Title: Corticotropin-releasing hormone analogs
Abstract: This invention relates to peptide analogs of corticotropin-releasing hormone. Particularly, the invention provides analogs wherein the 38.sup.th amino acid from the N-terminus is D-Nle, i.e. [D-Nle.sup.38 ]-CRH peptide.
Patent Number: 6,849,600 Issued on 02/01/2005 to Wei, et al.
| Inventors:
|
Wei; Edward T. (Berkeley, CA);
Carlson; Kurt W. (Sausalito, CA)
|
| Assignee:
|
The Regents of the University of California, Berkeley (Berkeley, CA)
|
| Appl. No.:
|
106588 |
| Filed:
|
March 25, 2002 |
| Current U.S. Class: |
514/12; 514/2; 514/9; 530/300; 530/317; 530/324; 930/21; 930/270 |
| Intern'l Class: |
A61K 038/12; A61K038/17; C07K014/47 |
| Field of Search: |
514/2,12,9
530/300,317,324
930/21,270
|
References Cited [Referenced By]
U.S. Patent Documents
| 4677191 | Jun., 1987 | Tanaka et al.
| |
| 4728721 | Mar., 1988 | Yamamoto et al.
| |
| 4801612 | Jan., 1989 | Wei et al.
| |
| 5116868 | May., 1992 | Chen et al.
| |
| 5137871 | Aug., 1992 | Wei.
| |
| 5480869 | Jan., 1996 | Wei et al.
| |
| 5777073 | Jul., 1998 | Rivier.
| |
| 5824771 | Oct., 1998 | Rivier | 530/306.
|
| 5844074 | Dec., 1998 | Rivier.
| |
| 5869450 | Feb., 1999 | Wei et al.
| |
| 6319900 | Nov., 2001 | Wei et al.
| |
Other References
Rivier et al. Single Point D-Substituted Corticotropin-Releasing Factor
Analogues: Effects on Potency and Physicochemical characteristics. J. Med.
Chem. 36: 2851-2859, 1993.*
Gulyas et al. Potent, structurally constrained agonists and competitive
antagonists of corticotropin-releasing factor. Proc. Natl. Acad. Sci. USA
92: 10575-10579, Nov. 1995.*
Beyermann et al. A Single-Point Slight Alteration Set as a Tool for
STructure-Activity Relationship Studies of Ovine Corticotropin Releasing
Factor. J. Med. Chem. 39: 3324-3330, 1996.*
Carlson, et al., "Inhibition of Mouse Melanoma Cell Proliferation by
Corticotropin-Releasing Hormone and its Analogs," AntiCancer Research, 21:
1173-1180 (2001).
Dautzenberg and Hauger, "The CRF peptide family and their receptors: yet
more partners discovered," Trends in Pharm. Sci., vol. 23, 2002, pp.
71-77.
Koerber et al., "Constrained Corticotropin-Releasing Factor (CRF) Agonists
and Antagonists with i-(i+3) Glu-Xaa-pXbb-Lys Bridges," J. Med. Chem.,
vol. 41, 1998, pp. 5002-5011.
Komreich et al., "Alanine Series of ovine Corticotropin Releasing Factor
(oCRF): A Structure-Activity Relationshiop Study," J. Med. Chem., vol. 35,
1992, pp. 1870-1876.
Slominski et al., "Characterization of corticotropin-releasing hormone
(CRH) in human skin," J. Clin. Endocrin. Metab., vol. 83, 1998, pp.
1020-1024.
Slominski et al., "Expression of proopiomelanocortin (POMC)-derived
melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone
(ACTH) peptides in skin of basal cell carcinoma patients," Hum. Pathol.,
vol. 30, 1999, pp. 208-215.
Slominski et al., "Proopiomelanocortin, corticotropin releasing hormone and
corticotropin releasing hormone receptor genes are expressed in human
skin," FEBS Letters, vol. 374, 1999, pp. 113-116.
Stein, J.H., editor, Internal Medicine, Chapter 216, "Psoriasis," 1998, pp.
1300-1302.
Theohardies et al., "Corticotropin-releasing hormone induces skin mast cell
degranulation and increased vascular permeability, a possible explaination
for its proinflammatory effects," Endocrinology, vol. 139, 1998, pp.
403-413.
Tjuvajev et al., "Anti-Neoplastic Properties of Human Corticotropin
releasing Factor: Involvement of the Nitric Oxide Pathway," In Vivo, vol.
12, 1998, pp. 1-10.
Wei and Thomas, "Correlation of Neuroendocrine and Anti-Edema Activites of
Alanine-Corticotropin-Releasing Factor Analogs," European Journal of
Pharmacology, vol. 263, 1994, pp. 319-321.
Wei et al., "D-Amino Acid-Substituted Analogs of Corticotropin-Releasing
Hormone (CRH) and Urocortin with Selective Agonist Activity at CRH.sub.1
and CRH.sub.2.beta. Receptors," Peptides, vol. 19, 1998, pp. 1183-1190.
|
Primary Examiner: Saoud; Christine J.
Attorney, Agent or Firm: Townsend & Townsend & Crew LLP
Claims
What is claimed is:
1. A peptide analog of a Corticotropin-Releasing Hormone (CRH) having the
amino acid sequence of human CRH (SEQ ID NO:1), wherein the 38th amino
acid from the N-terminus is D-Nle.
2. A peptide analog of claim 1 wherein the 20th amino acid from the
N-terminus is D-Glu.
3. A peptide analog of claim 1 wherein amino acids 30 to 33 from the
N-terminus are cyclized.
4. A peptide analog of claim 2 wherein amino acids 30 to 33 from the
N-terminus are cyclized.
5. A pharmaceutical composition comprising a pharmaceutically effective
amount of a peptide analog of claim 1 and a pharmaceutically acceptable
carrier.
6. A pharmaceutical composition of claim 5 comprising from about 0.01% by
weight to about 50% by weight of said peptide analog.
7. A pharmaceutical composition of claim 5 in unit dosage form.
8. A pharmaceutical composition of claim 5 formulated for sustained release
of said peptide analog.
9. The peptide acetyl-cyclo(30-33)[D-Phe12, D-Glu20, Nle21, Glu30, D-His32,
Lys33, D-Nle38]-CRH(4-41).
10. A pharmaceutical composition comprising a pharmaceutically effective
amount of a peptide of the formula acetyl-cyclo(30-33)[D-Phe12, D-Glu20,
Nle21, Glu30, D-His32, Lys33, D-Nle38]-CRH(4-41) and a pharmaceutically
acceptable carrier.
Description
FIELD OF THE INVENTION
This invention relates to peptide analogs of corticotropin-releasing
hormone and their use for treatment of proliferative disorders of the skin
and other tissues.
BACKGROUND OF THE INVENTION
Corticotropin-releasing hormone (CRH, also called CRF or corticoliberin)
was first characterized as a 41-residue peptide isolated from ovine
hypothalami by Vale et al. (1981). Subsequently, the sequence of human-CRH
was deduced from cDNA studies and shown to be identical to that of
rat-CRH. Caprine, bovine, porcine, and white sucker fish CRH have also
been characterized. The sequences of CRH from hoofed animals are
considerably different from the human sequence, but the pig and fish
sequences differ from the human/rat sequence by only 2 out of 41 residues.
Peptides with sequences homologous to that of mammalian CRH are found in
cells of certain frog skins and in the urophysis of fish. The sequence of
sauvagine, a 40-amino acid peptide isolated from the skins of Phyllomedusa
frogs, was reported several years before Vale's description of ovine-CRH.
The structure of sucker fish urotensin I was reported just months after
the description of ovine-CRH and resulted from an independent line of
inquiry by Lederis's group in Canada. Although sauvagine and urotensin I
are known to induce release of adrenocorticotropin (ACTH) from the
pituitary, the primary function of these peptides remains unknown.
In humans, CRH regulates, via release of proopiomelanocortin, ACTH
secretion from the anterior pituitary and has several direct actions on
central and peripheral tissues. CRH has also been found to have direct
anti-inflammatory properties. More recently, evidence has been provided
that mammalian skin cells both produce CRH and express functional CRH
receptors (Slominski et al., FEBS Lett., 374, pp. 113-116, 1995; Slominski
et al., J. Clin. Endocrinol. Metab., 83, pp. 1020-1024, 1998; Slominski et
al., Hum. Pathol., 30, pp. 208-215, 1999), although it was not known
whether locally produced CRH had an additional role in the physiology of
the skin other than as a vasodilator and inhibitor of thermal
injury-induced edema.
Some therapeutic methods and uses for CRH are described by Wei et al. in
U.S. Pat. No. 4,801,612, issued Jan. 31, 1989, entitled "Method of
Inhibiting Inflammatory Response," and U.S. Pat. No. 5,137,871, issued
Apr. 26, 1994, entitled "Treatment to Reduce Edema for Brain and
Musculature Injury." These patents describe the use of CRH to decrease the
leakage of blood components into tissues produced by various adverse
medical conditions, and thus to treat a patient for injury to or disease
of the brain, central nervous system or musculature in which edema is a
factor.
U.S. Pat. No. 5,869,450, issued Feb. 9, 1999, to Wei et al., describes CRH
analogs in which the fifth amino acid from the N-terminus is D-Pro or in
the case of urocortin or sauvagine where the fourth amino acid from the
N-terminus is D-Pro or D-Ser. These analogs have an anti-inflammatory
activity and a disassociated ACTH response.
Cyclic CRH agonists have been described by Rivier et al. (U.S. Pat. Nos.
5,844,074 and 5,824,771). These CRH analogs, modified by cyclization of
residues 30-33 of CRH via a glutamic acid-lysine bridge, are more potent
than native CRH in the release of ACTH and have lower molecular weights
than native CRH. The elimination of residues 1-3 at the N-terminus of CRH
has been shown to not alter biological activities or ACTH-release potency.
(Kornreich et al., J. Med. Chem., 35, pp. 1870-1876, 1992; Koerber et al.,
J. Med. Chem., 41(25), pp. 5002-5011, 1998.)
Tjuvajev et al. in In Vivo, 12, pp.1-10, 1998 reported a series of in vivo
and in vitro studies evaluating the anti-neoplastic potential of CRH in
W256 rat mammary carcinoma. Using magnetic resonance imaging (MRI) and
direct measurements of tumor and peritumoral brain water content they
found that CRH treatment (100 micrograms/kg subcutaneously twice a day for
3 days) caused significant inhibition of growth of
intracerebrally-injected W256 tumor cells. CRH also exhibited
antiproliferative effects in in vitro cultures of W256 cells. The
antiproliferative effects of CRH in W256 cells are believed to involve
activation of nitric oxide synthase (NOS) and L-arginine-NO pathways.
In U.S. Pat. No. 6,319,900, Wei and Slominski disclose that CRH and members
of the CRH superfamily, in which the 20.sup.th amino acid is replaced with
a D-amino acid, have anti-proliferative activity.
Human trials of CRH for the treatment of peritumoral brain edema have been
initiated and preliminary data indicate that CRH reduces brain edema
associated with tumor metastases. However, the limiting factor on the use
of CRH has been the known blood-pressure lowering property of CRH. CRH
causes relaxation of smooth muscles surrounding blood vessels
(vasodilation) resulting in a lowering of blood-pressure. The resultant
hypotension is sufficiently dangerous to limit the dosages of CRH that can
be administered to humans. Overcoming this dose-limiting toxicity by
design of CRH superfamily peptide analogs that have less blood-pressure
lowering activity should improve the therapeutic index and provide useful
anti-proliferative therapeutics.
SUMMARY OF THE INVENTION
This invention provides novel members of the corticiotropin-releasing
hormone superfamily and peptide analogs thereof wherein the 38.sup.th
amino acid from the N-terminus is D-Nle, i.e. [D-Nle.sup.38 ]-CRH peptide.
In one embodiment, in addition to D-Nle.sup.38, the 20.sup.th amino acid
from the N-terminus is a D-amino acid, i.e. [D-aa.sup.20, D-Nle.sup.38
]-CRH peptide. In an alternative D-Nle.sup.38 embodiment, amino acids 30
to 33 from the N-terminus are cyclized. i.e. cyclo(30-33)[D-Nle.sup.38
]-CRH peptide. Particularly preferred are those CRH peptides of general
formula cyclo(30-33)[D-Glu.sup.20, D-Nle.sup.38 ] CRH peptide. A
representative member of this family is the peptide
acetyl-cyclo(30-33)[D-Phe.sup.12, D-Glu.sup.20, Nle.sup.21, Glu.sup.30,
D-His.sup.32, Lys.sup.33, D-Nle.sup.38 ]-CRH(4-41).
Also provided are pharmaceutical compositions of the novel analogs
formulated with pharmaceutically effective amounts of CRH peptide and
pharmaceutically acceptable carriers. Such compositions may have from
about 0.01% by weight to about 50% by weight of peptide, preferably from
0. I% to about 10%. The compositions may be formulated for oral,
parenteral or topical delivery as appropriate for the indication under
treatment. Topical delivery modalities include intrabuccal, intranasal,
intraocular, transdermal, and rectal. The compositions may be provided in
unit dosage form or formulated for sustained release of peptide. A
preferred pharmaceutical composition contains a pharmaceutically effective
amount of a peptide of the formula acetyl-cyclo(30-33)[D-Phe.sup.12,
D-Glu.sup.20, Nle.sup.21, Glu.sup.30, D-His.sup.32, Lys.sup.33,
D-Nle.sup.38 ]-CRH(4-41) and a pharmaceutically acceptable carrier.
The peptides and their compositions are useful for treating proliferative
disorders of the skin or other tissues, such as cancer and inflammatory
dermatoses, particularly in human subjects. Cancers amenable to treatment
with CRH peptides include, but are not limited to melanoma and squamous
cell carcinoma; inflammatory dermatoses include, but are not limited to
eczema and psoriasis. The peptide or peptides may be administered in an
amount of from about 0.001 mg to about 1.0 mg/kg/day of patient body
weight. In particular administration of an effective amount of a peptide
of the formula acetyl-cyclo(30-33)[D-Phe.sup.12, D-Glu.sup.20, Nle.sup.21,
Glu.sup.30, D-His.sup.32, Lys.sup.33, D-Nle.sup.38 ]-CRH(4-41) is
contemplated.
DEFINITIONS
The term "CRH peptide" as used herein refers to an analog of a member of
the CRH superfamily having one or more replacement amino acids.
The term "CRH superfamily" includes those peptides recognized in the art as
belonging to the CRH family due to sequence similarities and similar
biological activities. These include, but are not limited to, the peptides
illustrated in Table 1. Thus, the CRH superfamily includes the CRH
peptides originating with or derived from a number of species, e.g., rat,
human, pig, sheep, cow, and fish, and also includes sauvagine, urotensin I
and urocortin. A more comprehensive list of CRH superfamily peptides has
recently been compiled (Trends in Pharmacological Sciences, 23:2, 71-77
(2002)), incorporated by reference herein.
TABLE 1
Peptides of the Corticotropin-Releasing Hormone Superfamily
SEQ.
ID
NO. PEPTIDE SPECIES SEQUENCE.sup.ab
1 CRH Human/rat SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMEII
2 CRH Pig SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMENF
3 CRH Sucker SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKMMEIF
fish
4 CRH Sheep/Goat SQEPPISLDL TFHLLREVLE MTKADQLAQQ AHSNRKLLDIA
5 CRH Cow SQEPPISLDL TFHLLREVLE MTKADQLAQQ AHNNRKLLDIA
6 Urotensin I Sucker NDDPPISIDL TFHLLRNMIE MARIENEREQ AGLNRKYLDEV
fish
7 Urotensin I Carp NDDPPISIDL TFHLLRNMIE MARIENEREQ AGLNRKYLDEV
8 Urotensin I Maggy SEEPPMSIDL TFHMLRNMIH RAKMEGEREQ ALINRNLLDEV
sole
9 Urotensin I European SEDPPMSIDL TFHMLRNMIH MAKMEGEREQ AQINRNLLDEV
flounder
10 Urocortin Rat DDPPLSIDL TFHLLRTLLE LARTQSQRER AEQNRIIFDSV
11 Urocortin Human DNPSLSIDL TFHLLRTLLE LARTQSQRER AEQNRIIFDSV
12 Sauvagine Frog >EGPPISIDL SLELLRKMIE IEKQEKEKQQ
AANNRLLLDTI
.sup.a The carboxyl termini of these peptides are amidated.
.sup.b Single letter abbreviations for amino acids: S, T, P, A, G; Ser,
Thr, Pro, Ala, Gly; M, L, I, V; Met; Leu, Ile, Val; E, D, N, Q; Glu, Asp,
Asn, Gln; R, K, H; Arg, Lys, His; F, Y, W, Phe, Tyr, Trp; >E;
pyroglutamyl
DETAILED DESCRIPTION OF THE INVENTION
This invention provides novel CRH peptides and their use for treating both
benign and malignant cell proliferative disorders of the skin and other
tissues. For example, the peptides of the instant invention can be used to
treat diseases such as psoriasis, skin cancer and melanoma. Certain
embodiments of the instant invention describe topical administration of
corticotropin-releasing hormone (CRH) peptides to the affected area, while
other embodiments describe parenteral delivery of the CRH peptides. The
peptides themselves are synthetically derived, highly potent, and
spatially constrained analogs of the naturally occurring hormone CRH and
members of the CRH superfamily. U.S. Pat. No. 5,844,074, hereby
incorporated by reference in its entirety, describes methods useful for
the synthesis of these peptides.
The CRH peptides of this invention are based upon our discovery that
replacing the thirty-eighth amino acid with a D-Nle provides improved
anti-neoplastic and anti-cell proliferative activities without inducing
significant hypotension. This substitution may be combined with the
further substitution of a D-amino acid at position 20 in the case of the
41 amino acid containing peptides of the CRH superfamily or replacing the
19th residue of CRH superfamily peptides having 40 amino acid residues
with a D-amino acid residue. In a preferred embodiment the D-amino acid is
D-Glu. Also known in the art are CRH peptides having cyclic bonds, such as
between the residues in the 30 and 33 position, which may be a disulfide
linkage (between two Cys residues) but preferably is an amide-bond (i.e.,
a lactam bridge). Exemplary cyclic analogs are described in U.S. Pat. No.
5,844,074, issued Dec. 1, 1998, to Rivier, incorporated herein by
reference. Such cyclic analogs may be suitable in practicing the subject
invention, provided such cyclic analogs also have a D-Nle at position 38.
CRH and related CRH peptides have been found to inhibit Cloudman cell
proliferation in vitro at picomolar levels. This effect is
concentration-dependent and is inhibited by the non-selective CRH receptor
antagonist, .alpha.-helical-CRH(9-41). The rank order of potency of CRE
and CRH-related peptides provided insight into the CRH receptor subtype
mediating the anti-proliferative effect. Replacement of residue 20 of CRH
with a D-amino acid reduced the potency of CRH at the CRH2 receptor while
activity at the CRH1 receptor was retained. The hypotensive activity of
[D-Glu.sup.20 ]-CRH relative to CRH is diminished, but suppressive effects
on melanoma cell proliferation are retained. Two novel CRH peptides with
D-amino acid substitutions are almost ten-fold more potent than CRH in
suppressing cell proliferation possibly through activation of the CRH1
receptor. Similarly, CRH peptides were found to inhibit in vivo growth of
B 16 melanoma. Agonist activation of the CRH1 receptor signaling system in
malignant melanocytes thus presents itself as a target for melanoma
therapy. Further details may be found in Carlson, et al., Anticancer
Research 21:1173-1180 (2001), incorporated herein by reference.
Since the peptides of this invention inhibit abnormal cell proliferation,
they are useful in a number of different therapeutic applications.
Specific tissues for which clinical usage of these peptides is
contemplated include skin, as well as its adnexal structures such as hair
follicle and sebaceous glands, and other epithelial tissues (eyelids,
nasal membranes, oropharyngeal membranes, upper respiratory tract,
esophagus, lower digestive tract), skeletal muscle, smooth muscle, cardiac
muscle, blood vessels of the brain, blood vessels of the lungs and
kidneys, and endometria. Where the tissues are not readily reached by
topical administration (such as by creams and the like as further
described hereinafter), then alternative modes of administration, such as
oral or parenteral, may be used.
For example, therapeutic uses of these peptides include administration to
treat disseminated cancers, including melanoma, squamous cell carcinoma,
breast cancer, and uterine cancer, premalignant lesions such as lentigo
maligna, actinic keratosis, and, for non-cancerous conditions, such as
psoriasis, eczema, alopecia areata, hypertrichosis or keloids. The
epithelial cells and keratinocytes are cells that line the base of the
epidermis and form new cells which cover the surface of the body. These
cells have a high metabolic activity and turnover; moreover, they
participate in the inflammatory response, as they actively secrete
cytokines and attract other inflammatory cells from the body (white blood
cells). A disruption of keratinocyte activity is prominent in inflammatory
dermatoses, of which psoriasis is a primary example. Other related
conditions are eczema and various forms of dermatitis. Thus, an agent
which inhibits keratinocyte proliferation may be useful for therapy of
inflammatory dermatoses. For example, the basic lesion in psoriasis is
hyperproliferation of keratinocytes in the epidermis. The turnover rate of
these cells may be ten times more rapid than usual, and maturation of the
cells is abnormal. (J. H. Stein, editor, Internal Medicine, chapter 216,
"Psoriasis," pp. 1300-1302, 1998.)
The hair follicle-sebaceous gland unit of the skin, part of the "adnexa" or
appendages of the skin is of pharmacological interest for these reasons:
(a) a large peptide such as a CRH peptide, with a molecular weight of four
to five thousand daltons, can get to targets because it can be formulated
to sit on the skin and penetrate along the hair shaft to the base of the
hair follicle and to the sebaceous gland;
(b) proliferation of hair follicle cells can result in hypertrichosis, so a
peptide like CRH may have value as a means for stopping excessive hair
growth;
(c) proliferation of lymphocytes at the base of the hair follicle, a
condition called lymphocytosis, poses a much serious problem, alopecia
areata, in which there is excessive hair loss. This frequently occurs in
women under stress and causes a strong emotional response, as the hair
comes off in clumps and is cosmetically disfiguring. Current treatment, a
steroid cream, is of limited effectiveness;
(d) proliferation of the epithelial cells of the sebaceous gland during
puberty and other conditions of excessive dihydrotestosterone production
contributes to the condition known as acne.
Typically a therapeutically effective dosage of a CRH peptide is at least
about 0.01% w/w up to about 50% w/w or more, preferably more than 0.1% w/w
of the active compound. The active ingredient may be administered at once,
or may be divided into a number of smaller doses to be administered at
intervals of time or as a controlled release formulation. The term
"controlled release formulation" encompasses formulations that allow the
continuous delivery of a CRH peptide to a subject over a period of time,
preferably several days to weeks. Such formulations may administered
subcutaneously or intramuscularly and allow for the continual steady
release of a predetermined amount of drug in the subject over time. The
controlled-release formulation of CRH peptide may be, for example, a
formulation of drug-containing polymeric microcapsules, such as those
described in U.S. Pat. Nos. 4,677,191 and 4,728,721, incorporated herein
by reference. Alternatively, the controlled-release formulation of CRH
peptide may employ an osmotic pump, e.g. the Alzet pump, commercially
available from Alza, Palo Alto, Calif. The dosage of CRH peptide released
by the sustained-release formulation is preferably about 5-300
microgram/kg/day, and more preferably 10-50 microgram/kg/day. In another
embodiment the controlled release formulation may be provided in a
transdermal (or other tissue) delivery device, employing, for example
concentration gradients or iontophoresis to drive delivery of the CRH
peptide.
The precise dosage and duration of treatment will be a function of the
condition being treated and may be determined empirically using known
testing protocols or by extrapolation from in vivo or in vitro test data.
Concentrations and dosage values may also vary with the age of the
individual treated. For any particular subject, specific dosage regimens
should be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising the
administration of the formulations and that the concentration ranges set
forth herein are exemplary only and are not intended to limit the scope or
practice of the claimed invention.
The CRH peptides may be suspended in micronized or other suitable form and
may be derivatized to produce a more soluble active product or to produce
a prodrug. The form of the resulting mixture depends upon a number of
factors, including the intended mode of administration and the solubility
of the compound in the selected carrier or vehicle. The effective
concentration is sufficient for ameliorating the proliferative condition
and may be empirically determined.
Preferable concentrations are in the range of 0.01% w/w to about 25% w/w,
more preferably 1% w/w to 25% w/w, yet more preferably greater than about
1% w/w to about 10% w/w, and most preferably greater than 1% w/w up to
about 5% w/w. Aqueous suspensions and formulations contain 1% w/w or more.
Suitable therapeutic formulations include solutions, suspensions, emulsions
and the like and may be formulated as creams, gels, ointments, emulsions,
solutions, elixirs, lotions, suspensions, tinctures, pastes, foams,
aerosols, irrigations, sprays, suppositories, bandages, or in any other
form suitable for delivery of the CRH peptide to the target tissue,
including oral, parenteral, and topical formutions. Processes for
producing acceptable formulations are known to the skilled artisan and are
disclosed, inter alia, in Remington: The Science and Practice of Pharmacy,
Mack Publishing Co, 1995, incorporated by reference herein.
Pharmaceutical and cosmetic carriers or vehicles suitable for
administration of the CRH peptides provided herein include any such
carriers known to those skilled in the art to be suitable for the
particular mode of administration. In addition, the CRH peptides may be
formulated as the sole pharmaceutically active ingredient in the
composition or may be combined with other active ingredients. The active
compound is included in the carrier in an amount sufficient to exert a
therapeutically useful effect in the absence of serious toxic effects on
the treated individual. The effective concentration may be determined
empirically by testing the compounds using in vitro and in vivo systems,
including the animal models described herein.
For topical administration, the CRH peptides may be formulated as gels,
creams, lotions, solids, solutions or suspensions, or aerosols.
Compositions for treating human skin are formulated for topical
application with an anti-proliferative effective amount of one or more of
the peptides selected as described herein, in an effective concentration
range (by weight), between about 0.1% and 80%, preferably 0.1 to 50%, more
preferably greater than about 1% up to about 50% or more in a cream,
ointment, lotion, gel, solution or solid base or vehicle known in the art
to be nontoxic and dermatologically acceptable or suitable for application
to the mucosa. Aqueous suspensions are preferably formulated at
concentrations greater than about 1% w/w, more preferably 2% w/w.
To formulate a composition, the weight fraction of CRH peptide is
dissolved, suspended, dispersed or otherwise mixed in a selected vehicle
at an effective concentration such that the proliferative condition is
relieved or ameliorated. Generally, emollient or lubricating vehicles that
help hydrate the skin are preferred to volatile vehicles, such as ethanol
that dry the skin. Examples of suitable bases or vehicles for preparing
compositions for use with human skin are petrolatum, petrolatum plus
volatile silicones, lanolin, cold cream, and hydrophilic ointment.
The choice of an acceptable vehicle is largely determined by the mode of
application and tissue to be treated. Suitable pharmaceutically and
dermatologically acceptable vehicles for topical application include
lotions, creams, solutions, gels, tapes and the like. Generally, the
vehicle is either organic in nature or an aqueous emulsion and capable of
having the selected peptide, which may be micronized, dispersed, suspended
or dissolved therein. The vehicle may include pharmaceutically-acceptable
emollients, skin penetration enhancers, coloring agents, fragrances,
emulsifiers, thickening agents, and solvents.
Suitable lotions contain an effective concentration of one or more of the
peptides. The effective concentration is preferably effective to deliver
an anti-proliferative amount, typically at a concentration of between
about 0.1-50% w/w or more of one or more of the CRH peptides provided
herein. The lotions may also contain from 1% to 50% w/w, preferably from
3% to 15% w/w of an emollient and the balance water, a suitable buffer, a
C.sub.2 or C.sub.3 alcohol, or a mixture of water of the buffer and the
alcohol. Any emollients known to those of skill in the art as suitable for
application to human skin may be used.
Suitable creams are formulated to contain concentration effective to
deliver an anti-proliferative effective amount of the CRH peptide to the
treated tissue, typically from about 0.1%, preferably from about 1% to
about 50%, preferably between about 5% and 15% of one or more of the CRH
peptides provided herein. The creams may also contain from 5% to 50%,
preferably from 10% to 25%, of an emollient and the remainder is water or
other suitable non-toxic carrier, such as an isotonic buffer. The cream
may also contain a suitable emulsifier, at a level from 3% to 50%,
preferably from 5% to 20%.
Solutions and suspensions for topical administration are formulated to
contain an amount of one or more CRH peptides effective to deliver an
anti-proliferative amount, typically at a concentration of between about
0.01 and about 50% w/w, preferably at least 1% w/w, of one or more of the
peptides. The balance may be water, a suitable organic solvent or other
suitable solvent or buffer. Suitable organic solvents include propylene
glycol, polyethylene glycol (M.W. 200-600), polypropylene glycol (M.W.
425-2025), glycerine, sorbitol esters, 1,2,6-hexanetriol ethanol,
isopropanol, diethyltartrate, butanediol and mixtures thereof. Such
solvent systems may also contain water.
Solutions or suspensions useful for local application may include any of
the following components: a sterile diluent, such as water for injection,
saline solution, fixed oil, polyethylene glycol, glycerine, propylene
glycol or other synthetic solvent; antimicrobial agents, such as benzyl
alcohol and methyl parabens; antioxidants, such as ascorbic acid and
sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic
acid (EDTA); buffers, such as acetates, citrates and phosphates; and
agents for the adjustment of tonicity such as sodium chloride or dextrose.
Liquid preparations may be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass, plastic or other suitable material.
Suitable carriers may include physiological saline or phosphate buffered
saline (PBS), and the suspensions and solutions may contain thickening and
solubilizing agents, such as glucose, polyethylene glycol, and
polypropylene glycol and mixtures thereof. Liposomal suspensions may also
be suitable as pharmaceutically acceptable carriers. These may be prepared
according to methods known to those skilled in the art.
Suitably prepared solutions and suspension may also be topically applied to
the eyes and mucosa. Solutions, particularly those intended for ophthalmic
use, may be formulated as 0.01%-10% w/w isotonic solutions, pH about 5-7,
with appropriate salts, and preferably containing one or more of the CRH
peptides at a concentration of about 0.1% w/w to 50% w/w or more. Suitable
ophthalmic solutions are known (see, e.g. U.S. Pat. No. 5,116,868,
incorporated herein, which describes ophthalmic irrigation solutions).
Gel compositions can be formulated by simply admixing a suitable thickening
agent to the previously described solution or suspension composition.
Examples of suitable thickening agents have been previously described with
respect to the lotions. The gelled compositions contain an effective
amount of one or more of a CRH peptide, typically at a concentration of
between about 0.1 and about 50% w/w, preferably from 10% to 50% w/w, of an
organic solvent; from 0.5% to 20% w/w, preferably from 1% to 10% w/w of
the thickening agent; the balance being water or other aqueous carrier.
EXAMPLES
The following Examples are offered to illustrate, but not to limit the
claimed invention.
Example 1
CRH Peptide Synthesis
CRH and CRH peptides used in these Examples have or are based upon the
human-rat CRH sequence. For urocortin, the human sequence was used. CRH,
urocortin, sauvagine, and .alpha.-helical-(9-4l)-CRH were obtained from
Phoenix Pharmaceuticals, Belmont, Calif. [D-Pro.sup.5 ]-CRH, [D-Glu.sup.20
]-CRH, acetyl-cyclo(30-33)[D-Phe.sup.12, D-Glu.sup.20, Nle.sup.18,
Glu.sup.30, D-His.sup.32, Lys.sup.33, D-Nle.sup.38 ]-CRH(4-41), and
acetyl-cyclo(30-33)[D-Phe.sup.12, Nle.sup.18, D-Glu.sup.20, Nle.sup.21,
Glu.sup.30, D-Ala.sup.32 ]-urotensin 1(4-41) were custom synthesized by
Dr. Janos Varga (California Peptide Research, Inc., Napa, Calif.) using
standard solid-phase techniques. The purities of the synthesized peptides,
as determined by HPLC in two buffer systems, were 95 to 99% and the
principal peak of the mass spectrum for each peptide corresponded with the
calculated average mass. For all peptides the amino acid analysis gave the
expected ratio of amino acids. The peptides were stored in vacuo at room
temperature. Peptides were weighed and dissolved in sterile water
containing 0.5% bovine serum albumin (BSA) to a concentration of 10 mM and
frozen at -70.degree. C. 10 mM stock solutions of peptides were diluted on
experimental days with incubation medium for subsequent in vitro studies.
Example 2
In Vitro Assay
S91 mouse Cloudman cells (cell line M3) were obtained from the American
Type Culture Collection. Hams F-10 medium, fetal bovine serum, and horse
serum were obtained from Gibco BRL. Cloudman cells were cultured in Ham's
F-10 medium supplemented with 5% horse serum, 2.5% fetal bovine serum, and
1% penicillin/streptomycin in a humidified incubator with 5% CO2 at
37.degree. C. Media were changed every second day.
Cells were plated at a concentration of .about.20,000 cells per well in
Ham's F-10 medium supplemented with 5% horse serum, 2.5% fetal bovine
serum and 1% penicillin/streptomycin into tissue-culture-12-well plates.
Test peptides or sterile water containing 0.5% BSA (vehicle control) were
added to culture medium prior to plating, and cells were allowed to adhere
overnight in a humidified incubator with 5% CO.sub.2 at 37.degree. C.
Culture medium containing peptides and culture medium containing vehicle
control were changed daily. For counting, cells were detached with 200
.mu.L of 0.25% trypsin. Detachment levels were verified under a light
microscope. After detachment, cell suspensions were transferred to 1.5-mL
Eppendorf tubes, vigorously pipetted for uniform suspension, and cell
number in 10 .mu.L of cell suspension was counted in a hemacytometer under
a light microscope.
CRH and CRH peptides were found to inhibit growth of Cloudman melanoma
cells in vitro in a concentration-dependent manner. Significant
anti-proliferative effects were observed with CRH at the higher
concentrations by the end of the first 24-hr measurement period, and
maximum suppression occurred about 96 hr after treatment. The EC50 of CRH
after 96 hr was 9 (SE 3) pM. No enhancement of the anti-proliferative
effects was observed after 96 hr of CRH treatment, at least out to the end
of the study period, which was 14 days. Other members of the CRH peptide
family--sauvagine (frog), urotensin I (fish), and urocortin (human)--were
also evaluated, as were three synthetic analogs of CRH, for
anti-proliferative effects on Cloudman cells. Like CRH, the three
CRH-related peptides sauvagine, urocortin, and the urotensin analog
acetyl-cyclo(30-33)[D-Phe.sup.12, Nle.sup.18, D-Glu.sup.20, Nle.sup.21,
D-Ala.sup.32 ]-urotensin I(4-41) were highly effective in suppressing
proliferation of Cloudman cells (Table 2). Two new potent CRH analogs
[D-Glu.sup.20 ]-CRH and
acetyl-cyclo(30-33)[D-Phe.sup.12,D-Glu.sup.20,Nle.sup.21, Glu.sup.30,
D-His.sup.32, Lys.sup.33, D-Nle.sup.38 ]-CRH(4-41) (Peptide IV of Table
2), were found to significantly inhibit proliferation. The synthetic
analog acetyl-cyclo(30-33)[D-Phe.sup.12, Nle.sup.18, D-Glu.sup.20,
Nle.sup.21, Glu.sup.30, D-Ala.sup.32 ]-urotensin I(4-41) (Peptide V of
Table 2) was the most potent, with an EC50 10-fold lower than that of CRH.
[D-Pro.sup.5 ]-CRH, a CRH2 receptor-selective agonist was less active than
CRH with an EC50 10-fold greater than CRH. The rank order potencies of the
natural peptides of the CRH family
--CRH.about.urocortin.about.sauvagine--suggests that the
anti-proliferative effects were mediated by CRH1 receptors.
TABLE 2
EC.sub.50 values for CRH peptides versus Cloudman melanoma
Peptide EC.sub.50 (+/- Std. Error) picomolar
I. CRH 8.6 (2.9)
II. [D-Glu.sup.20 ]-CRH 16 (4.6)
III. [D-Pro.sup.5 ]-CRH 82 (14)
IV. Ac-[D-NIe.sup.38 ]-CRH(4-41) 0.48 (0.2)
V. Ac-[D-Ala.sup.32 ]-urotensin I 0.36 (0.1)
VI. Urocortin 9.5 (4.1)
VII. Sauvagine 7.8 (1.9)
Example 3
In Vivo Assay
Female C57B1/6 mice, weighing 20-25 g, were inoculated subcutaneously
(s.c.) with B 16 melanoma cells. Mice were randomized into different
groups and injected 3 to 7 days after inoculation with saline (control) or
acetyl-cyclo(30-33)[D-Phe.sup.12, D-Glu.sup.20, Nle.sup.21, Glu.sup.30,
D-His.sup.32, Lys.sup.22, D-Nle.sup.38 ]-CRH(4-41) s.c. each day for 5
days at 0.1 or 0.2 mg/kg acetyl-cyclo(30-33)[D-Phe.sup.12, D-Glu.sup.20,
Nle.sup.21, Glu.sup.30, D-His.sup.32, Lys.sup.33, D-Nle.sup.38
]-CRH(4-41). The tumor volume was estimated using a caliper-ruler; tumor
size (mm.sup.3) was calculated by multiplying the long axis by the square
of the short axis divided by 2. Inhibition of tumor volume was observed 12
and 14 days after inoculation.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in
light thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and scope of
the appended claims. All publications, patents, and patent applications
cited herein are hereby incorporated by reference in their entirety for
all purposes.
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 12
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: CRH peptide
<400> SEQUENCE: 1
Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala His
20 25 30
Ser Asn Arg Lys Leu Met Glu Ile Ile
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Sus sp.
<220> FEATURE:
<223> OTHER INFORMATION: CRH peptide
<400> SEQUENCE: 2
Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala His
20 25 30
Ser Asn Arg Lys Leu Met Glu Asn Phe
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Pterygoplichthys sp.
<220> FEATURE:
<223> OTHER INFORMATION: CRH peptide
<400> SEQUENCE: 3
Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala His
20 25 30
Ser Asn Arg Lys Met Met Glu Ile Phe
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Oreamnos americanus
<220> FEATURE:
<223> OTHER INFORMATION: CRH peptide
<400> SEQUENCE: 4
Ser Gln Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Glu Val Leu Glu Met Thr Lys Ala Asp Gln Leu Ala Gln Gln Ala His
20 25 30
Ser Asn Arg Lys Leu Leu Asp Ile Ala
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Bos taurus
<220> FEATURE:
<223> OTHER INFORMATION: CRH peptide
<400> SEQUENCE: 5
Ser Gln Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Glu Val Leu Glu Met Thr Lys Ala Asp Gln Leu Ala Gln Gln Ala His
20 25 30
Asn Asn Arg Lys Leu Leu Asp Ile Ala
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Pterygoplichthys sp.
<220> FEATURE:
<223> OTHER INFORMATION: Urotensin I peptide
<400> SEQUENCE: 6
Asn Asp Asp Pro Pro Ile Ser Ile Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Asn Met Ile Glu Met Ala Arg Ile Glu Asn Glu Arg Glu Gln Ala Gly
20 25 30
Leu Asn Arg Lys Tyr Leu Asp Glu Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Carassius sp.
<220> FEATURE:
<223> OTHER INFORMATION: Urotensin I peptide
<400> SEQUENCE: 7
Asn Asp Asp Pro Pro Ile Ser Ile Asp Leu Thr Phe His Leu Leu Arg
1 5 10 15
Asn Met Ile Glu Met Ala Arg Ile Glu Asn Glu Arg Glu Gln Ala Gly
20 25 30
Leu Asn Arg Lys Tyr Leu Asp Glu Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Hippoglossoides sp.
<220> FEATURE:
<223> OTHER INFORMATION: Urotensin I peptide
<400> SEQUENCE: 8
Ser Glu Glu Pro Pro Met Ser Ile Asp Leu Thr Phe His Met Leu Arg
1 5 10 15
Asn Met Ile His Arg Ala Lys Met Glu Gly Glu Arg Glu Gln Ala Leu
20 25 30
Ile Asn Arg Asn Leu Leu Asp Glu Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9
<211> LENGTH: 41
<212> TYPE: PRT
<213> ORGANISM: Platichthys flesus
<220> FEATURE:
<223> OTHER INFORMATION: Urotensin I peptide
<400> SEQUENCE: 9
Ser Glu Asp Pro Pro Met Ser Ile Asp Leu Thr Phe His Met Leu Arg
1 5 10 15
Asn Met Ile His Met Ala Lys Met Glu Gly Glu Arg Glu Gln Ala Gln
20 25 30
Ile Asn Arg Asn Leu Leu Asp Glu Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10
<211> LENGTH: 40
<212> TYPE: PRT
<213> ORGANISM: Rattus sp.
<220> FEATURE:
<223> OTHER INFORMATION: Urocortin peptide
<400> SEQUENCE: 10
Asp Asp Pro Pro Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr
1 5 10 15
Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln
20 25 30
Asn Arg Ile Ile Phe Asp Ser Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11
<211> LENGTH: 40
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Urocortin peptide
<400> SEQUENCE: 11
Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr
1 5 10 15
Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln
20 25 30
Asn Arg Ile Ile Phe Asp Ser Val
35 40
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 12
<211> LENGTH: 40
<212> TYPE: PRT
<213> ORGANISM: Xenopus sp.
<220> FEATURE:
<223> OTHER INFORMATION: Sauvagine peptide
<400> SEQUENCE: 12
Glu Gly Pro Pro Ile Ser Ile Asp Leu Ser Leu Glu Leu Leu Arg Lys
1 5 10 15
Met Ile Glu Ile Glu Lys Gln Glu Lys Glu Lys Gln Gln Ala Ala Asn
20 25 30
Asn Arg Leu Leu Leu Asp Thr Ile
35 40
*
