U.S. patent application number 09/730464 was filed with the patent office on 2001-09-06 for composition and method for dermal and transdermal administration of a cytokine.
This patent application is currently assigned to PharmaDerm Laboratories LTD.. Invention is credited to Attah-Poku, Sam Kwadwo, Foldvari, Marianna.
Application Number | 20010019712 09/730464 |
Document ID | / |
Family ID | 22085259 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010019712 |
Kind Code |
A1 |
Foldvari, Marianna ; et
al. |
September 6, 2001 |
Composition and method for dermal and transdermal administration of
a cytokine
Abstract
A composition for transdermal administration of a cytokine is
described. The composition includes a conjugate composed of a
cytokine, such as an interferon, and at least one fatty acid moiety
covalently attached to the cytokine. The conjugate has enhanced
cutaneous delivery relative to the cytokine alone.
Inventors: |
Foldvari, Marianna;
(Saskatchewan, CA) ; Attah-Poku, Sam Kwadwo;
(Saskatchewan, CA) |
Correspondence
Address: |
IOTA PI LAW GROUP
350 CAMBRIDGE AVENUE SUITE 250
P O BOX 60850
PALO ALTO
CA
94306-0850
US
|
Assignee: |
PharmaDerm Laboratories
LTD.
|
Family ID: |
22085259 |
Appl. No.: |
09/730464 |
Filed: |
December 4, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09730464 |
Dec 4, 2000 |
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09216500 |
Dec 18, 1998 |
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6165458 |
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60068873 |
Dec 26, 1997 |
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Current U.S.
Class: |
424/85.1 ;
424/85.2; 424/85.4 |
Current CPC
Class: |
A61K 9/0014 20130101;
A61K 47/542 20170801; A61K 47/02 20130101; A61K 38/1774 20130101;
A61K 38/1722 20130101; A61K 47/26 20130101; A61K 47/54
20170801 |
Class at
Publication: |
424/85.1 ;
424/85.2; 424/85.4 |
International
Class: |
A61K 045/00; A61K
038/21 |
Claims
It is claimed:
1. A pharmaceutical composition for dermal or transdermal
administration of a cytokine, comprising a conjugate composed of a
cytokine and at least one fatty acid moiety having between 12-24
carbon atoms covalently attached to the cytokine, said conjugate
having a substantially higher rate of skin penetration than the
cytokine alone.
2. The composition of claim 1, wherein said cytokine is selected
from the group consisting of interferons and interleukins.
3. The composition of claim 1, wherein said cytokine is selected
from the group consisting of interferon .alpha., interferon .beta.,
interferon .gamma., interleukin 1, interleukin 2 and interleukin
13.
4. The composition of claim 1, wherein said fatty acid is a
saturated fatty acid having between 12-24 carbon atoms.
5. The composition of claim 1, wherein said fatty acid is an
unsaturated fatty acid having between 12-20 carbon atoms.
6. The composition of claim 1, wherein said fatty acid is selected
from palmitic acid, behenic acid and lignoceric acid.
7. The composition of claim 3, wherein said fatty acid is palmitic
acid.
8. The composition of claim 1, wherein said cytokine is an
interferon .alpha. and said fatty acid is palmitic acid.
9. A method for dermal or transdermal administration of a cytokine,
comprising preparing a conjugate composed of said cytokine and,
covalently attached to the cytokine, at least one fatty acid moiety
having between 12-24 carbon atoms, said conjugate having a
substantially higher rate of skin penetration than the cytokine
alone, and applying said conjugate to the skin of a subject in a
pharmaceutically acceptable preparation.
10. The method of claim 9, wherein said cytokine is an interferon
or an interleukin.
11. The method of claim 10, wherein said cytokine is selected from
the group consisting of interferon .alpha., interferon .beta.,
interferon .gamma., interleukin 1, interleukin 2 and interleukin
13.
12. The method of claim 9, wherein said fatty acid is a saturated
fatty acid having between 12-24 carbon atoms.
13. The method of claim 12, wherein said fatty acid is selected
from palmitic acid, behenic acid and lignoceric acid.
14. The method of claim 9, wherein said fatty acid is an
unsaturated fatty acid having between 12-20 carbon atoms.
15. The method of claim 9, wherein said cytokine in interferon
.alpha. and said fatty acid is palmitic acid.
16. A method of treating an infection caused by human papilloma
virus in a subject, comprising administering topically at the site
of infection, a conjugate composed of said cytokine and, covalently
attached to the cytokine, at least one fatty acid moiety having
between 12-24 carbon atoms, said conjugate having a substantially
higher rate of skin penetration than the cytokine alone.
17. The method of claim 16, wherein said infection is genital warts
and said cytokine is interferon .alpha..
18. A method of enhancing an immune response to a vaccine,
comprising administrating topically to a patient receiving a
vaccine, a conjugate composed of a cytokine and, covalently
attached to the cytokine, at least one fatty acid moiety having
between 12-24 carbon atoms, said conjugate having a substantially
higher rate of skin penetration than the cytokine alone.
Description
[0001] This application claims the priority of U.S. Provisional
Application Ser. No. 60/068,873, filed Dec. 26, 1997, and which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a composition for
transdermal administration of a cytokine. The composition includes
a conjugate composed of a cytokine and at least one fatty acid
moiety covalently attached to the cytokine.
BACKGROUND OF THE INVENTION
[0003] The routine administration of therapeutic proteins and
peptides is hindered by the lack of a reliable and convenient mode
of delivery. The oral route is often impractical due to the
digestion of proteins in the gastrointestinal tract. Parenteral
administration is an alternative, although frequent injections are
required due to the short half-life of peptides and this can
decrease patient compliance.
[0004] Other potential routes of administration for proteins
include nasal, pulmonary, rectal, vaginal, ocular and transdermal.
The transdermal route offers some advantages in that the skin has
low proteolytic activity, so that metabolism of the protein during
transit through the skin is minimized thereby improving
bioavailability.
[0005] One problem with transdermal administration of proteins and
peptides is that they may exhibit very low permeability through the
skin due to their hydrophilicity and high molecular weight. One
approach to overcoming the low skin permeability is directed to
temporarily compromising the integrity or physicochemical
characteristics of the skin to enhance skin penetration, e.g.,
using a skin penetration enhancer, employing ultrasonic vibration,
removing the epithelial layer by suction or employing an electric
current (iontophoresis). These approaches have demonstrated the
feasibility of transdermal administration of proteins and peptides,
however are associated with skin irritation and/or other
disadvantages.
SUMMARY OF THE INVENTION
[0006] Accordingly, it is an object of the invention to provide a
composition for administration of a protein or peptide
transdermally. More specifically, it is an object of the invention
to provide a composition for transdermal administration of a
cytokine.
[0007] In one aspect, the invention includes a pharmaceutical
composition for dermal or transdermal administration of a cytokine.
The composition includes a conjugate composed of a cytokine and at
least one fatty acid moiety having between 12-24 carbon atoms
covalently attached to the cytokine. The conjugate has a
substantially higher rate of skin penetration than the cytokine
alone.
[0008] In one embodiment, the cytokine is an interferon or an
interleukin, and in a preferred embodiment, the cytokine is
interferon .alpha., interferon .beta., interferon .gamma.,
interleukin 1, interleukin 2 or interleukin 13.
[0009] The fatty acid to which the cytokine is attached is a
saturated fatty acid having between 12-24 carbon atoms or an
unsaturated fatty acid having between 12-20 carbon atoms. In
preferred embodiments of the invention, the fatty acid is palmitic
acid, behenic acid or lignoceric acid.
[0010] One preferred conjugate includes interferon .alpha. as the
cytokine and palmitic acid as the fatty acid.
[0011] In another aspect, the invention includes a method for
dermal or transdermal administration of a cytokine. The method
includes preparing a conjugate, as described above, and applying
the conjugate to the skin of a subject in a pharmaceutically
acceptable preparation.
[0012] In another aspect, the invention includes a method of
treating an infection caused by human papilloma virus in a subject
by administering topically at the site of infection, a conjugate as
described above. In one embodiment of the method, the infection to
be treated is genital warts and the cytokine in the conjugate is
interferon .alpha..
[0013] In another aspect, the invention includes a method of
enhancing an immune response to a vaccine, by administrating
topically to a patient receiving a vaccine, a conjugate composed of
a cytokine and, covalently attached to the cytokine, at least one
fatty acid moiety having between 12-24 carbon atoms.
[0014] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a synthetic reaction scheme for acylation of a
cytokine;
[0016] FIG. 2 shows a synthetic reaction scheme for acylation of
interferon with palmitic acid;
[0017] FIG. 3 shows the nucleotide sequence of interferon .alpha.2b
(SEQ ID No. 1)
[0018] FIGS. 4A-4B are capillary electrophoresis electropherograms
showing the time dependence of derivatization of interferon .gamma.
with palmitic acid (FIG. 4A) and the effect of protein:reagent
ratio on the derivatization (FIG. 4B);
[0019] FIGS. 5A-5B are plots of mobility, determined by capillary
electrophoresis, as a function of cytokine:fatty acid ester ratio
(FIG. 5A) and time (FIG. 5B) for interferon .alpha.2b derivatized
with palmitic acid (FIG. 5A) and oleic acid (FIG. 5B, closed
triangles);
[0020] FIG. 6A is a chromatographic profile of palmitoylated
interferon .alpha.2b on Sephadex G-25:
[0021] FIG. 6B is a SDS-PAGE pattern of the corresponding
chromatographed fractions of FIG. 6A after silver staining;
[0022] FIG. 6C is a SDS-page profile of palmitoylated interferon
.alpha.2b synthesized under various conditions;
[0023] FIGS. 7A-7B are plots showing binding to human keratinocytes
of interferon .alpha.2a as a function of concentration of
interferon .alpha.2a (FIG. 7A) and of interferon .alpha.2a
derivatized with behenic acid (closed circles) and lauric acid
(closed diamonds) and interferon .alpha.2a treated with DMSO
(closed squares) (FIG. 7B);
[0024] FIG. 8A is a plot showing in vitro percutaneous absorption
through human skin as a function of time of conjugates of
interferon .alpha.2b and palmitic acid (closed diamonds), oleic
acid (open triangles), myristic acid (open diamonds), stearic acid
(open circles) and lauric acid (open squares) and of
liposomally-entrapped interferon .alpha.2b (closed circles) and
interferon .alpha.2b alone (closed squares); and
[0025] FIG. 8B is a bar graph showing in vitro cutaneous absorption
into human skin after 24 hours of the formulations shown in FIG.
8A, where absorption into whole skin and into skin after removal of
the stratum corneum is reported for each formulation.
DETAILED DESCRIPTION OF THE INVENTION
[0026] I. Preparation of the Conjugate
[0027] As discussed above, the conjugate of the invention is
composed of a cytokine and a fatty acid moiety covalently attached
to the cytokine. As used herein, a cytokine includes any immune
system protein that is a biological response modifier. Generally,
cytokines coordinate antibody and T cell immune system interactions
and amplify immune reactivity and include monokines synthesized by
macrophages and lymphokines produced by activated T lymphocytes and
natural killer cells. Monokines include interleukin 1, tumor
necrosis factor, .alpha. and .beta. interferons and
colony-stimulating factors. Lymphokines include interleukins,
interferon .gamma., granulocyte macrophage colony-stimulating
factor and lymphotoxin. Cytokines are also synthesized by
endothelial cells and fibroblasts.
[0028] FIG. 1 shows a synthetic reaction scheme for derivatizing a
protein, in particular a cytokine, having amino positions available
for covalent attachment, with a fatty acid. In the first step of
the process, the N-hydroxysuccinimide ester of the fatty acid is
prepared by mixing the fatty acid with N-hydroxysuccinimide in a
suitable solvent in the presence of dicyclohexylcarbodiimide. The
fatty acid ester is then isolated by recrystallization or other
technique. In the second step, the fatty acid ester is mixed with
the protein to react with available amino groups to yield the fatty
acid linked to the protein through an amide bond.
[0029] It will be appreciated that other reaction schemes are
suitable to derivatize a protein with a fatty acid. For example,
the amide bond formation can be done more selectively by blocking
and de-blocking certain groups on the protein. The protein can also
be derivatized with the fatty acid through formation of an ester
bond.
[0030] In studies performed in support of the invention, interferon
.alpha., more specifically, interferon .alpha.2b, interferon
.alpha.2a and interferon .gamma., were derivatized with various
fatty acids according to the scheme set forth in FIG. 1. The
procedure is suitable for derivatization of other proteins, such as
IL-4, IL-12 and GM-CSF.
[0031] A reaction scheme for fatty acylation of interferon with
palmitic acid is illustrated in FIG. 2. Fatty acylation of
interferon .alpha. by this reaction forms an amide bond which is
stable for dosage form development and in biological environments.
As described in Example, 1, the first step in the synthesis is to
prepare N-hydroxysuccinimide-palmit- ate, which, in the second step
of the process, is reacted with interferon in a suitable solvent,
such as dimethylsulfoxide or dimethylformamide.
[0032] Interferon .alpha.2b is a hydrophilic protein with nine
lysine amino acids, which, with reference to FIG. 3, are at
positions 31, 49, 70, 83, 112, 121, 131, 134 and 164. These lysine
amino acids, in addition to the amino terminal, are available for
potential covalent attachment of fatty acids. Interferon .alpha.2b
has disulfide bonds between residues 1 and 19 and between residues
29 and 138 (Wetzel, Nature, 289:606, 1981), and only the latter
disulfide bond is critical for maximal antiviral activity
(Morehead, et al., Biochemistry, 23:2500, 1984). Three structurally
distinct domains are important for activity: 10-35, 78-107 and
123-166 (Fish, et al., J. Interferon Res., 9:97, 1989).
[0033] As noted above, interferon .alpha. has nine lysine residues,
as well as the terminal cysteine, for potential acylation.
Depending on the availability of these positions for acylation and
on the reaction conditions, one or more positions can be
derivatized with a fatty acid. The three dimensional structure of
interferon .alpha. has been constructed by computer modeling for
the primary amino acid sequence of consensus interferon .alpha.
(Korn, et al., J. Interferon Res., 14:1, 1994). The model indicates
that the conformationally accessible regions for derivatization
within interferon .alpha. are domains 29-35, 79-95 and 123-140.
Thus, at least the four lysine residues within these regions
(positions 31, 83, 131 and 134), plus the terminal amino acid, are
conformationally available to bind with a fatty acid.
[0034] Because the reaction shown in FIG. 2 is a non-specific
acylation synthesis, it is expected that some of the lysine
.epsilon.-amino groups and the terminal amino group on the protein
will be acylated. The actual fatty acid-derivatized interferon is
likely a mixture containing interferon .alpha. acylated to various
degrees, i.e., mono-palmitate, di-palmitate, etc. For the purpose
of the studies reported herein, the different fractions were not
separated or purified. However, it will be appreciated that the
fractions can be separated if desired in order to optimize activity
and rate of transdermal penetration of the conjugate.
[0035] The degree of derivatization appears to be time dependent,
as evidenced by the electropherogram in FIG. 4A. The trace in FIG.
4A was obtained by capillary electrophoresis and the methodology is
set forth in the methods section below. The trace shows that after
2 and 18 minutes of reaction time with palmitic acid, the migration
time of the palmitoylated interferon changed from 7 minutes to 7.8
minutes, respectively. Smaller changes in migration time up to 1
hour of incubation was observed. After 1 hour of reaction time, no
further change in migration was observed.
[0036] The effect of protein:N-hydroxysuccinimide ester of palmitic
acid ratio on palmitoylation was evaluated using capillary
electrophoresis. As seen in FIG. 4B, at low ratios of
protein:palmitic acid a more heterogeneous population of
derivatized protein was formed, as evidenced by the broader peaks
with lower mobility. At a ratio of 1:10 or higher a reproducible
population of palmitoylated interferon .alpha.2b with an
electrophoretic mobility of 9.5 minutes was obtained.
[0037] FIG. 5A is a plot which corresponds to the trace of FIG. 4B
and shows the mobility of the interferon (.alpha.2b-palmitic acid
conjugate as a function of protein:fatty acid ester (palmitic acid
esterified with N-hydroxysuccinimide) ratio. The fatty acid ester
has a mobility of about 23 and conjugation with interferon
.alpha.2b at a 1:1 ratio decreasing the mobility to about 17. The
mobility decreases slowly thereafter with increasing protein:fatty
acid ester ratio.
[0038] FIG. 5B shows mobility as a function of time for the
N-hydroxysuccinimide ester of oleic-acid (closed triangles) and for
the oleic acid-interferon .alpha.2b conjugate prepared in a 50/50
v/v mixture of distilled water/DMSO and a protein:fatty acid ester
ratio of 1:25 (closed diamonds). After about 30 minutes of
incubation time, the mobility of the conjugate is about 17, with a
slow continuous decrease in mobility with longer reaction time.
[0039] Further in support of the invention, interferon .alpha.2b
and interferon .alpha.2a were derivatized as described above with
fatty acids having between 12 and 24 carbon atoms. The conjugates
prepared and the molar ratio of interferon .alpha. to the
N-hydroxysuccinimide fatty acid ester are shown in Table 1. The
mobility values shown in Table 1 were determined by capillary
electrophoresis, as set forth in the methods section below.
1TABLE 1 Fatty Acid Cytokine-Fatty Mobility in Cytokine (No.
Carbons) Acid.sup.1 Ratio SDS Gel.sup.2 Interferon .alpha.2b Lauric
Acid 1:20 .sup. nd.sup.3 (C12) Interferon .alpha.2b Myristic Acid
1:20 nd (C14) Interferon .alpha.2b Palmitic Acid 1:20 nd (C16)
Interferon .alpha.2b Stearic Acid 1:20 nd (C18) Interferon
.alpha.2b Oleic Acid (C18, 1:20 nd unsaturated) Interferon
.alpha.2a Lauric Acid 1:25 12.532 (C12) Interferon .alpha.2a
Myristic Acid 1:25 12.533 (C14) Interferon .alpha.2a Palmitic Acid
1:25 12.608 (C16) Interferon .alpha.2a Stearic Acid 1:25 12.636
(C18) Interferon .alpha.2a Oleic Acid (C18, 1:25 12.627
unsaturated) Interferon .alpha.2a Arachidic Acid 1:25 nd (C20)
Interferon .alpha.2a Behenic Acid 1:25 nd (C22) Interferon
.alpha.2a Lignoceric Acid 1:25 nd (C24) Interferon .alpha.2a none
-- 13.085 (control) Interferon .alpha.2a in none -- 13.213 DMSO
(control) .sup.1Ratio of cytokine to N-hydroxysuccinimide fatty
acid ester. .sup.2Determined by capillary electrophoresis. .sup.3nd
= not determined
[0040] II. Characterization of the Conjugates
[0041] The conjugates composed of interferon .alpha. and various
fatty acids, prepared as described above, were characterized by
electrophoresis (polyacrylamide gel electrophoresis (PAGE)) and
were characterized for antiviral activity and receptor binding
activity.
[0042] 1. Gel Electrophoresis
[0043] A chromatographic profile of interferon .alpha.2b acylated
with palmitic acid on Sephadex G-25 column is shown in FIG. 6A. The
intactness of the interferon .alpha.2b after lipid modification is
evident and the individual column (Sephadex G25) fractions are
shown in the SDS-PAGE pattern of FIG. 6B. Lane 1 in the profile is
for a Bio-Rad molecular weight standard; lane 2 is for an
interferon .alpha.2b standard and lanes 3-9 correspond to fractions
taken at 1.5-5.5 ml from the Sephadex column (FIG. 6A).
[0044] FIG. 6C is a SDS-PAGE profile comparing interferon
.alpha.2b-palmitate conjugates prepared under various conditions.
Lane 1 in the profile is a molecular weight standard; lane 2 is
interferon .alpha.2b incubated in DMF; lane 3 corresponds to a
conjugate of interferon .alpha.2b and palmitic acid prepared in
DMF; lane 4 corresponds to interferon .alpha.2b incubated in DMSO;
lane 5 corresponds to a conjugate of interferon .alpha.2b and
palmitic acid prepared in DMSO; lane 6 is an interferon .alpha.2b
standard, 100 ng; and lane 7 is an interferon .alpha.2b standard,
50 ng.
[0045] A comparison of the bands in lanes 3 and 5 shows that the
yield of palmitoyl-interferon .alpha.2b prepared in DMSO was 15-20%
higher than when the conjugate was prepared in DMF. Lanes 2 and 4
in FIG. 6C compare the effect of the two solvents, DMSO and DMF,
respectively, on the protein alone. No differences in the bands are
apparent, indicating that the neither solvent has a negative effect
on the protein. The PAGE bands for the conjugate indicate a 6-10%
increase in molecular weight of interferon .alpha. after
acylation.
[0046] 2. Antiviral Activity
[0047] The palmitate-interferon .alpha.2b conjugate prepared as
described above was evaluated for antiviral activity to determine
whether acylated cytokines in general retain biological activity.
Antiviral activity was evaluated according the procedure described
in Example 2, where the cytopathic effect inhibition assay using
Georgia Bovine Kidney (GBK) cells and vesicular stomatitis virus
(VSV) as the challenge virus. The results are shown in Table 2.
2 TABLE 2 Antiviral Activity (% of interferon-.alpha.2b) conjugate
prepared conjugate prepared in DMSO.sup.1 in DMF.sup.1 Interferon
.alpha.2b.sup.1 100% 100% palmitoyl-interferon .alpha.2b 50% 0%
.sup.1Interferon .alpha. treated under the same conditions as the
protein undergoing acylation. .sup.2Palmitoyl-interferon .alpha.
acylated in dimethylformamide (DMF) or in dimethylsulfoxide
(DMSO).
[0048] The antiviral activity of interferon .alpha.2b was
unaffected when the protein was treated to the conditions of the
acylation reaction, except for addition of palmitic acid, in both
dimethylformamide (DMF) and dimethylsulfoxide (DMSO). That is, 100%
of the antiviral activity of interferon .alpha. was preserved.
Acylation of the cytokine with palmitic acid in the solvent DMF
resulted in a complete loss of activity. When the reaction was
carried out in DMSO a 50% preservation of antiviral activity was
achieved.
[0049] The loss in activity may be in part attributed to
experimental conditions, and the assay was modified for greater
control and accuracy. The GBK cells in 96-well microtiter plates
were dosed with 50 .mu.l interferon .alpha.2b reference solution of
a conjugate sample. After incubation overnight the cells were
infected with VSV virus. After incubation, washing, fixing and
staining, the plates were read by a spectrophotometer to determine
the antiviral activity of the compounds. The results, shown in
Table 3, indicate enhanced activity of the novel derivatives
compared to the parent protein.
3 TABLE 3 Sample Antiviral Activity Interferon .alpha.2b 100% (INF
.alpha.2b) Lauroyl-INF .alpha.2b 210% Myristol-INF .alpha.2b 175%
stearoyl-INF .alpha.2b 190% oleyl-INF .alpha.2b 200%
[0050] In another experiment using the revised method, antiviral
activity of interferon .alpha.2a derivatized with behenic and
lignoceric acid was measured. The conjugate including behenic acid
retained nearly 100% of the interferon .alpha.2a activity and the
conjugate with lignoceric acid retained about 30% of interferon
.alpha.2a antiviral activity.
[0051] Table 4 shows the antiviral activity of conjugates prepared
with interferon .gamma..
4 TABLE 4 Antiviral Activity Fatty Acid (% of interferon .gamma.)
Lauric Acid 25% Myristic Acid 20% Palmitic Acid 22% Stearic Acid
40% Oleic Acid 10% Arachidic Acid 2% Behenic Acid 8% Lignoceric
Acid 9%
[0052] As noted above, the conjugates used in the studies reported
herein were not separated or purified into single acyl-protein
fractions. There may be an optimum degree of fatty acylation for
maximum retention of biological activity of the cytokine--for
example, a di-palmitoyl interferon .alpha. may have a higher, or
lower, biological activity than tri-palmitoyl interferon .alpha..
Separation of the fractions for analysis can be readily performed
by those of skill in the art to determine such an optimum, as
evidenced by the work of Hashimoto, et al (Pharm. Res., 6:171,
1989). Nonetheless, partial loss of antiviral activity does not
exclude the possibility that other functions of interferon .alpha.
are unchanged or perhaps increased. In fact, some cytokine
functions do not involve receptor binding and can act directly on
intercellular signaling pathways (Baron et al., JAMA, 266:1375,
1991). Also, partial loss of antiviral activity may be
inconsequential or at least offset in view of the enhanced skin
penetration, discussed below.
[0053] 3. Receptor Binding
[0054] Binding of the conjugates composed of interferon .alpha.2a
and behenic acid or lauric acid was determined in an assay using
human keratinocytes, as described in Example 3. The results are
shown in FIGS. 7A-7B, where FIG. 7A shows binding of iodinated
interferon .alpha.2a to human keratinocytes as a function of
concentration of interferon .alpha.2a. The binding of interferon
.alpha.2a is concentration dependent and saturation of binding was
not evident at 40 ng interferon .alpha.2a. Scatchard analysis
indicated the dissociation constant was 5.1.times.10.sup.-10M, with
1579 receptors per human keratinocyte cell (see insert in FIG.
7A).
[0055] FIG. 7B shows binding of conjugates of interferon .alpha.2a
derivatized with behenic acid (closed circles) and lignoceric acid
(closed circles) and, as a control, of interferon .alpha.2a treated
with DMSO (closed squares) as a function of amount of interferon
.alpha.2a. The behenic acid-interferon .alpha.2a conjugate had a
binding comparable to that of the protein alone treated with
DMSO.
[0056] 4. Solubility
[0057] The relative hydrophobicity of the conjugates described
above were determined by measuring the partition coefficient of
each conjugate into stratum corneum. Powdered stratum corneum,
prepared as described in Example 4, was incubated with radiolabeled
interferon .alpha.2a and the lipid derivatized conjugates, prepared
as described above, and the ratio of uptake (Kp) into the powdered
stratum corneum to that remaining in the saline preparation was
determined (Example 4). The results are shown in Table 5.
5 TABLE 5 Conjugate Kp interferon .alpha.2a 3.360 lauric
acid-interferon .alpha.2a 4.404 myristic acid-interferon .alpha.2a
4.541 palmitic acid-interferon .alpha.2a 5.071 stearic
acid-interferon .alpha.2 4.508 oleic acid-interferon .alpha.2a
5.044 arachidic acid-interferon .alpha.2a 5.079 behenic
acid-interferon .alpha.2A 3.555 lignoceric acid-interferon
.alpha.2a 3.730 DMSO treated interferon .alpha.2a 3.906
[0058] The results show that the fatty acid derivatization of
interferon increases the uptake relative to the parent protein,
indicating an increase in hydrophobicity and greater affinity for
the skin.
[0059] A similar study was conducted for interferon .alpha.2b and
conjugates of interferon .alpha.2b, where the partition coefficient
was determined in the conventional octanol/water system, at
octanol/phosphate buffered saline ratios of 1:7 and 1:25. The
results are shown in Table 6.
6TABLE 6 p value (paired Test System Conjugate Kp t-test)
octanol/saline (1:7) interferon .alpha.2b 0.0348 lauric
acid-interferon .alpha.2b 0.0737 0.103 myristic acid-interferon
.alpha.2b 0.0691 0.001 palmitic acid-interferon .alpha.2b 0.0364
0.800 stearic acid-interferon .alpha.2b 0.0531 0.024 oleic
acid-interferon .alpha.2b 0.0329 0.540 octanol/saline (1:25)
interferon .alpha.2b 0.0373 lauric acid-interferon .alpha.2b 0.0434
0.423 myristic acid-interferon .alpha.2b 0.0487 0.201 palmitic
acid-interferon .alpha.2b 0.0337 0.634 stearic acid-interferon
.alpha.2b 0.0263 0.142 oleic acid-interferon .alpha.2b 0.0475
0.265
[0060] 5. Cutaneous Absorption
[0061] The rate and extent of skin penetration of the conjugates
was determined in vitro according to the procedure described in
Example 5. In these studies, interferon .alpha.2b and the palmitoyl
derivative of interferon .alpha.2b were iodinated by the
lactoperoxidase method set forth in Example 3. A preparation of
each test compound was placed on full thickness human skin mounted
in a diffusion cell and the downstream reservoir of the cell was
monitored for 24 hours for amount of interferon .alpha.2b.
[0062] After 24 hours, the skin was removed from the test cells and
the radioactivity associated with the skin was determined by gamma
counting. These results are shown in Table 7 under the column
headed "whole skin counts". The skin was then stripped ten times
with Scotch.TM. tape and the radioactivity associated with each
strip was determined separately. These values are reported in Table
7 in the column headed "stratum corneum". The skin after stripping
was counted again to obtain the counts associated with the viable
skin layers (epidermis, dermis and subcutaneous tissues), and this
data is in the third column of Table 7. The skin stripping
technique was validated by sectioning paraffin embedded stripped
skin and viewing under a light microscope for complete removal of
the stratum corneum.
7TABLE 7 In vitro cutaneous absorption of interferon .alpha.2b and
palmitoyl-interferon .alpha.2b into human breast skin Stratum Whole
Skin Corneum Viable Layers Preparation .mu.g/cm.sup.2, n = 6
.mu.g/cm.sup.2, n = 6 .mu.g/cm.sup.2, n = 6 interferon .alpha.2b
0.41 .+-. 0.11 0.20 .+-. 0.08 0.23 .+-. 0.09 (1.8% .+-. 0.5%)
(0.98% .+-. 0.39%) palmitoyl- 2.11 .+-. 1.22 0.23 .+-. 0.14 1.88
.+-. 1.16 interferon .alpha.2b (11.5% .+-. 6.7%) (10.3% .+-.
6.4%)
[0063] The results in Table 7 show that both the cutaneous and
percutaneous absorption of the acylated cytokine was 5-6 fold
greater than that of the cytokine alone. The amount of acylated
interferon .alpha.2b and of interferon .alpha.2b in whole skin
after 24 hours of treatment was 2.11.+-.1.22 .mu.g/cm.sup.2 and
0.41.+-.0.11 .mu.g/cm.sup.2, respectively. This represents
11.5.+-.6.7% and 1.8%.+-.0.5% of total drug applied, respectively.
In the viable skin layers the difference in absorption between the
derivatized protein and the parent protein was 8-10 fold,
1.88.+-.1.16 .mu.g/cm.sup.2 (10.3%.+-.6.4%) and 0.228.+-.0.91
.mu.g/cm.sup.2 (0.98%.+-.0.39%).
[0064] The calculated percutaneous absorption parameters for the
preparations reported in Table 7 are shown in Table 8.
Approximately two times higher flux was detected for the conjugate
compared to the non-fatty acylated protein. The total amount of
drug diffused in 24 hours was also about two times higher for the
conjugate.
8TABLE 8 In vitro percutaneous Absorption of interferon .alpha.2b
and palmitoyl-interferon .alpha.2b through human breast skin
.sup.125I-interferon .sup.125I-palmitoyl- Parameters .alpha.2b
interferon .alpha.2b Steady state flux (ng/cm.sup.2/h).sup.1 1.47
2.71 Permeability coefficient (cm/h).sup.2 1.65 .times. 10.sup.-5
3.03 .times. 10.sup.-5 Diffusion coefficient (cm.sup.2/sec).sup.3
6.85 .times. 10.sup.-12 5.45 .times. 10.sup.-12 Total amount
diffused in 24 h: 23.8 .+-. 17.4 42.7 .+-. 25.70 (ng/cm.sup.2)
.sup.1Determined by regression analysis of the linear portion of
cumulative amount of drug diffused (Q) vs. time (t) curve.
.sup.2Permeability coefficient (P) was calculated from Fick's first
law: (dQ/dt).sup.ss = J.sup.ss = P.DELTA.C; where P = Kp D/h
[J.sup.ss = steady state flux; .DELTA.C = concentration difference
between donor and receiver compartments; Kp = partition coefficient
between skin and the preparation] .sup.3Diffusion coefficient was
calculated from D = h.sup.2/6L; where h = thickness of the stratum
corneum (0.001 cm); L = lagtime (sec).
[0065] The cutaneous and percutaneous absorption into and through
skin was also measured in vitro for conjugates of interferon
.alpha.2b and lauric acid, myristic acid, palmitic acid, stearic
acid and oleic acid, prepared as described above. A preparation of
liposomes having entrapped interferon .alpha.2b was also tested.
The results are shown in FIGS. 8A-8B, where in FIG. 8A the amount
of interferon .alpha.2b absorbed percutaneously is reported, e.g.,
the quantity of interferon .alpha.2b in the downstream receiving
volume after 24 hours. FIG. 8B shows the amount of interferon
.alpha.2b in the skin after 24 hours.
[0066] The figures show that fatty acylation of the cytokine
enhanced percutaneous absorption significantly when compared to
liposomally-entrapped interferon .alpha.2b (closed circles) and
interferon .alpha.2b alone (closed squares). As seen in FIG. 8A,
the conjugate with palmitic acid (closed triangles) had the highest
percutaneous absorption, followed by oleic acid (open triangles),
myristic acid (open diamonds), stearic acid (open circles) and
lauric acid (open squares). Interferon .alpha.2b entrapped in
liposomes (closed squares) and the control formulation of
interferon .alpha.2b alone (closed squares) had the lowest
cutaneous penetration rates.
[0067] FIG. 8B is a bar graph showing the amount of interferon
.alpha. in whole skin and in the viable skin, that is, skin after
removal by tape stripping of the stratum corneum for the
formulations with interferon .alpha.2b shown in FIG. 8A and for two
formulations with interferon .alpha.2a; behenic acid-interferon
.alpha.2a and lignoceric acid-interferon .alpha.2a.
[0068] The in vitro skin penetration results show that fatty
acylation of a cytokine is effective to significantly increase the
skin penetration of the cytokine. "Significantly is increase" or
"substantially higher rate of skin penetration" as used herein
means that the skin penetration, that is cutaneous or
transcutaneous penetration, is increased by at least two-fold, more
preferably three-fold, over the skin penetration of the cytokine
alone.
[0069] III. Method of Use
[0070] In another aspect, the invention includes a method of
transdermally delivering a cytokine by preparing a conjugate of the
cytokine as described above and applying the conjugate to the skin.
In a preferred embodiment of this aspect, the conjugate is composed
of interferon .alpha. and a fatty acid having between 12-24 carbon
atoms and is administered topically for treatment of genital warts
caused by human papilloma virus.
[0071] The conjugate is typically applied to the skin in a
pharmaceutically acceptable preparation, by which is meant any
preparation or device suitable for maintaining the conjugate in
contact with the skin. For example, such a preparation can be a
simple saline solution containing the conjugate that is gelled with
a suitable gelling agent, such as a cellulose derivative, to a
viscosity suitable for application. In general, topical gels,
creams or ointments are preferred, however non-rate limiting
transdermal devices that can incorporate the conjugate are also
suitable.
[0072] Preferred cytokines for use in the invention are interferons
and interleukins. The interferons are a group of immunoregulatory
proteins synthesized by T lymphocytes, fibroblasts and other types
of cells following stimulation with viruses, antigens, mitogens,
double-stranded DNA or lectins. Interferons have immunomodulatory
functions and enhance the ability of macrophages to destroy tumor
cells, viruses and bacteria. Interferons are classified as .alpha.
and .beta., which have antiviral properties, and as .gamma. which
is known as immune interferon. The .alpha. and .beta. interferons
share a common receptor, and .gamma. interferon has its own
receptor. Interferons .alpha. and .beta. are synthesized mainly by
leukocytes and fibroblasts and are acid stable. Interferon .gamma.
is acid labile and is formed mainly by T lymphocytes stimulated by
antigen or mitogen, but is also secreted by natural killer
cells.
[0073] The ability of interferons to prevent infection of
noninfected cells is species specific, it is not virus specific.
Essentially all viruses are subject to the inhibitory action of
interferons. Interferons induce formation of a second inhibitory
protein that prevents viral messenger RNA translations.
[0074] Interferon .alpha.2b (recombinant) is a 18.4 kDa molecular
weight polypeptide consisting of 165 amino acids. Interferon
.alpha. shows multiple biological effects including antiviral,
antiproliferative and immunomodulatory. The mechanism of action is
through binding to specific cell surface receptors. The binding
induces protein kinase and 2'5'-oligoadenylate synthetase (Clemens,
Br. J. Clin. Pract., 42:5, 1988). These enzymes can inhibit protein
synthesis in the cell and therefore can prevent a virus from
replicating (Pestka, et al., Ann. Rev. Biochem., 56:727, 1987).
IV. EXAMPLES
[0075] The following examples illustrate methods of preparing,
characterizing, and using the acylated cytokine conjugate of the
present invention. The examples are in no way intended to limit the
scope of the invention.
[0076] A. Materials
[0077] Interferon .alpha.2b was provided by Schering-Plough
Research Corporation, Kenilworth, N.J. Interferon .alpha.2a and
interferon .gamma. were obtained from (Roche Biosciences). The
fatty acids lauric acid, myristic acid, palmitic acid, stearic
acid, arachidic acid, lignoceric acid, oleic acid and behenic acid
were obtained from Sigma Chemical Co. (St. Louis, Mo.).
N-hydroxysuccinimide was obtained from Sigma Chemical Co.
[0078] B. Methods
[0079] 1. Page
[0080] Polyacrylamide gel electrophoresis (PAGE) in the presence of
sodium dodecyl sulfate (SDS) was carried out in a Mini-Protean II
(BioRad, Missisauga, Ontario, Canada) apparatus according to
Laemmli (Nature, 227:680, 1970). The gel consisted of a running gel
containing 14% (w/v) acrylamide and a stacking gel containing
5-acrylamide. The gel thickness was 1.0 mm. The electrophoresis
buffer was 25 mM Tris, 192 mM glycine, 0.01% (w/v) SDS, pH 8.6.
Electrophoresis was carried out at 200 V constant voltage. The
electrophoresis was conducted for 45 minutes. After
electrophoresis, the gels were silver stained to detect the protein
(Foldvari, et al., Biochem Cell Biol., 68:499, 1990).
[0081] 2. Capillary Electrophoresis
[0082] Capillary electrophoresis studies were performed using a
P/ACE System 5500 (Beckman Instruments, Fullerton, Calif.) with
diode array detector and System Gold Software. Free-zone
electrophoresis was carried out using an uncoated capillary (57
cm.times.75 .mu.m) at 23.degree. C. and 20 KV with a 5 second
pressure injection. The running buffer was 0.6% w/v sodium borate
(Na.sub.2B.sub.4O.sub.7.10H.sub.2O) and 0.5% boric acid, pH 8.75.
The detector was used at 200-300 nm. Prior to use, the capillary
was washed with NaOH (0.1M) for 10 minutes and for 1 minute between
each run.
Example 1
Preparation of Palmitoyl Derivative of Interferon .alpha.2b
[0083] Palmitoyl derivatives of interferon .alpha.2b were
synthesized according to the scheme shown in FIG. 2, where the
N-hydroxysuccinimide ester of palmitic acid (NHS-P) was synthesized
as follows. Equal molar amounts of palmitic acid and
N-hydroxysuccinimide were mixed together in ethyl acetate followed
by addition of dicyclohexylcarbodimide (DCI). The mixture was
stirred overnight at 4.degree. C. Dicyclohexylurea was filtered out
and NHS-P was recrystallized from the filtrate by the addition of
ethanol at 4.degree. C. .sup.1H-NMR studies on NHS-P confirmed the
expected structure (results not shown).
[0084] The palmitoyl derivative of interferon .alpha.2b was
prepared follows. NHS-P was dissolved in DMF or DMSO and added at
25:1 molar ratio to the PBS buffer (7.5 mM Na.sub.2HPO.sub.4, 2.5
mM NaH.sub.2PO.sub.4, 151.2 mM NaCl) containing interferon
.alpha.2.beta. at pH 7.2. The mixture was kept at room temperature
for 3 hours with occasional gentle agitation. After the reaction,
DMF or DMSO was removed under vacuum and the residue was
redissolved in sterile distilled water.
[0085] The palmitoyl-interferon .alpha.2b derivative was separated
from free fatty acid by chromatography on Sephadex G-25 column
(Pharmacia, Uppsala, Sweden). The yield of palmitoyl-interferon
.alpha.2b was dependent on the starting concentration, where a 25
.mu.g batch and a 100 .mu.g batch yielded 50.2% and 84.0%,
respectively, as determined by the densitometry of the
palmitoyl-interferon bands of the column fractions. Fractions
containing protein were pooled, freeze-dried and reconstituted with
sterile distilled water before use.
[0086] A portion of each fraction was used for polyacrylamide gel
electrophoresis (PAGE) and silver staining according to the
procedure described above in the Methods section, and SDS-PAGE
profiles of palmitoyl-interferon .alpha.2b are shown in FIGS.
6A-6B.
Example 2
Antiviral Activity of the Conjugate
[0087] Antiviral activity of palmitoyl derivatives of interferon -a
was determined by the cytopathic effect inhibition assay using
Georgia Bovine Kidney (GBK) cells, which are sensitive to human
interferon .alpha., and vesicular stomatitis virus (VSV) as the
challenge virus (Ohmann, et al., J. Gen. Virol., 65:1485, 1984).
The reference standard was interferon .alpha.2b, specific activity
2.24.times.10.sup.8 IU/mg. The results are shown in Table 2.
Example 3
Conjugate Receptor Binding
[0088] A. Iodination of Interferon
[0089] Iodination of interferon .alpha. and conjugates of
interferon was carried out using the lactoperoxidase method
(Sarkar, et al., Methods Enzymol., 119:263, 1986). Briefly, 2 mCi
.sup.125I, obtained from Amersham Corporation (Oakville, Ontario,
Canada), was neutralized by adding 3 volumes of 0.03 N HCl and the
total was made up to 25 .mu.l with 0.2 M sodium phosphate buffer pH
7.2. The following were added to the mixture: 50 .mu.l Enzymobeads
(Bio-Rad), 15 .mu.l freshly made 2% .beta.-D-glucose in 0.1 M
sodium phosphate buffer, pH 7.2, 10 .mu.l interferon (approximately
10 .mu.g protein) The reaction mixture was incubated for 20 minutes
at room temperature. The reaction was stopped by adding 25 .mu.l of
1 M sodium azide and incubating for 15 minutes. Finally, 125 .mu.l
of saturated L-tyrosine in PBS was added and the mixture
transferred onto a Sephadex G25 column. Fractions containing the
protein were pooled.
[0090] In another method, the iodination mixture was transferred
onto Bio-Spin columns (exclusion limit 6,000) (Bio-Rad) and
iodinated protein recovered by a brief low speed centrifugation. To
remove any possible residue of unbound iodine the protein
preparation was dialyzed overnight against 1 mM sodium iodide in
PBS. This procedure removed practically all acid soluble iodine as
determined by trichloroacetic acid precipitation.
[0091] The final preparations of .sup.125I-interferon .alpha.2A and
.sup.125I-palmitoyl-interferon a had specific activities of
2.05.times.10.sup.7 cpm/.mu.g and 1.94.times.10.sup.7 cpm/.mu.g
protein, respectively. The iodinated interferon .alpha. and
palmitoyl-interferon .alpha. were examined by PAGE for intactness,
and the protein concentration was determined by densitometry.
[0092] B. Receptor Binding
[0093] A single cell suspension of human keratinocyte cells
(isolated from patients undergoing mammoplasty within one day of
surgery) from a confluent culture was prepared and resuspended at
2.times.10.sup.6 cells/mL in KSF-medium. Two mL of KSF-medium was
added to each well of a 6-well flat bottom tissue culture plate and
incubated at 37.degree. C. until the cells in each well reached
confluency. .sup.126I-interferon .alpha.2a conjugates, prepared as
described above, at concentrations between 0.5-40 ng and incubated
at 4.degree. C. for 5 hours on a shaker. The medium was aspirated
from each well to gamma counting tubes and washed three times with
1 mL of cold HBSS. The .sup.125I-interferon sample wells were
scraped using cell scrapers and examined using an inverted
microscope. The cell suspension was transferred to the gamma
counting tubes and the wells were washed three time with 0.5 mL of
HBSS and transferred to the same tubes. One mL of HBSS was added to
every well to wash the wells and the HBSS was transferred to
another tube. The radiation of each tube was counted using a gamma
counter. The cells in the cell control well were detached using
0.25% trypsin. The cells were counted and evaluated to detect
viability by trypan blue exclusion. The results are shown in FIGS.
7A-7B.
Example 4
Measurement of Partition Coefficients
[0094] Human skin was cut into 1.times.1 cm squares and placed into
60.degree. C. water for 1 minute. The epidermis was separated with
forceps. The peeled skin pieces were placed epidermis side down on
filter paper saturated with 1% trypsin solution and incubated at
room temperature for 1 hour. Then the digested epidermis was washed
with water. The stratum corneum pieces were blot dried with tissue
and further dried in a freeze dryer overnight. The stratum corneum
pieces were ground to form powder using liquid nitrogen. The
portion that can pass through a 60-mesh but not 80-mesh sieve was
collected for partition coefficient determination.
[0095] Five milligrams of powdered stratum corneum was weighed into
each vial. Fifty .mu.l of fatty acid derivatized
.sup.125I-interferon .alpha. in phosphate buffered saline was added
to cover the stratum corneum. Empty vials without powdered stratum
corneum were used as controls. The mixture was incubated for 24
hours at 32.degree. C. with gently shaking followed by
centrifugation at 14,000 rpm for 5 seconds. The supernatant was
counted by gamma counting. The powder was washed three times by
adding 50 .mu.l phosphate buffered saline. After washing, the
stratum corneum powder left in the vial was counted.
[0096] The partition coefficient (Kp) was calculated as the ratio
of (cpm.sub.psc/weight of psc)/(cpm.sub.PBS/volume of PBS)
(psc=powdered stratum corneum; PBS=phosphate buffered saline). The
values are shown in Table 5.
Example 5
In Vitro Cutaneous and Percutaneous Absorption
[0097] The rate of diffusion of palmitoyl-interferon .alpha.2b
across full thickness human breast skin (freshly obtained from
plastic surgery and kept at -20.degree. C. until used within 1
week) was investigated using Teflon.RTM., Flow-Thru Diffusion Cells
(Crown Glass Co. Inc., Somerville, N.J.) (Bronaugh and Stewart, J.
Pharm. Sci., 74:64 1985), which have a surface area for diffusion
of 0.32 cm.sup.2. The diffusion cells were operated with a
continuous perfusion fluid flow of PBS pH of 7.2 on the downstream
side in order to maintain sink conditions. The flow rate of the
perfusion fluid was 3 mL per hour.
[0098] The diffusion cells were mounted in a diffusion cell heater
(Crown Glass Co. Inc.) to maintain the temperature at 37.degree. C.
with circulating water. Each cell was connected to a fraction
collector and each experiment was conducted for a continuous period
of 24 hours over which time samples were collected at
intervals.
[0099] The test preparations consisted of 0.1 mL solution [PBS
buffer] or 0.1 g methylcellulose 1500 cP [2.5%] gel hydrated with
PBS labeled with .sup.125I-palmitoyl-interferon .alpha.2b. The test
preparations were instilled into the cells at concentration of
20.times.10.sup.6 IU (89.3 .mu.g) of palmitoyl-interferon .alpha.2b
per g or mL product. The average amount of interferon applied was
20.7 .mu.g/cm.sup.2 skin surface area. The quantity of
palmitoyl-interferon .alpha.2b in the collected fractions was
determined by gamma counting and the results are shown in Table
7.
[0100] After 24 hours, the skin was removed from the diffusion cell
and rinsed thoroughly with cold (4.degree. C.) PBS (3.times.15 mL)
and the skin was blotted with tissue paper. The skin surface was
swabbed with a cotton tip applicator dipped into PBS containing
0.5% Tween 80 two times to remove surface-bound drug. Care was
taken not to disturb the stratum corneum. The skin was carefully
folded (epidermal sides together) to avoid contamination of dermal
side and placed into glass tubes. The radioactivity associated with
the skin was determined by gamma counting and was considered to be
the "whole skin" counts. The skin was then stripped ten times with
a Scotch tape and the radioactivity associated with each strip was
determined separately. The skin after the stripping was counted
again in a clean tube to obtain the counts associated with the
viable skin (epidermis, dermis and subcutaneous tissue). The skin
stripping technique was validated by sectioning the paraffin
embedded stripped skin to observe the complete removal of the
stratum corneum in the light microscope (results not shown).
[0101] Trichloroacetic acid (TCA) precipitation was used to
determine free and bound iodine label in percutaneous fractions and
skin homogenate prepared from treated skin samples. TCA was added
to each sample to 5% w/v concentration and was incubated at
4.degree. C. overnight. The supernatants and pellets were analyzed
by gamma counting after centrifugation in a Beckman Microfuge at
14,000 rpm for 15 minutes. The experiments with TCA precipitation
from skin homogenates (after tape stripping) and fractions showed
that 40-50% of radioactivity was precipitated from both interferon
.alpha.2b and palmitoyl-interferon .alpha.2b, indicating that
protein, not just the free iodine label, was present. The results
are shown in Table 7.
[0102] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention.
Sequence CWU 1
1
1 1 165 PRT Homo sapiens 1 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85
90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met
Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Asp Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu
165
* * * * *