazoline, tetryzolin,
tramazoline, xylo-metazoline or oxyinetazoline; n-sympatholytics
having an imidazoline structure, for example phentolamine or
tolazoline, or centrally active antihypertensives having an
imidazoline structure, for example clonidine, tolonidine or
flutonidine; or vasodilatators having a hydrazino group, for
example dihydralazine, hydralazine or picodralazine.
[0145] A phospholipid (II) that is mixed with an amphipatic
compound (I) with surfactant of biological activity, such as a
drug, for example, can have the following formula 8
[0146] in which one of the radicals R1 and R2 represents hydrogen,
hydroxy or C1-C4-alkyl, and the other radical represents a long
fatty chain, especially an alkyl, alkenyl, alkoxy, akenyloxy or
acyloxy, each having from 10 to 24 carbon atoms, or both radicals
R1 and R2 represent a long fatty chain, especially an alkyl,
alkenyl, alkoxy, alkenyloxy or acyloxy each having from 10 to 24
carbon atoms, R3 represents hydrogen or C1-C4-alkyl, and R4
represents hydrogen, optionally substituted C1-C7-alkyl or a
carbohydrate radical having from 5 to 12 carbon atoms or, if both
radicals R1 and R2 represent hydrogen or hydroxy, R4 represents a
steroid radical, or is a salt thereof. The radicals R1, R2, R3, and
R4 are typically selected so as to ensure that lipid bilayer
membrane is in the fluid lamellar phase during practical
application and is a good match to the drug of choice.
[0147] In a phospholipid of the formula 5, R1, R2 or R3 having the
meaning C1-C4-alkyl is preferably methyl, but may also be ethyl,
n-propyl, or n-butyl.
[0148] Alkyl R1 or R2 is preferably straight-chained with an even
number of 10 to 24 carbon atoms, for example n-decyl, n-dodecyl
(lauryl), n-tetradecyl (myristyl), n-hexadecyl (cetyl), n-octadecyl
(stearyl), n-eicosyl (arachinyl), n-docosyl (behenyl) or
n-tetracosyl (lignoceryl).
[0149] Alkenyl R1 and/or R2 is preferably straight-chained with an
even number of 12 to 24 carbon atoms and a double bond, for example
9-cis-dodecenyl (lauroleyl), 9-cis-tetradecenyl (myristoleyl),
9-cis-hexadecenyl (palmitoleinyl), 9-cis-octadecenyl
(petroselinyl), 6-trans-octadecenyl (petroselaidinylj,
9-cis-octadecenyl (oleyl), 9-trans-octadecenyl (elaidinyl),
9-cis-eicosenyl (gadoleinyl), 9-cis-docosenyl (cetoleinyl) or
n-9-cis-tetracosoyl (nervonyl).
[0150] Alkoxy R1 and/or R2 is preferably straight-chained with an
even number of 10 to 24 carbon atoms, for example n-decyloxy,
n-dodecyloxy (lauryloxy), n-tetradecyloxy (myristyloxy),
n-hexadecyloxy (cetyloxy), n-octadecyloxy (stearyloxy),
n-eicosyloxy (arachinyloxy), n-docosoyloxy (behenyloxy) or
n-tetracosoyloxy (lignoceryloxy).
[0151] Alkenyloxy R1 and/or R2 is preferably straight-chained with
an even number of 12 to 24 carbon atoms, for example
9-cis-dodecenyloxy (lauroleyloxy), 9-cis-tetradecenyloxy
(myristoleyloxy), 9-cis-hexadecenyloxy (palmitoleinyloxy),
6-cis-octadecenyloxy, (petroselinyloxy), 6-trans-octadecenyloxy
(petroselaidinyloxy), 9-cis-octadecenyloxy (oleyloxy),
9-trans-octadecenyloxy (elaidinyloxy), and 9-cis-eicosenyl
(gadoleinyloxy), 9-cis-docosenyl (cetoleinyloxy) or
n-9-cis-tetracosoyl (nervonyloxy).
[0152] Acyloxy R1 and/or R2 is preferably straight-chained with an
even number of 10 to 24 carbon atoms, for example alkanoyloxy or
alkenoyloxy, preferably n-decanoyloxy, n-dodecanoyloxy
(lauroyloxy), n-tetradecanoyloxy (myristoyloxy), n-hexadecanoyloxy
(palmitoyloxy) I n-octadecanoyloxy (stearoyloxy), n-eicosanoyloxy
(arachinoyloxy), n-n-docosoanyloxy (behenoyloxy) and
n-tetracosanoyloxy (lignoceroyloxy).
[0153] Alkenoyloxy R1, and/or R2 is preferably straight-chained
with an even number of 10 to 20 carbon atoms, for example
9-cis-dodecenyloxy (lauroleoyloxy), 9-cis-tetradecenoyloxy
(myristoleoyloxy), 9-cis-hexadecenoyloxy (palmitoleinoyloxy),
6-cis-octadecenoyloxy (petroselinoyloxy), 6-trans-octadecenoyloxy
(petroselaidinoyloxy), 9-cis-octadecenoyloxy (oleoyloxy),
9-trans-octadecenoyloxyelaidinoyloxy), and 9-cis-eicosenoyloxy
(gadoleinoyloxy), 9-cis-docosenoyloxy (cetoleinoyloxy) and
9-cis-tetracosenoyloxy (nervonoyloxy).
[0154] Optionally substituted C1-C7-alkyl R4 is, for example,
methyl, ethyl, isopropyl, n-propyl, isobutyl or n-butyl, which can
be substituted by acidic groups, for example, carboxy or sulpho, by
acidic and basic groups, for example, carboxy and amino, the amino
group being in the alpha-position to the carboxy group, by free or
etherified hydroxy groups, it being possible for two etherified
hydroxy groups to be bonded to one another by a bivalent
hydrocarbon radical, for example methylene, ethylene, ethylidene,
1,2-propylene or 2,2-propylene; or by halogen, for example chlorine
or bromine, by lower alkoxycarbonyl, for example methoxy-or
ethoxy-carbonyl, or by lower alkanesulphonyl, for example
methanesulphonyl.
[0155] Substituted C1-C7-alkyl R4 is, for example, carboxy-lower
alkyl, for example carboxymethyl, 2-carboxyethyl or
3-carboxy-n-propyl, (omega-amino-omega-carboxy-lower alkyl, for
example 2-amino-2-carboxyethyl or 3-amino-3-carboxy-n-propyl,
hydroxy-lower alkyl, for example 2-hydroxyethyl or
2,3-dihydroxypropyl, lower alkoxy-lower alkyl, for example methoxy-
or ethoxy-methyl, 2-methoxyathyl or 3-methoxy-n-propyl, lower
alkylenedioxy-lower alkyl, for example 2,3-ethylenedioxypropyl or
2,3-(2,2-propylene)-dioxy-propyl, or halo-lower alkyl, for example
chloro- or bromo-methyl, 2-chloro- or 2-bromo-ethyl, 2- or
3-chloro- or 2- or 3-bromo-n-propyl.
[0156] Substituted C1-C7-alkyl R4 is preferably ethyl substituted
by tri-lower alkylammonium, for example trimethyl- or
triethyl-ammonium, for example 2-trimethylammonium-ethyl or
2-ammonium-ethyl, or is, for example
omega-amino-omega-carboxy-lower alkyl, for example
2-amino-2-carboxyethyl.
[0157] A carbohydrate radical R4 having from 5 to 12 carbon atoms
is, for example, a natural monosaccharide radical that is derived
from a pentose or hexose present in the form of aldose or ketose.
Detailed definitions of most relevant carbohydrate radicals
(pentoses, hexoses, disaccharides, etc.) is given in the patent EP
0 475 160 by the same applicant.
[0158] A steroid radical R4 is, for example, a sterol radical that
is esterified by the phosphatidyl group by way of the hydroxy group
located in the 3-position of the steroid nucleus.
[0159] A sterol radical is, for example, the lanosterol,
sitosterol, coprostanol, cholestanol, glycocholic acid, ergosterol
or stigmasterol radical, preferably the cholesterol radical.
[0160] If R4 represents a steroid radical, R1 and R2 are preferably
hydroxy and R3 is hydrogen.
[0161] Phospholipids of the formula 5 can be in the form of free
acids or in the form of salts. Salts are formed by reaction of the
free acid of the formula 11 with a base, for example a dilute,
aqueous solution of alkali metal hydroxide, for example lithium,
sodium or potassium hydroxide, magnesium or calcium hydroxide, a
dilute aqueous ammonia solution or an aqueous solution of an amine,
for example a mono-, di- or tri-lower alkylamine, for example
ethyl-, diethyl- or triethyl-amine,
2-hydroxyethyl-tri-C.sub.1-C.sub.4-alkyl-amine, for example
choline, and a basic amino acid, for example lysine or
arginine.
[0162] A phospholipid of the formula 5 has especially two acyloxy
radicals R1 and R2, for example alkanoyloxy or alkenoyloxy, for
example lauroyloxy, myristoyloxy, palmitoyloxy, stearoyloxy,
arachinoyloxy, oleoyloxy, linoyloxy or linoleoyloxy, and is, for
example, natural lecithin (R3=hydrogen, R4=2 -trimethylammonium
ethyl) or cephalin (R3=hydrogen, R4=2-ammonium ethyl) having
different acyloxy radicals R1 and R2, for example egg lecithin or
egg cephalin or lecithin or cephalin from soya beans, synthetic
lecithin or cephalin having different or identical acyloxy radicals
R1 and R2, for example 1-palmitoyl-2-oleoyl lecithin or cephalin or
dipalmitoyl, distearoyl, diarachinoyl, dioleoyl, dilinoyl or
dilinoleoyl lecithin or cephalin, natural phosphatidyl serine
(R3=hydrogen, R4=2-amino-2-carboxyethyl) having different acyloxy
radicals R1 and R2, for example phosphatidyl serine from bovine
brain, synthetic phosphatidylserine having different or identical
acyloxy radicals R1 and R2, for example dioleoyl-, dimyristoyl- or
dipalmitoyl-phosphatidyl serine, or natural phosphatidic acid (R3
and R4=hydrogen) having different acyloxy radicals R1 and R2.
[0163] A phospholipid of the formula 5 is also a phospholipid in
which R1 and R2 represent two identical alkoxy radicals, for
example n-tetradecyloxy or n-hexadecyloxy (synthetic ditetradecyl
or dihexadecyl lecithin or cephalin), R1 represents alkenyl and R2
represents acyloxy, for example myristoyloxy or palmitoyloxy
(plasmalogen, R3=hydrogen, R4=2 -trimethylammonium ethyl), R1
represents acyloxy and R2 represents hydroxy (natural or synthetic
lysolecithin or lysocephalin, for example 1-myristoyl- or
1-palmitoyl-lyso-lecithin or -cephalin; natural or synthetic
lysophosphatidyl serine, R3=hydrogen, R4=2-amino-2-carboxyethyl- ,
for example lysophosphatidyl serine from bovine brain or
1-myristoyl- or 1-palmitoyl-lysophosphatidyl serine, synthetic
lysophosphatidyl glycerine, R3=hydrogen,
R4=CH.sub.2OH--CHOH--CH.sub.2--, natural or synthetic
lysophosphatidic acid, R3=hydrogen, R4=hydrogen, for example egg
lysophosphatidic acid or 1-lauroyl-, 1-myristoyl- or
1-palmitoyl-lysophosphatidic acid).
[0164] A lipid that is analogous to abovementioned phospholipid and
can replace the latter is, for example, a lysolecithin analogue,
for example 1-lauroyl-1,3-propanediol-3-phosphoryl choline, a
monoglyceride, for example monoolein or monomyristin, a
cerebroside, a ganglioside, or a glyceride that does not contain a
free or esterified phosphoryl or phosphonyl group in the
3-position, for example a diacylglyceride or
1-alkenyl-1-hydroxy-2-acylglyceride, having the mentioned acyl or
alkenyl groups in which the 3-hydroxy group is etherified by one of
the mentioned carbohydrate radicals, for example a galactosyl
radical, for example monogalactosyl glycerine.
[0165] The lipids and certain surfactants mentioned hereinbefore
and hereinafter having a chiral carbon atom can be present both in
the form of racemic mixtures and in the form of optically pure
enantiomers in the pharmaceutical compositions that can be prepared
and used according to the invention.
[0166] The said at least one third amphipatic substance in said
combination, which acts as a drug, can be an adrenocorticostatic, a
.beta.-adrenolytic, an androgen an antiandrogen, an antiparasitic,
an anabolic, an anaesthetic, an analgesic, an analeptic, an
antiallergic, an antiarrhythmic, an antiarterosclerotic, an
antiasthmatic, a bronchospasmolytic, an antibiotic, an
antidrepressive, an antipsychotic, an antidiabetic, an antidot, an
antiemetic, an antiepileptic, an antifibrinolytic, an
anticonvulsive, an anticholinergic, an enzyme, a coenzyme or
corresponding inhibitor, an antihistaminic, an antihypertonic, a
biological inhibitor of drug activity, an antihypotonic, an
anticoagulant, an antimycotic, an antimyasthenic, an agent against
Morbus Parkinson or Morbus Alzheimer, an antiphlogistic, an
antipyretic, an antirheumatic, an antiseptic, a respiratory
analeptic or a respiratory stimulant, a broncholytic, a
cardiotonic, a chemotherapeutic, a coronary dilatator, a
cytostatic, a diuretic, a ganglium-blocker, a glucocorticoid, an
antiflew agent, a haemostatic, a hypnotic, an immunoglobuline or
its fragment, an immunologically active substance, a bioactive
carbohydrate, a bioactive carbohydrate derivative, a contraceptive,
an anti-migraine agent, a mineralo-corticoid, a
morphine-antagonist, a muscle relaxant, a narcotic, a
neurotherapeutic, a neuroleptic, a neurotransmitter or its
antagonist, a small peptide, a small peptide derivative, an
ophthalmic, a sympaticomimetic or a sympathicolytic, a
para-sympaticomimetic or a para-sympathicolytic, a psoriasis drug,
a neurodermitis drug, a mydriatic, a psychostimulant, a rhinologic,
a sleep-inducing agent or its antagonist, a sedating agent, a
spasmolytic, tuberculostatic, an urologic agent, a vasoconstrictor
or vasodilatator, a virustatic, a wound-healing substance, or a
combination of aforesaid agents.
[0167] When a drug is used as said at least one second or third
component, its content is preferably chosen to be between 0.1 rel.%
and 60 rel.% compared to the total mass of all three said
substances forming said extended surfaces. Somewhat narrower, and
more preferred, choice is to use between 0.5 rel.% and 50 rel.% and
most favourably between 1 rel.% and 40 rel.% compared to the total
mass of all three said substances that form said extended
surfaces.
[0168] Said at least one third substance in amphipat combination,
which solves the outlined problems, can be a low molecular weight
immunomodulator, a bio-catalyst, a co-enzyme, a hormone, or a low
molecular weight agonist or antagonist of some biologically
important substance action.
[0169] Any low to intermediate weight polypeptide with membrane
destabilising properties is also useful in the context of this
invention, if included into said combinations in suitable form and
concentration.
[0170] A list of potential ingredients that can be used for
preparing pharmaceutical formulations according to the present
invention is given in Cosmetic Ingredient Review (CIR Compendium),
which is regularly published in Washington, D.C., and in
appropriate Food and Drug Administration or other national
regulatory agency publications, including the list of GRAS
(Generally Recognised As Safe) compounds.
[0171] It is furthermore an explicit aim of the document, to teach
the use of amphipat combinations, as described herein, as drug
carriers, drug depots, or for other kind of medicinal or biological
application. For the purpose the required extended surfaces are
advantageously provided in the form of membranes formed by the at
least one first substance, the at least one second and the at least
one third substance, which together surround miniature droplets.
The substance with a biological activity, such as a drug, is then
mainly associated with said droplets at the surface or else is
mainly incorporated into the droplet to be carried by the droplet
to the place where the biologically active substance is supposed to
act.
[0172] Relatively detailed recommendations for preparing
compositions, as advocated in this application, are given in EP 0
475 160 and U.S. Pat. No. 6,165,500, which are herewith included by
reference. When filtration is use to prepare aggregate suspensions,
filter material with pore diameters between 0.01 .mu.m and 0.1
.mu.m, more preferably with pore diameters between 0.02 .mu.m and
0.3 .mu.m and even more advisable with pore diameters between 0.05
.mu.m and 0.15 .mu.m are used for homogenisation.
[0173] The present patent application moreover teaches suitable
methods for preparing combinations such that solve the outlined
problems by providing suitable formulations of biologically,
cosmetically and/or pharmaceutically active agents, comprising the
steps of: a) selecting the at least one first and the at least one
second substance which together form extended surfaces, when in
contact with said liquid suspending medium, such that said extended
surfaces formed by the at least one first and the at least one
second substance are more adaptable than the at least one first
substance alone and the surfaces formed by the at least one second
substance alone are not extended; alternatively; b) selecting the
at least one first and the at least one third substance which
together form extended surfaces, when in contact with said medium,
such that said extended surfaces formed by the at least one first
and the at least one third substance are more adaptable than the at
least one first substance alone and the surfaces formed by the at
least one third substance alone are not extended, if this substance
self-aggregates; and c) generating said combination of at least one
first, at least one second, and at least one third substance, such
that the surface of resulting at least three component combination
is even more adaptable than the surfaceprepared from at least one
first and one second substance alone or of the surfaces formed by
the at least one first and one third substance alone, bringing the
combination of at least two or all three said substances into
suspension by means of controlled mechanical fragmentation,
preferably in the presence of or before being mixed with the at
least one third substance, such that said third substance is
incorporated at least partly in said extended surface formed by
controlled mechanical fragmentation to obtain final
preparation.
[0174] It is particularly preferred to use filtration, pressure
change or mechanical homogenisation, or else shaking, stirring, or
mixing as said means of controlled mechanical fragmentation. The
desirable intermediary or final characteristics of the liquid
medium used to prepare aggregate suspension are defined in previous
paragraphs of this section.
[0175] The present patent application furthermore teaches methods
based on use of said at least quaternary mixtures containing at
least one active agent selected from the group comprising
anti-diabetic agents, growth factors, immunomodulators, enzymes,
recognition molecules, adrenocorticostatics, adrenolitics,
androgens, antiandrogens, antiparasitics, anabolics, anaesthetics,
analgesics, analeptics, antiallergics, antiarrhythmics,
antiarterosclerotics, antiasthmatics, bronchospasmolytics,
antibiotics, antidrepressiva, antipsychotics, antidots,
antiemetics, antiepileptics, antifibrinolytics, anticonvulsiva,
anticholinergics, enzyme, coenzymes or corresponding inhibitors,
antihistaminics, antihypertonics, biological inhibitors of drug
activity, antihypotonics, anticoagulants, antimycotics,
antimyasthenics, agents against Morbus Parkinson or Morbus
Alzheimer, antiphlogistics, antipyretics, antirheumatics,
antiseptics, respiratory analeptics or respiratory stimulants,
broncholytics, cardiotonics, chemotherapeutics, coronary
dilatators, cytostatics, diuretics, ganglium-blockers,
glucocorticoids, antiflew agents, haemostatics, hypnotics,
immunologically active substances, contraceptives, anti-migraine
agents, mineralo-corticoids, morphine-antagonists, muscle
relaxants, narcotics, neurotherapeutics, neuroleptics,
neurotransmitters or their antagonists, peptides, peptide
derivatives, ophthalmics, sympaticomimetics or sympathicolytics,
para-sympaticomimetics or para-sympathicolytics, anti-psoriasis
drugs, neurodermitis drugs, mydriatics, psychostimulants,
rhinologics, sleep-inducing agents or their antagonists, sedating
agents, spasmolytics, tuberculostatics, urologics, vasoconstrictors
or vasodilatators, virustatics, wound-healing substances, or a
combination of aforesaid agents.
[0176] Aforesaid method can rely on either using the recommended at
least three amphiphilic substances as such, or else dissolved in a
physiologically compatible polar fluid, comprising water or
water-miscible fluids, or in solvation-mediating agent, together
with a polar solution. Use of co-solvents is also possible.
[0177] A preferred, particularly practical method for preparing
said aggregate formulations contains at least one surfactant or
surfactant-like amphipat, which destabilises bilayer membrane, and
at least one more membrane destabilising, biologically active
ingredient or an additional surfactant in said polar solution.
[0178] In the case of need, the method can include the formation of
said surfaces induced by addition of one or more formulation or
aggregate components into a fluid phase, e.g. by using evaporation
from a reverse phase, injection or dialysis, or even by additional
mechanical stress.
[0179] Furthermore, it may be-preferred to use preparation method
in which the formation of said surfaces is induced by filtration,
the filtering material having pores between 0.01 .mu.m and 0.8
.mu.m wide. The choice of most convenient or favourable pore
diameter depends on the desired final aggregate dimensions and also
on the anticipated or achieved suspension flux through a filter.
Higher flux rates produce stronger shear and relatively smaller
final vesicle diameter, suspension viscosity also being
important.
[0180] When filtration is used to manufacture aggregate
suspensions, it may be convenient to use several filters
sequentially or in parallel. In the former case, pore diameters in
different filters can vary in diameter.
[0181] An preferred advantageous method for preparing suspensions
according to the present invention is such that ensures said agents
and carriers to associate, at least partly, after the formation of
said extended surfaces.
[0182] For better convenience, said extended surfaces, with which
agent molecules are made to associate, may be prepared just before
the application of the formulation. If desired, and possible, this
can be done from a suitable concentrate or a lyophylisate.
[0183] It is practically convenient to use a single container
comprising the selected pharmaceutical composition based on the
combination of substances as described in previous text. It is also
convenient to make said container a part of a package.
[0184] The present patent application moreover teaches a method for
generating a therapeutic effect on a warm blood creature by
applying a suitably selected pharmaceutical composition onto or
into a leaving creature's body, whereby the selection of a suitable
combination of substances is made according to the claims of this
document.
[0185] Special application of the method described herein is to
choose such administration volume that ensures control over the
applied medicament dose and the outcome of therapeutic
application.
[0186] It may be preferred, and practically valuable, to load a
suspension of drug-free aggregates with the drug, via association,
during the day prior to an administration, preferably within 360
min, more preferably within 60 min and most preferably within 30
min time window before the administration of resulting formulation
in or on the body.
[0187] The method of treatment done according to the present
invention typically involves administration of at least one dose of
the pharmaceutical composition with therapeutic activity on or in a
warm blood animal.
[0188] Last but not least, the present invention teaches a method
for finding suitable compositions, as described herein. This method
comprising the steps of: a) determining the flux of aggregates in a
suspension associated with a drug through pores in a well-defined
barrier, or various barriers, as a function of the driving force or
the driving pressure, which acts across the barrier; b) describing
the data within the framework of a suitable model such that fits
the characteristic flux vs. pressure or penetrability vs. pressure
curve; c) to deduce the characteristic system parameters, such as
p* and P.sub.max, in particular; d) employing said parameters to
optimise or characterise the formulation for application. Eq. (*)
is recommended as, and is claimed herein to be, particularly
suitable for describing and analysing such data.
PRACTICAL EXAMPLES
[0189] The following examples illustrate the invention without
setting or delineating its limits. All temperatures are in degree
Celsius. Carrier diameters are in nanometers, pressures in Pascal
(Pa) and other units correspond to standard SI system. Ratios and
percentages are given in moles, unless otherwise stated.
[0190] All measurements were done at room temperature, except when
specified otherwise. For aggregate adaptability/barrier transport
resistance measurements the test temperature was constant to within
plus/minus 2 degrees. For aggregate size measurements the
temperature accuracy was plus/minus 0.1 degree. The pH value of the
bulk suspension was determined with a commercial (gel) electrode.
All substances were used as received and were of p.a. quality,
unless stated otherwise. Molar masses were taken to be identical to
the published reference data.
[0191] Determination of Barrier Transport Resistance and aggregate
Adaptability. Barrier resistance to the transport of test vesicle
suspension in earlier patent applications by the same applicant was
called "permeation" resistance. In this document, more precise term
"penetration" resistance is used to stress the fact that vesicles
do not diffuse (=permeate) through but rather penetrate
barriers.
[0192] In first approximation one relies on simple experimental
method (SEM) and takes barrier transport resistance (in arbitrary
units) to be proportional to the pressure (in arbitrary units)
needed to drive a suspension of relatively large vesicles through a
0.2 micrometer filter with good efficacy. (In our experience, a
porous filter acts as a permeability barrier when the average pore
diameter is at least 40% to 50%, for the vesicles bigger and
smaller than 150 nm, respectively, and more preferably is at least
100% smaller than the average vesicle diameter in the tested
suspension.) Barrier transport resistance is then given in relative
units of 1 to 10 elsewhere (in EP 0 475 160 and U.S. Pat. No.
6,165,500) and in this document whenever reference is made to a 0.2
micrometer filter. Barrier penetrability, which in older
publications is called permeability, is identified with inverse
barrier resistance value. Aggregate adaptability is a direct
function of the former value, as is explained e.g. in Critical
Reviews in Therapeutic Drug Carrier Systems 13:257-388 (1996) or in
Adv. Drug Delivery Rev. 18:349-378 (1996).
[0193] Use of relative penetrability and barrier resistance values
is also convenient. These values are given by the ratio of the
penetrability/permeability or of the corresponding barrier
resistance values measured with a given suspension and its
supporting medium (e.g. water), e.g.: (relative) Penetrability
.eta. P.sub.rel=P.sub.suspension/P- .sub.medium. Similar use of the
trans-barrier flux data, measured with constant driving pressure,
provides more direct but still relative measure of barrier
penetrability/permeability for different formulations. Theoretical
explanation for such comparisons and calculations is given in
Critical Reviews in Therapeutic Drug Carrier Systems 13:257-388
(1996).
[0194] To get an absolute Barrier Transport Resistance or
Penetrability data, and to interpret these values in molecular
terms, an improved analytical method is needed, which is described
in brief in Definitions sections (see especially e.q. (*)). To get
absolute penetrability--and thus aggregate adaptability--data,
transbarrier flux is first measured serially. (This can be done as
is described in this document or in Biochim. Biophys. Acta 1368:
201-215 (1998).) Barrier resistance/penetrability value for the
test suspension is then calculated from the flux vs. pressure data
using e.q. (*), following the description given in previous
sections. From calculated resistance/penetrability value, a
convenient parameter that describes the adaptability of mixed
aggregates is deduced, e.g. by assuming: a.sub.a=1/p*.
[0195] Aggregate adaptability is thus identified with the inverse
pressure difference needed to attain a predefined, practically
relevant fraction of maximum achievable flux-pressure ratio; using
50-60% maximum penetrability criterion gives reasonable results.
Specifically, all p* values given in this document correspond to
57% of P.sub.max-value. If the maximum penetrability for a given
suspension-barrier combination cannot be measured, the
penetrability of a barrier to the medium in which the tested
aggregates are suspended is used as surrogate: P.sub.max=f x
Suspending medium flux/Driving pressure. Proportionality factor is
then typically taken to be up to 3-times (and more often up to
2-times) smaller than 57%, to allow for trivial friction
effects.
[0196] Exemplary results are given in FIGS. 4 and 5. The latter
figure also graphically illustrates the meaning of parameters "p*"
(in pressure units, and proportional to the barrier transport
resistance) and "Maximum penetrability" (=Pmax; in flux per
pressure units, and indicative of barrier porosity).)
[0197] Aggregate size determination. The average aggregate (most
often vesicle) diameter was measured with the dynamic light
scattering (for a few samples with a Malvern Zeta-Sizer instrument
and for the majority of samples with the instrument with an ALV
5000 correlator. Cumulant analysis method and an implementation of
software package "Contin" were used for analysing the correlation
curves obtained with Zeta-Sizer. To analyse the ALV measurements
the software delivered by the manufacturer (cumulant analysis or
"Contin") was employed.
Examples 1-120
[0198]
1 Composition: 37.74-84.5 mg Phosphatidylcholine from soy-bean
(SPO, .about.85% purity, MFS) introduced as an ethanolic solution
SPC/EtOH = 1/1 V/V and containing approx. 10% charged phospholipid
(presumably anionic phosphatidylglycerol) 187-34.9 mg Polysorbate
(Tween 80, pharmaceutical grade; MDS.sub.1) 5.6-20 rel. mol %
Sodium dodecylsulphate (SDS, p.a.; MDS.sub.2) replacing
phospholipid to the given amount add 1 ml Isotonic phosphate buffer
(pH = 7.2)
[0199] Objective: to test the synergism between membrane
destabilising, and thus aggregate adaptability increasing, activity
of two different surfactants, used in a combination with a lipid,
as the basic membrane forming system component.
[0200] Suspension preparation. To prepare a series with changing
lipid/surfactant ratio in the range 1/1 to 9/1, the necessary
amounts of phospholipid and surfactant are pipetted into buffer to
yield 10% lipid suspensions. These are first stirred at room
temperature for 5 days and then pre-filtered through a 0.8
micrometer polycarbonate filter to narrow down the starting
aggregate diameter. The average vesicle diameter (2r.sub.ves) is
determined and confirmed to exceed at least 2-fold the nominal
diameter of pores in the test filter (2r.sub.pore), which is
approximately 0.2 micrometer. This is done with the dynamic light
scattering e.g. by using a Malvern Zeta-Sizer instrument.
[0201] Transport (pore penetration) capability Transport resistance
is equated with the volume of test suspension that does not pass
through a 0.2 micrometer filter in a sterile holder. (A
ready-to-use, commercially available "blue" filter unit of
Sartorius (G6ttingen, Germany) is used for the test.) This reveals
that transport resistance decreases with increasing Tween content
when the relative SPC content is lower than 6/1 (SPC/Tw). The trend
is enhanced by the presence of sodium dodecylsulphate in the mixed
lipid aggregates. The latter shifts the minimum amount of Tween
needed to cross the semi-permeable barrier to increasingly lower
relative concentration values.
[0202] For example, when 12 mol-% of SPC in the mixed amphipat
aggregates is replaced by SDS, the suspension can be pushed through
a barrier with 0.2 micrometer pores practically without transport
resistance even when the relative SPC/Tween concentration is as low
as 15/1. Increasing SDS content further does not improve the
situation, as measured in this test series.
[0203] In contrast, reducing SDS content to and below 10 mol-%
relative to SPC shows a clear deterioration of penetration ability
of the resulting quaternary suspension. Rather low transport
resistance is now measured for SPC/Tween 7/1 (in case of 10 mol-%
SDS concentration) and for SPC/Tween 4/1, when SDS concentration is
between approx. 2 mol-% and 5 mol-%, as can be seen from FIG. 6. In
contrast, maximum barrier resistance value of 10 is found for the
suspensions without SDS and/or with little Tween and SDS, the
properties of which approach those of plain, single component
liposomes, which also have characteristic resistance value of
10.
[0204] Post-test determination of vesicle diameter confirms that
vesicles are still at least 1.3-times greater than the nominal pore
diameter.
Examples 121-129
[0205]
2 Composition: 14.2 mg Polysorbate (Tween 80) 85.8 mg
Phosphatidylcholine from soy-bean (SPC), as with examples 1-120
0-17.5 rel. mol % Sodium dodecylsulphate (SDS), relative to SPC and
replacing phospholipid to the given amount add 1 ml Isotonic
phosphate buffer (pH = 7.2)
[0206] Objective: as with examples 1-120, to test the synergism of
different surfactant action on extended surface aggregate
properties.
[0207] Suspension preparation. The method used to prepare vesicle
suspension was the same as in examples 1-120. The only notable
difference between both test series was the somewhat greater
average diameter and polydispersity of the vesicles used in
examples 121-129.
[0208] Transport ability (pore penetration capability and
adaptability) of aggregate suspension. To characterise the
resistance of semi-permeable barrier to suspension flux
(=transbarrier flux), the same method as in examples 1-120 was
used. The resistance was measured as a function of relative SDS
concentration in bilayer, to determine minimum amount of this
latter surfactant that is needed to maximise suspension flux across
the barrier and minimises the barrier transport resistance value.
Experimental data suggest that the threshold limit is around 6
mol-%, with some uncertainty in the 2-6 mol % region. This is
consistent with the results of first test series (examples 1-120)
except in that the measured resistance values are now somewhat
higher. This is explicable by different starting vesicle diameter
and polydispersity. The results are given in following table.
3TABLE 1 The effect of SDS, as the second surfactant (MDS.sub.2) in
addition to Tween 80 (MDS.sub.1; 10 mol % rel. to SPC), on the
resistance of mixed lipid membranes containing phosphatidylcholine
(SPC; MFS), as the basic building block, to the passage through a
semi-permeable barrier with pores, which were at least .about.50%
smaller than the average aggregate diameter. SDS/SPC Barrier
transport resistance [mol/mol] [rel. units, as defined in SEM]
0/100, reference Tween Tfs 10 2/98 4 4/96 10 6/94 1.88 8/92 1.75
10/90 1.50 25/175 1.12 15/85 0.75 35/165 0.44
Examples 130-131
[0209]
4 Composition: [ 52.1 mg Phosphatidylcholine from soy-bean (SPC),
actual amount = 52.2 mg - Na Chol amount in mg 45.2 mg Polysorbate
(Tween 80) 5, 10, 15 mol % Sodium cholate = Na Chol (relative to
SPC in the suspension) add 1 ml Isotonic phosphate buffer (pH =
7.2)
[0210] Objective: as with examples 1-120, but using a different
charged surfactant (cholate instead of SDS).
[0211] Suspension preparation. The starting suspension was prepared
as in previous examples. However, to make vesicles in the test
formulation more uniform before actual measurements, the starting
suspension was pre-filtered through 80 nm pore filters. This
yielded vesicles with approx. 120 nm diameter, as determined with
the dynamic light scattering using ALV 5000 correlator and a
personal computer.
[0212] Vesicle transport ability (pore penetration
capability/adaptability- ). The actual transport test was done with
relatively narrow pore (30 nm) filters, using different pressures
applied on the filter to characterise the penetrability of such
semi-permeable filter to the test suspension. This revealed fairly
comparable penetration ability for the vesicles with 10 mol-% and
15 mol-% cholate, exceeding the pore penetration ability, and thus
the adaptability, of the vesicles with merely 5 mol-% of cholate as
the third membrane destabilising component (cf. FIG. 3). These
results indicate that incorporation of the second surfactant into
mixed lipid bilayers does not increase membrane adaptability
proportionally, as one would expect on the basis of model results
shown in FIG. 7.
Examples 132-138
[0213]
5 Composition: See Table 2 Phosphatidylcholine from soy-bean (SPC)
See Table 2 Ketoprofen, sodium (KT); See Table 2 Tween 80, see
Table 2 Add 1 ml Phosphate buffer (pH = 7.2)
[0214] Objective: to test the synergistic effect of a membrane
destabilising drug (KT) combined with a surfactant (Tween 80) in a
lipid (SPC) suspension in terms of mixed aggregate adaptability and
relative capability to cross semipermeable barriers.
[0215] Test suspension preparation. The stated phospholipid and
drug amounts were brought into suspension using mechanical
homogenisation. That resulting average aggregate diameter was
around 100 nm.
[0216] Vesicle transport ability (pore penetration
capability/adaptability- ). The efflux of the test suspension from
a vessel pressurised with nitrogen gas was measured as a function
of the time to determine the pressure dependency of material
transport through the 20 nm pore filter in front of an opening in
the measuring vessel. From the measured flux data, the effective
"barrier penetrability", which defines the adaptability of the
tested mixed amphipat vesicles, was calculated as is described in
the main text body. The measured curves were also analysed in terms
of the pressure p*, needed to achieve 57% of maximum possible
suspension flux/pressure ratio. The result of the test series
indicate that both ketoprofen and Tween can act as a membrane
destabilising component. Consequently, either of these two system
ingredients improves the ability of test suspension to penetrate
barriers compared with simple phosphatidylcholine, reference
liposomes in a suspension without KT or Tween 80. When a
combination of said membrane destabilising components is used,
extended surface aggregate adaptability is increased to the value
measured with proper non-ionic Tween-based Transfersome.RTM.
suspension, with surfactant concentration much higher than that
used in the quaternary mixture. Data given in Table 2 justify the
conclusion. They are also compared with those pertainint to simple
buffer fluid (Ref. fluid) in which the mixed SPC/KT/Tween vesicles
were suspended.
6TABLE 2 Experimental and fit results for the pore penetration
experiments done with various quaternary suspensions of a
phospholipid (SPC; MFS), a drug (KT; MDS.sub.1), and Tween 80
(MDS.sub.2) co-suspended in a buffer; TL = total lipid Tween 80
Ketoprofen p* P.sub.max Adaptability [mol % of SPO] [mol % of TLI]
[MPa] [10.sup.-11 m Pa.sup.-1 .multidot. sec.sup.-1] a.sub.a,
[MPa.sup.-1] 0 (Liposomes) 0 >3 Not measurable (<0.3) 0 25
2.41 .+-. 0.15 Not measurable 0.415 0 33 1.66 .+-. 0.07 345 .+-. 37
0.602 10 33 0.25 .+-. 0.03 230 .+-. 17 4.000 50 0 0.20 .+-. 0.01
227 .+-. 3 5.000 0 (=Ref. Fluid) 0 Not applicable 613 .+-. 15 Not
applicable
Examples 139-142
[0217]
7 Composition: .quadrature.75.0 mg Phosphatidylcholine from
soy-bean (SPC), used as a saturated ethanolic solution SPC amount =
75 mg-Brij 98 content given in Table 3 25.0 mg Ketoprofen, sodium
(KT) See Table 3 Brij 98 Add 1 ml Phosphate buffer (pH = 7.2)
[0218] Objective: to test adaptability/pore penetrability
supporting activity of a different surfactant (Brij) combined with
a membrane destabilising drug (KT) in lipid (SPC) extended surface
aggregates.
[0219] Suspension preparation was essentially the same as in
examples 132-135.
[0220] Vesicle transport ability (pore penetration
capability/adaptability- ). In order to test whether or not the
increased adaptability of SPC/KT ternary suspensions is a unique
feature of Tween, as the fourth component, the effect of another
surfactant was investigated. In order to avoid undesired
electrostatic interactions between the anionic KT and such
additive, the uncharged Brij 98 (oleoyl-chain, 20 oxyethylene units
per molecule) was chosen. The penetrability of resulting
SPC/KT/Brij 3/1/0-0.323 w/w/w mixtures was finally calculated using
eq. (*)
[0221] The results for similar series measured with Brij 98 are
given in Table 3.
8TABLE 3 Fit results, based on e.q. (*), for the transbarrier flux
of suspensions containing a lipid (SPC; MFS), a drug (KT;
MDS.sub.1) and Brij 98 (MDS.sub.2) in different relative
concentrations, SPC and Brij together representing the total lipid
(TL) Brij 98 KT p* P.sub.max Adaptability [mol % of SPC] [mol % of
TL] [MPa] [10.sup.-11 m Pa.sup.-1 .multidot. sec.sup.-1] a.sub.a,
[MPa.sup.-1] 0 33 1.66 .+-. 0.07 345 .+-. 37 0.602 2.5 33 0.56 .+-.
0.07 266 .+-. 28 1.786 5.0 33 0.29 .+-. 0.07 191 .+-. 30 3.448 7.5
33 0.32 .+-. 0.06 171 .+-. 21 3.125 .sctn.The quoted error only
accounts for analytical and not for experimental data uncertainty,
which for example 16 exceeds 80%
Examples 143-146
[0222]
9 Composition: 100 mg Total lipid (TL, including SPC and Tween 80)
See Table 4 Phosphatidylcholine from soy-bean (SPC) See Table 4
Tween 80 See Table 4 Diclofenac See Table 4 Ethanol (EtOH) 5.25
Benzyl alcohol Add 1 g 154 mM Phosphate buffer, pH = 7.2
[0223] Objective: to test the effects of a surfactant (Tween 80)
and a drug (diclofenac), as two membrane destabilising amphipats,
and of a short-chain alcohol (ethanol) as an additional--and
potentially the second membrane destabilising amphipat.
[0224] Vesicle preparation was done essentially as described in
example 8 of WO 98/17255, but a more modern version of barrier
penetration assay was used to assess vesicle aggregate
adaptability. Vesicles with a similar overall composition but
lacking ethanol were tested (cf. examples. The results are given in
FIG. 8 and in Table 4.
[0225] Due to the limited measuring range of pore penetration
assay, it was only possible to obtain a rough estimate for the
adaptability of extended surface aggregates tested in this test
series. The estimated p*-value of the preparations containing
ethanol were lowered to .about.1.6 MPa from .about.4.8 MPa measured
in the absence of this alcohol. (It must be kept in mind, however,
that experimental variability in these tests was at least 50%, as
the standard deviations given in Table 4 only stem from the fit
routine.) The direction of the change is reasonable, but the
calculated absolute difference in p* is not significant.
10TABLE 4 Results of driving pressure and aggregate adaptability
analysis for the test. Tween 80 EtOH Diclofenac p* P.sub.max
Adaptability Ex [mol % of SPC] [w-%] [w-% of TL] [MPa] [10.sup.-11m
Pa.sup.-1 .multidot. sec.sup.-1] a.sub.a, [MPa.sup.-1] 143 0 0 10
(4.8 .+-. 1.6) Not measurable 0.208 144 0 9 10 2.4 .+-. 0.04 402
.+-. 17 0.417 145 0 9 20 146 10 9 10 .sup..sctn.The quoted error
only accounts for analytical and not for experimental data
uncertainty, which for example 16 exceeds 80%.
[0226] Data given in FIG. 8 and in Table 4 imply that ethanol makes
the tested lipid aggregates more adaptable. The effect is much
smaller, however, than in case of using a surfactant, such as Tween
80 (see Table 2).
[0227] Simple use of a membrane destabilising drug (diclofenac) and
of a short-chain alcohol, as membrane softening agents disclosed in
the prior art, thus only produces extended surface aggregates with
an adaptability significantly inferior to that of the formulations
disclosed in the present invention.
[0228] Ethanol containing, diclofenac loaded vesicles, indeed, are
more adaptable than the ethanol-free vesicles. However, even the
former vesicles have a much higher p* value, and therefore are far
less adaptable, than the ternary mixtures of phosphatidylcholine, a
non-ionic surfactant (Tween 80) and ketoprofen described in Table
2; the beneficial effect of a surfactant-like membrane
destabilising component, such as Tween 80, is directly reflected in
the lower p* value and/or in a higher flux of the modified
formulation through a barrier. This conclusion is practically
inaffected if the latter formulation contains ethanol.
[0229] It therefore stands to reason that at least two membrane
destabilising components should be present in an aggregate with
extended surface in adequate quantities to maximise the
adaptability of extended surface aggregates. Mere use of a lipid,
ethanol and a drug, as is disclosed in the prior art, is
insufficient for reaching the goal.
* * * * *