U.S. patent application number 10/716578 was filed with the patent office on 2004-05-27 for methods of altering the binding affinity of a peptide to its receptor.
Invention is credited to Anderson, Wesley R. JR., Ansari, Aslam M., Ekwuribe, Nnochiri N., Price, Christopher H., Radhakrishnan, Balasingam.
Application Number | 20040102381 10/716578 |
Document ID | / |
Family ID | 22465087 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040102381 |
Kind Code |
A1 |
Ekwuribe, Nnochiri N. ; et
al. |
May 27, 2004 |
Methods of altering the binding affinity of a peptide to its
receptor
Abstract
The present invention relates to amphiphilic drug-oligomer
conjugates capable of traversing the blood-brain barrier ("BBB")
and to methods of making and using such conjugates. An amphiphilic
drug-oligomer conjugate comprises a therapeutic compound conjugated
to an oligomer, wherein the oligomer comprises a lipophilic moiety
coupled to a hydrophilic moiety. The conjugates of the invention
further comprise therapeutic agents such as proteins, peptides,
nucleosides, nucleotides, antiviral agents, antineoplastic agents,
antibiotics, etc., and prodrugs, precursors, derivatives and
intermediates thereof, chemically coupled to amphiphilic
oligomers.
Inventors: |
Ekwuribe, Nnochiri N.;
(Cary, NC) ; Radhakrishnan, Balasingam; (Chapel
Hill, NC) ; Price, Christopher H.; (Chapel Hill,
NC) ; Anderson, Wesley R. JR.; (Raleigh, NC) ;
Ansari, Aslam M.; (Durham, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
22465087 |
Appl. No.: |
10/716578 |
Filed: |
November 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10716578 |
Nov 19, 2003 |
|
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09134803 |
Aug 14, 1998 |
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6703381 |
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Current U.S.
Class: |
424/85.4 ;
424/130.1; 424/94.6; 514/10.8; 514/10.9; 514/11.1; 514/11.3;
514/11.5; 514/11.6; 514/11.7; 514/11.8; 514/11.9; 514/12.3;
514/12.6; 514/13.3; 514/14.2; 514/18.5; 514/3.2; 514/5.2;
514/7.7 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 5/10 20180101; A61P 43/00 20180101; A61K 47/60 20170801; A61P
5/02 20180101; A61P 5/18 20180101; A61P 5/14 20180101 |
Class at
Publication: |
514/012 ;
424/130.1; 424/094.6 |
International
Class: |
A61K 038/23; A61K
038/42; A61K 038/33 |
Claims
What is claimed is:
1. A method for altering the binding affinity of a peptide to its
receptor, comprising conjugating the peptide to an amphiphilic
oligomer comprising a lipophilic moiety coupled to a hydrophilic
moiety.
2. The method according to claim 1 further characterized in that
the binding affinity is increased.
3. The method according to claim 1 further characterized in that
the binding affinity is reduced.
4. The method of claim 1, wherein the peptide is a peptide or
protein.
5. The method of claim 4, wherein the peptide is selected from the
group consisting of: enkephalin, adrenocorticotropic hormone,
adenosine deaminase, ribonuclease, alkaline phosphatase,
angiotensin, antibodies, arginase, arginine deaminease,
asparaginase, caerulein, calcitonin, chemotrypsin, cholecystokinin,
clotting factors, dynorphins, endorphins, enkephalins,
erythropoietin, gastrin-releasing peptide, glucagon, hemoglobin,
hypothalmic releasing factors, interferon, katacalcin, motilin,
neuropeptide Y, neurotensin, non-naturally occurring opioids,
oxytocin, papain, parathyroid hormone, prolactin, soluble CD-4,
somatomedin, somatostatin, somatotropin, superoxide dismutase,
thyroid stimulating hormone, tissue plasminogen activator, trypsin,
vasopressin, and analogues and fragments of such peptides.
6. The method of claim 4 wherein the peptide is
[met.sup.5]enkephalin.
7. The method of claim 1, wherein the lipophilic moiety is selected
from the group consisting of fatty acids, C.sub.1-26alkyls; and
cholesterol.
8. The method of claim 1, wherein the hydrophilic moiety is
selected from the group consisting of sugars or PEG.sub.1-7.
9. The method of claim 1, wherein the receptor is an opioid
receptor.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 09/134,803, filed Aug. 14, 1998, allowed, the
disclosure of which is incorporated herein by reference in its
entirety.
1. BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to amphiphilic oligomer
conjugates capable of traversing the blood-brain barrier ("BBB")
and to methods of making and using such conjugates. The conjugates
of the invention comprise therapeutic agents such as proteins,
peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, etc., and prodrugs, precursors,
derivatives and intermediates thereof, chemically coupled to
amphiphilic oligomers.
[0004] 2. Description of the Related Art
[0005] In the field of pharmaceutical and therapeutic invention and
the treatment of disease states and enhancement of physiological
conditions associated with the CNS, a wide variety of therapeutic
agents have been developed, including proteins, peptides,
nucleosides, nucleotides, antiviral agents, antineoplastic agents,
antibiotics, etc., and prodrugs, precursors, derivatives and
intermediates thereof.
[0006] Additionally, the many known neuroactive peptides offer
additional possibilities for useful therapeutic agents. Such
neuroactive peptides play important biochemical roles in the CNS,
for example as neurotransmitters and/or neuromodulators. Delivery
of this diverse array of peptides to the CNS provides many
opportunities for therapeutic benefit. For example, delivery of
endogenous and synthetic opioid peptides, such as the enkephalins,
can be used to effect analgesia.
[0007] However, a number of obstacles currently limit the use of
many compounds for use as CNS therapeutic agents.
[0008] First, the brain is equipped with a barrier system. The
brain barrier system has two major components: the choroid plexus
and the blood-brain barrier (BBB). The choroid plexus separates
cerebrospinal fluid (CSF) from blood and the BBB separates brain
ISF from blood.
[0009] The BBB has about 1000 times more surface area than the
choroid plexus and is the primary obstacle to delivery of
therapeutic compounds to the CNS. The BBB acts as a selective
partition, regulating the exchange of substances, including
peptides, between the CNS and the peripheral circulation. The
primary structure of the BBB is the brain capillary endothelial
wall. The tight junctions of brain capillary endothelial cells
prevent circulating compounds from reaching the brain ISF by the
paracellular route. Furthermore, recent work suggests the existence
of a physiological barrier at the level of the basal lamina, in
addition to the barrier provided by the tight junctions. Kroll et
al., Neurosurgery, Vol. 42, No. 5, p.1083 (May 1998). Other unique
characteristics of the BBB include lack of intracellular
fenestrations and pinocytic vesicles and a net negative charge on
the luminal surface of the endothelium. Id.
[0010] The mechanisms by which substances may traverse the BBB may
generally be divided into active and passive transport mechanisms.
Lipophilic molecules readily traverse the BBB by passive transport
or diffusion through the endothelial plasma membranes. In contrast,
hydrophilic molecules, such as peptides, typically require an
active transport system to enable them to cross the BBB. Certain
larger peptides, such as insulin, have receptors on the luminal
surface of the brain capillaries which act as active transcytosis
systems.
[0011] Diffusion of many therapeutic compounds, such as peptides,
across the BBB is also inhibited by size. For example, cyclosporin,
which has a molecular weight of .about.1200 Daltons (Da), is
transported through the BBB at a much lower rate than its lipid
solubility would predict. Such divergence between lipid solubility
and BBB permeation rates is probably due to steric hinderances and
is common where the molecular weight of a compound exceeds 800-1000
Da.
[0012] A further barrier to peptide delivery to the CNS is
metabolic instability. In particular, before peptides injected into
the blood reach the CNS, they must survive contact with enzyme
degrading enzymes in the blood and in the brain capillary
endothelium. BBB enzymes are known to degrade most naturally
occurring neuropeptides. Orally administered peptides face
additional barriers discussed below. Metabolically stablized
peptides may exhibit increased resistance to certain enzymes;
however, it has not been possible to protect peptides from the wide
range of peptide-degrading enzymes present in the blood and
BBB.
[0013] Another difficulty inherent in delivering peptides to the
BBB is that successful transcytosis is a complex process which
requires binding at the lumenal or blood side of the brain
capillary endothelium, movement through the endothelial cytoplasm,
and exocytosis at the ablumenal or brain side of the BBB. Peptides
may bind to the lumenal membrane of the brain capillary endothelium
or undergo binding and endocytosis into the intracellular
endothelial compartment without being transported into the CNS.
[0014] In any event, many currently existing drug substances,
especially peptides, are unable to overcome these structural and
metabolic barriers to enter the BBB in sufficient quantities to be
efficacious. There is therefore a need for pharmaceutical
compositions which can (1) withstand degradative enzymes in the
blood stream and in the BBB and (2) which can penetrate through the
BBB in sufficient amounts and at sufficient rates to be
efficacious.
[0015] Many attempts have been made in the art to deliver
therapeutic compounds, such as peptides, to the CNS with varying
levels of success. Such attempts can generally be grouped into two
categories: invasive and pharmacological.
[0016] Invasive delivery strategies include, for example,
mechanical procedures, such as implantation of an intraventricular
catheter, followed by pharmaceutical infusion into the ventricular
compartment. Aside from general considerations relating to the
invasiveness of mechanical procedures, a major difficulty with
mechanical approaches is the lack of peptide distribution. For
example, injection of peptides into the CSF compartment results in
very little distribution beyond the surface of the brain. This lack
of distribution is due in part to rapid exportation of peptides to
the peripheral circulation.
[0017] Another invasive strategy for delivering therapeutic
compounds to the CNS is by intracartoid infusion of highly
concentrated osmotically active substances, such as mannitol or
arabinose. Their high local concentration causes shrinkages of the
brain capillary endothelial cells, resulting in a transient opening
of the tight junctions which enable molecules to traverse the BBB.
Such procedures have considerable toxic effects, including
inflammation, encephalitis, etc. Furthermore, such procedures are
not selective: the opening of the tight junctions of the BBB
permits many undesirable substances to cross the BBB along with the
therapeutically beneficial molecule. For a recent review of osmotic
opening and other invasive means for traversing the BBB, see Kroll,
Robert A. Neurosurgery, Vol. 42, No. 5, May 1998.
[0018] While the risks involved in these invasive procedures may be
justified for life-threatening conditions, they are generally not
acceptable for less dramatic illnesses. There is therefore a need
for less invasive, non-mechanical and safer means for enabling
therapeutic compounds to cross the BBB.
[0019] As noted above, lipophilic substances can generally diffuse
freely across the BBB. Accordingly, a common pharmacological
strategy for enabling peptides to traverse the BBB is to chemically
modify the peptide of interest to make it lipid-soluble.
Hydrophilic drug substances have been derivatized with short chain
or long chain fatty acids to form prodrugs with increased
lipophilicity.
[0020] Prodrugs are biologically inert molecules which require one
or more metabolic steps to convert them into an active form. A
difficulty with the prodrug approach to crossing the BBB is that
the cleavage necessary to yield an active drug may not occur with
sufficient efficiency and accuracy to produce an efficacious amount
of the drug.
[0021] There is therefore a need for modified stable therapeutic
compounds, such as peptides, which are capable of traversing the
BBB but which retain all or part of their efficacy without
requiring metabolic steps to convert them into an active form.
[0022] A further difficulty with lipidized prodrugs is that they
pass in and out of the CNS so readily that they may never reach
sufficient concentration in the CNS to achieve their intended
function. For example, previous attempts have been made to engineer
enkephalin conjugates which can traverse the BBB. See Partridge,
W.M., "Blood-Brain Barrier Transport and Peptide Delivery to the
Brain," Peptide-Based Drug Design: Controlling Transport and
Metabolism, p. 277 (1995). However, these strategies required the
subcutaneous delivery of frequent and massive doses of peptide to
induce analgesia. Frequent and/or massive dosing is inconvenient to
the patient and may result in serious side effects.
[0023] There is therefore a need in the art for means for enabling
therapeutic agents, such as peptides, to cross the BBB in a
controlled manner which permits accumulation of sufficient
quantities of the therapeutic in the brain to induce the desired
therapeutic effect.
[0024] Another pharmacological method for delivering peptides
across the BBB is to covalently couple the peptide of interest to a
peptide for which a specific receptor-mediated transcytosis system
exists. For example, it is theoretically possible to attach
.beta.-endorphin, which is not normally transported through the
BBB, to insulin to be transported across the BBB by insulin
receptor-mediated transcytosis. Upon entry into the brain
interstitial space, the active peptide (.beta.-endorphin) is then
released from the transport vector (insulin) to interact with its
own receptor.
[0025] However, the difficulty with this system is designing a
chimeric molecule which can become detached upon entry into the
interstitial space; to the inventor's knowledge, this has not yet
been achieved. Additionally, the poor stoichiometry of the
neuropeptide to the carrier molecule limits the mass of the target
peptide. Furthermore, receptor-mediated cellular transport systems
typically have physiologically limited transport capacity. This is
a rate-limiting factor which can prevent entry of pharmaceutically
active amounts of peptide.
[0026] There is therefore a need in the art for means for enabling
therapeutic substances, such as peptides, to cross the BBB by
diffusion so as to avoid the limitations inherent in
receptor-mediated transport.
[0027] Other pharmacological strategies include using an active
fragment of a native peptide; modification of a native peptide to
increase blood-brain barrier (BBB) transport activity; and delivery
of a gene encoding the neuropeptide to the brain.
[0028] Oral administration is a desirable and convenient route of
administration; however, orally delivered peptides must overcome a
series of barriers before they can enter the blook stream. Such
peptides must survive proteolysis and the acidic environment of the
stomach, gastric and pancreatic enzymes, exo- and endopeptidases in
the intestinal brush border membrane.
[0029] There is therefore a need for orally administered peptides
which can also resist proteolytic enzymes in the blood and BBB and
which can traverse the BBB in sufficient quantities to provide
broad distribution of drugs into the entire brain parenchyma.
[0030] Methionine-enkephalin and leucine-enkephalin are naturally
occurring analgesic pentapeptides. These peptides and their analogs
are known to act as neurotransmitters or modulators in pain
transmission. Their analgesic properties are short in duration.
When administered by intracerebroventricular injection, the
duration of their action is also transient.
[0031] These properties make the enkephalins attractive compounds
for use as therapeutic agents, for mediating analgesia and
providing a viable alternative to morphine. However, in order to
deliver enkephalkins across the BBB, they must be protected against
rapid degration by aminopeptidases and enkephalinases. Furthermore,
since enkephalins are hydrophilic peptides, they must be modified
to provide them with increased lipophilic characteristics before
they can passively diffuse across the BBB into the CNS.
[0032] The attractive therapeutic properties of enkephalins have
been known for some time, and many investigators have attempted to
enhance the ability of enkephalins to traverse the BBB.
[0033] Schroder et al., Proc. lnt. Symp. Control Rel. Biact.
Material, Vol. 23, p. 611 (1996) teaches that Dalargin, a
leu-enkephalin analogue can be incorporated in nanoparticles formed
by polymerization of butylanoacrylate. The particles are coated
with polysorbate, a penetration enhancer. Analgesic activity is
obtained after intravenous administration. Unlike the present
invention, however, the Schroder peptide must be chemically bound
to the polymeric material or to the polysorbate. The formulation is
therefore a physical mixture of active drug and polymeric
material.
[0034] Tsuzuki et aL, Biochem. Pharm. Vol. 41, p. R5 (1991) teaches
that analogues of leu- enkephalin can be derivatized with
adamantane moiety to obtain lipophilic enkephalin that shows an
antinociceptive effect after subcutaneus administration.
Modification at the N-terminus abolishes activity while the
derivative at the C-terminus through ester bond retains activity.
It is postulated that the activity is obtained after cleavage of
the adamantane moiety. The derivative is therefore a prodrug, a
concept not consistent with aspects of the present invention in
which the therapeutic conjugate retains the activity of the native
peptide.
[0035] Prokai-Tatra, J. M. Chem, Vol. 39, p. 4777 (1996) teaches
that a leucine-enkephalin analogue can be modified with chemical
delivery system which is based on a retrometabolic drug design. The
enkephaklin analogue is derivatized with a dihydropyridine moiety
at the N-terminus and a lipophilic moiety at the C-terminus. After
intravenous administration of the conjugate, analgesic response is
observed. It is postulated that the lipophilic modification at the
C-terminus enables penetration into the CNS, while the
dihydropyridine moiety undergoes oxidative transformation to
generate a charged moiety which restricts the peptides from
effluxing into the circulatory system. Cleavage of the peptide from
this moiety restores the observed analgesic activity. The
derivatized peptide is inactive and regains activity only after
metabolic transformation. The product is therefore a pure prodrug,
requiring metabolic transformation to transform it into an active
form.
[0036] U.S. Pat. No. 4,933,324 to Shashoua teaches that certain
natural fatty acids can be conjugated to neuroactive drugs. A
highly unsaturated fatty acid of twenty-two (22) carbon chain
length is particularly preferred. Administration of the conjugate
shows absorption into the brain. As is the case with adamantane
conjugation, this approach requires metabolic transformation of the
prodrug conjugate of enkephalin to restore the activity of the
enkephalin peptide.
[0037] There is therefore a compelling need in the art for
pharmaceutically acceptable and effective therapeutic/diagnostic
compositions capable of traversing the BBB without substantial loss
or diminution of their therapeutic or diagnostic character.
2. SUMMARY OF THE INVENTION
[0038] The present invention broadly relates to therapeutic and/or
diagnostic drug-oligomer conjugates wherein a drug molecule is
covalently bonded to an oligomer to form an amphiphilic conjugate.
In one aspect, the oligomer comprises at least one lipophilic
moiety and at least one hydrophilic moiety, and the size and nature
of the amphiphilic and lipophilic moieties is so selected as to
impart an amphiphilic nature to the resulting conjugate.
[0039] The present invention relates generally to amphiphilic
drug-oligomer conjugates capable of traversing the BBB and to
methods of making and using such conjugates.
[0040] In one aspect, the therapeutics are neuroactive drugs,
proteins, peptides and especially enkephalin analogues. The
conjugates are stable in the environment of the bloodstream and
resist degradation by the enzymes of the BBB and in the CNS.
Furthermore, the conjugates readily traverse the BBB.
[0041] In one aspect, the drug-oligomer conjugates produce their
intended pharmacological effect without undergoing metabolic
cleavage of the oligomer.
[0042] In another aspect, the lipophile and hydrophile are
connected by a labile, hydrolyzable bond. When the bond is
hydrolyzed in the CNS, the hydrophile remains attached to the
drug.
[0043] The amphiphilic oligomers are composed of lipophilic and
hydrophilic moieties. The lipophilic moieties are preferably
natural fatty aids or alkyl chains. Preferably, the fatty- acid
moiety is a straight chain molecule having (saturated or
unsaturated) carbon atoms and suitably ranges from four (4) to
twenty-six (26) carbon atoms. Most preferably, the fatty acid has
from fourteen (14) to twenty-two (22) carbon atoms.
[0044] The hydrophilic moieties are preferably small segment of
polyethylene glycol (PEG), preferably having 1-7 PEG units, and
more preferably 1-5 PEG units. The length and composition of the
lipophilic moieties and the hydrophilic moieties may be adjusted to
obtain desired amphiphilicity.
[0045] In another aspect, a cholesterol or adamantane moiety is
substituted for straight chain fatty acid portion of the
oligomers.
[0046] Examples of preferred oligomers are as follows:
CH.sub.3(CH.sub.2).sub.n(OC.sub.2H.sub.4).sub.mOH (Formula 1);
[0047] wherein n=3 to 25 and m=1 to 7;
CH.sub.3(CH.sub.2).sub.n(OC.sub.2H.sub.4).sub.mOCH.sub.2CO.sub.2H
(Formula 2);
[0048] wherein n=3 to 25 and m=1 to 6;
CH.sub.3(CH.sub.2).sub.nCX(OC.sub.2H.sub.4).sub.mOH (Formula
3);
[0049] wherein n=3 to 25, m=1 to 7 and X=O;
R--(OC.sub.2H.sub.4).sub.mCH.sub.2CO.sub.2H (Formula 4)
[0050] wherein m=0 to 5 and R=cholesterol or adamantane; or
R--OCO(C.sub.2H.sub.4O).sub.mCH.sub.2CO.sub.2H (Formula 5);
[0051] wherein m=0 to 5;
CH.sub.3(CH.sub.2--CH.dbd.CH).sub.6(CH.sub.2).sub.2CH.sub.2(OC.sub.2H.sub.-
4).sub.mOH (Formula 6);
[0052] wherein m=0 to 7;
CH.sub.3(CH.sub.2--CH.dbd.CH).sub.6(CH.sub.2).sub.2CX(OC.sub.2H.sub.4).sub-
.mOH (Formula 7);
[0053] wherein m=1 to 7 and X=N or 0.
[0054] Other unsaturated fatty acid moieties which can be used
according to the present invention include oleic, linoleic, and
linolenic.
[0055] For example, in one aspect, the lipophile and hydrophile are
connected by hydrolyzable bonds. It is prefered to provide
hydrolyzable bonds between the fatty acid and hydrophilic moieties.
This permits hydrolysis to occur after penetration into the CNS,
thus releasing the active peptides with the hydrophilic group still
attached to the peptide. As a result, the peptide acquires a more
hydrophilic character and efflux to the circulatory system is
thereby hindered.
[0056] Exemplary conjugates having non-hydrolyzable bonds are as
follows: 1
[0057] In another aspect, the lipophile and hydrophile are
connected by hydrolizable bonds.
[0058] For example: 2
[0059] In one aspect, the covalent bond between the oligomer and
the drug is preferably amide (a carboxy group of the oligomer is
linked to an amine group of the peptide drug), or carbamate (an
chloroformate group of the oligomer is linked to an amine group of
the peptide drug). In general, the derivitizable amine group of the
peptide is the amine of the N-terminus or a nucleophilic amino
residue, usually found on the epsilon amino residue of a lysine
residue.
[0060] In another aspect, an ester (a carboxy group of the peptide
is covalently coupled to a hydroxyl group of the oligomer or a
carboxy group of the oligomer is covalently coupled to a hydroxyl
group of the drug), amide (a carboxy group of the oligomer is
linked to an amine group of the drug) or carbamate (an
chloroformate group of the oligomer is linked to an amine group of
the drug) bond is provided for non-peptide drugs.
[0061] For the enkephalin analogues, the preferred peptides are
leu-enkephalin lysine and met-enkephalin lysine. The amino side
chain of the lysine is preferably utilized in bonding.
[0062] The amphiphilic drug-oligomer conjugate may comprise
multiple oligomers of differing compositions.
[0063] In another aspect, the amphiphilic drug-oligomer conjugates
moieties are configured as follows:
[0064]
R--OCH.sub.2CH.sub.2OCH.sub.2C(O)OCH.sub.2CH.sub.2CH.sub.2OCH.sub.2-
CH.sub.2CH.sub.2NH--Enkephalin; or
[0065]
R--OCH.sub.2CH.sub.2OCH.sub.2C(O)OCH.sub.2CH.sub.2NH--Enkephalin
.
[0066] Wherein R=alkyl.sub.1-26, cholesterol or amantane.
[0067] In another aspect of the amphiphilic oligomer moieties are
sugar moieties coupled to natural fatty acids and segments of
polyethylene glycol. The PEG moiety serves to increase the
amphiphilicity of the fatty sugar. Examples of arrangements
including sugar moieties are provided in FIGS. 1A-1C.
[0068] In another aspect, PEG is used as a spacer group in the
amphiphilic conjugate and the length and number of the PEG moieties
can be varied to refine the amphiphilicity of the conjugate.
Increasing the number of PEGs increases the hydrophilicity of the
conjugate.
[0069] In another aspect of the invention, a proline or alanine is
added to the N-terminus of the peptide. In a preferred aspect, a
proline or alanine is added to the N-terminus of an enkephalin
peptide and the oligomer moiety is coupled to the N-terminus of the
proline or alanine residue.
[0070] After absorption into the central nervous system, the esters
of the fatty sugar are hydrolysized leaving a hydrophilic moiety.
Efflux is hindered and the brain aminopeptidases cleave the proline
or alanine portion leaving the peptide to regain full activity.
[0071] The invention also provides a pharmaceutical composition
comprising an amphiphilic drug-oligomer conjugate and a
pharmaceutically acceptable carrier.
[0072] In another aspect, a pharmaceutical composition is provided
comprising (1) a mixture of an enkephalin conjugate according to
the present invention wherein the enkephalin peptide has proline or
alanine added to its N-terminus and an enkephalin conjugate
according to the present invention which does not have a proline or
alanine added to the N-terminus, and (2) a pharmaceutical carrier.
This aspect provides a faster acting sustained dose of
enkephalin.
[0073] The invention also provides methods of administering a
conjugate of the invention.
[0074] The invention further provides assays, both in vitro and in
vivo, for testing the efficacy of the conjugates of the
invention.
[0075] Other objects and further scope of applicability of the
present invention will become apparent from the detailed
description given hereafter. It should be understood that the
detailed description and specific examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only, since various changes and modifications will
become apparent to those skilled in the art from the detailed
description.
[0076] 2.1 DEFINITIONS
[0077] As used herein, the term "lipophilic" means the ability to
dissolve in lipids and/or the ability to penetrate, interact with
and/or traverse biological membranes.
[0078] As used herein, the term, "lipophilic moiety" or "lipophile"
means a moiety which is lipophilic and/or which, when attached to
another chemical entity, increases the lipophilicity of such
chemical entity, e.g., fatty acid, cholesterol.
[0079] As used herein, the term "hydrophilic" means the ability to
dissolve in water.
[0080] As used herein, the term "hydrophilic moiety" or
"hydrophile" refers to a moiety which is hydrophilic and/or which
when attached to another chemical entity, increases the
hydrophilicity of such chemical entity, e.g., sugars, PEG.
[0081] As used herein, the term "amphiphilic" means the ability to
dissolve in both water and lipids.
[0082] As used herein, the term "amphiphilic moiety" means a moiety
which is amphiphilic and/or which when attached to a peptide or
non-peptide drug increases the amphiphilicity of the resulting
conjugate, e.g., PEG-fatty acid oligomer, sugar-fatty acid
oligomer.
[0083] As used herewith, the term "neuroactive drug" is used
broadly to encompass any peptide or other drug having an activity
within the CNS, e.g., enkephalin, enkephalin analogues.
[0084] As used herein the term "peptide" is intended to be broadly
construed as inclusive of polypeptides per se having molecular
weights of up to about 10,000, as well as proteins having molecular
weights of greater than about 10,000.
[0085] As used herein, the term "covalently coupled" means that the
specified moieties are either directly covalently bonded to one
another, or else are indirectly covalently joined to one another
through an intervening moiety or moieties, such as a bridge,
spacer, or linkage moiety or moieties.
[0086] As used herein, the term "drug" means a substance intended
for use in the diagnosis, characterization, cure, mitigation,
treatment, prevention or allaying the onset of a disease, disease
state, or other physiological condition or to enhance normal
physiological functioning in humans and/or in non-human
animals.
3. BRIEF DESCRIPTION OF THE FIGURES
[0087] FIGS. 1A-1C: Formulae 8-10; amphiphilic oligomers of the
present invention where in the lipophile is a sugar. In 1b and 1c,
PEG is used as a spacer group. In 1A-1C a proline residue is added
at the N-terminus of the enkephalin peptide.
[0088] FIG. 2: Compares the stability of the
cetyl-PEG2-enkephalin-lys conjugate (non-hydrolyzable) to
unconjugated enkephalin in rat brain homogenate.
[0089] FIG. 3: Compares the stability of the cetyl-PEG3-enkephalin
conjugate (non-hydrolyzable) to unconjugated enkephalin in rat
brain homogenate.
[0090] FIG. 4: Compares palmitate-PEG3-Enk conjugate (hydrolyzable)
to unconjugated enkephalin in rat brain homogenate.
[0091] FIG. 5a-5d: HPLC data showing extraction of conjugate from
homogenized rat brain.
[0092] FIG. 6: Graph demonstrating competitive binding between
cetyl-PEG.sub.2-enkephalin conjugate and naloxone, an Opioid .mu.
receptor agonist.
[0093] FIG. 7: Graphic comparison of analgesic effect of
cetyl-PEG.sub.2-enkephalin with clonidine (a morphine
substitute).
[0094] FIG. 8: Table showing results of receptor binding assays for
various conjugates according to the present invention.
[0095] FIG. 9: Exemplary synthetic scheme for an oligomer according
to the present invention.
[0096] FIG. 10: Exemplary synthetic scheme showing attachment of an
oligomer to an enkephalin peptide according to the present
invention.
4. DETAILED DESCRIPTION OF THE INVENTION
[0097] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
[0098] The present invention relates generally to amphiphilic
drug-oligomer conjugates capable of traversing the BBB and to
methods of making and using such conjugates.
[0099] The drugs are preferably neuro-active drugs, proteins,
peptides and especially enkephalin analogues. The conjugates are
stable in the environment of the bloodstream and resist degradation
by the BBB. The conjugates readily traverse the BBB.
[0100] In one aspect, the conjugates produce their intended
pharmacological effect without requiring metabolic cleavage of the
oligomer. When cleavage of the oligomer occurs, the drug retains
activity.
[0101] The amphiphilic oligomers are composed of lipophilic and
hydrophilic moieties. The lipophilic moieties are preferably
natural fatty aids or alkyl chains. The lipophilic moieties are
preferably small segments of PEG, having 1 to 7 PEG moieties, and
preferably having 1 to 5 PEG moieties. The length and composition
of the lipophilic moieties and the hydrophilic moieties may be
adjusted to obtain desired amphiphilicity. For example, the carbon
chains of the fatty acid or alkyl moieties may be lengthened to
increase lipophilicity, while PEG moieties may be lengthened to
increase hydrophilicity.
[0102] Preferably, the fatty-acid moiety is a straight chain
molecule having saturated and unsaturated carbons and ranges from
four (4) to twenty-six (26) carbon atoms. Most preferably, the
fatty acid has from fourteen (14) to twenty-two (22) carbon
atoms.
[0103] A cholesterol or adamantane moiety can be substituted for
straight chain fatty acid as the lipophilic portion of the
oligomers.
[0104] Examples of preferred oligomers are as follows:
CH.sub.3(CH.sub.2).sub.n(OC.sub.2H.sub.4).sub.mOH (Formula 1);
[0105] wherein n=3 to 25 and m=1 to 7;
CH.sub.3(CH.sub.2).sub.n(OC.sub.2H.sub.4).sub.mOCH.sub.2CO.sub.2H
(Formula 2);
[0106] wherein n=3 to 25 and m=1 to 6;
CH.sub.3(CH.sub.2).sub.nCO(OC.sub.2H.sub.4).sub.mOH (Formula
3);
[0107] wherein n=3 to 25, and m=1 to 7;
R--(OC.sub.2H.sub.4).sub.mCH.sub.2CO.sub.2H (Formula 4)
[0108] wherein m=0 to 5 and R=cholesterol or adamantane; or
R--OCO(C.sub.2H.sub.4O).sub.mCH.sub.2CO.sub.2H (Formula 5);
[0109] wherein m=0 to 5;
CH.sub.3(CH.sub.2--CH.dbd.CH).sub.6(CH.sub.2).sub.2CH.sub.2(OC.sub.2H.sub.-
4).sub.mOH (Formula 6);
[0110] wherein m=1 to 7;
CH.sub.3(CH.sub.2--CH=CH).sub.6(CH.sub.2).sub.2CO(OC.sub.2H.sub.4).sub.mOH
(Formula 7);
[0111] wherein m=1 to 7.
[0112] Other unsaturated fatty acid moieties which can be used
according to the present invention include oleic, linoleic and
linolenic.
[0113] In certain instances, it is preferred to provide
hydrolyzable bonds between the polyethylene glycol and the fatty
acid moieties. This permits hydrolysis to occur after penetration
into the central nervous system, thus releasing the active peptides
with the polyethylene glycol group still attached to the peptide.
The peptides acquire a more hydrophilic character and efflux to
circulatory system is thereby hindered.
[0114] The covalent bond between the oligomer and the drug is
preferably amide (a carboxy group of the oligomer is linked to an
amine group of the peptide), or carbamate (an chloroformate group
of the oligomer is linked to an amine group of the peptide).
[0115] For non-peptide drug, the bond is preferably ester (a
carboxy group of the peptide is covalently coupled to a hydroxyl
group of the oligomer or a carboxy group of the oligomer is
covalently coupled to a hydroxyl group of the drug), amide (a
carboxy group of the oligomer is linked to an amine group of the
drug) or carbamate (a chloroformate group of the oligomer is linked
to an amine group of the drug). For the enkephalin analogues, the
preferred peptides are leu-enkephalin lysine and met-enkephalin
lysine. The amino residue of the lysine is preferably utilized in
bonding.
[0116] Other preferred amphiphilic moieties are sugar moieties,
coupled to natural fatty acids and segments of polyethylene glycol.
The PEG moiety serves to increase the amphiphilicity of the fatty
sugar.
[0117] The length and number of the PEG moieties can be varied to
refine the amphiphilicity of the conjugate. Increasing the number
of PEGs increases the hydrophilicity of the resulting oligomer.
[0118] In certain instances, it is preferred to modify the
N-terminus of an enkephalin with proline or alanine before
attaching the oligomer. After absorption into the central nervous
system, the esters of the fatty sugar are hydrolysized leaving
hydrophilic moiety. Easy efflux is hindered and the brain
aminopeptidases cleave the proline or alanine portion leaving the
peptide to regain full activity.
[0119] Where the hydrophilic moiety is a sugar, it is preferred
that the sugar is a monosaccharide. The sugar may be an amino sugar
or a non-amino sugar.
[0120] In another aspect, the oligomer is attached to the
C-terminus of the peptide drug. For example:
[0121]
R--OCH.sub.2CH.sub.2OCH.sub.2C(O)OCH.sub.2CH.sub.2CH.sub.2OCH.sub.2-
CH.sub.2CH.sub.2NH--Enkephalin; or
[0122]
R--OCH.sub.2CH.sub.2OCH.sub.2C(O)OCH.sub.2CH.sub.2NH--Enkephalin.
[0123] Wherein R=alkyl.sub.1-26, cholesterol or amantane.
[0124] In another aspect, the oligomer is attached at the
N-terminus of the peptide drug. For example: 3
[0125] It will be appreciated by one of skill in the art that the
oligomers may be attached at the carboxy terminus or at a
constituent.of an amino acid side chain, such as at the amino group
of lysine.
[0126] The present invention broadly relates to therapeutic and/or
diagnostic conjugates wherein the therapeutic and/or diagnostic
molecule is covalently bonded to an oligomer to form an amphiphilic
conjugate. In one aspect, the oligomer comprises at least one
lipophilic moiety and at least one hydrophilic moiety, and the size
and nature of the two moieties is so selected as to impart an
amphiphilic nature to the resulting conjugate.
[0127] Exemplary oligomers according to the present invention are
as follows:
CH.sub.3(CH.sub.2CH.dbd.CH)6(CH.sub.2).sub.2CH.sub.2(OC.sub.2H.sub.4).sub.-
mOH,
[0128] where m=1 to 7;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub.2CO(OC.sub.2H.sub.4).sub.m-
OH,
[0129] where m=1 to 7;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub.2CONHCH.sub.2CH.sub.2(OC.s-
ub.2H.sub.4).sub.mOH,
[0130] where m=1 to 6;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub.3(OC.sub.2H.sub.4).sub.mOC-
H.sub.2COOH,
[0131] where m=1 to 6;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub.2CO(OC.sub.2H4).sub.mOCH.s-
ub.2COOH,
[0132] where m=1 to 6;
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.8(OC.sub.2H.sub.4).sub.mOH-
,
[0133] where m=1 to 7;
CH.sub.3(CH.sub.2).sub.7CH.dbd.C
H)(CH.sub.2).sub.7CO(OC.sub.2H.sub.4).sub- .mOH,
[0134] where m=1 to 7;
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CONHCH.sub.2CH.sub.2(OC.s-
ub.2H.sub.4).sub.mOH,
[0135] where m=l to 6;
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.8(OC.sub.2H.sub.4).sub.mOC-
H.sub.2COOH,
[0136] where m=1 to 6;
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CO(OC.sub.2H.sub.4).sub.m-
OCH.sub.2CH.sub.2OH,
[0137] where m=1 to 6;
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7CH.sub.2-
(OC.sub.2H.sub.4).sub.mOH,
[0138] where m=l to 6;
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7CO(OC.su-
b.2H.sub.4).sub.mOH,
[0139] where m=1 to 7;
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7CONHCH.s-
ub.2CH.sub.2(OC.sub.2H.sub.4).sub.mOH,
[0140] where m=1 to 6;
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7CO(OC.su-
b.2H.sub.4).sub.mOCH.sub.2COOH,
[0141] where m=1 to 6;
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7CH.sub.2-
(OC.sub.2H.sub.4).sub.mOCH.sub.2COOH,
[0142] where m=1 to 6;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.7CH.sub.2(OC.sub.2H.sub.4)-
.sub.mOH
[0143] where m=1 to 7;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.7CO(OC.sub.2H.sub.4).sub.m-
OH,
[0144] where m=l to 7;
CH.sub.3(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.7CONHCH.sub.2CH.sub.2(OC.s-
ub.2H.sub.4).sub.mOH,
[0145] where m=1 to 6;
CH.sub.3(CH.sub.2CH.dbd.CH.sub.3(CH.sub.2).sub.7CO(OC.sub.2H.sub.4).sub.mO-
CH.sub.2COOH,
[0146] where m=1 to 6;
CH.sub.3(CH.sub.2CH.dbd.CH.sub.3(CH.sub.2).sub.7CH.sub.2(OC.sub.2H.sub.4).-
sub.mOCH.sub.2COOH,
[0147] where m=1 to 6.
[0148] 4.1 THERAPEUTIC COMPOUNDS
[0149] The invention thus comprehends various compositions for
therapeutic (in vivo) application, wherein the peptide component of
the conjugated peptide complex is a physiologically active, or
bioactive, peptide. In such peptide-containing compositions, the
conjugation of the peptide component to the oligomer may be by
direct covalent bonding or indirect (through appropriate spacer
groups) bonding, and the hydrophilic and lipophilic moieties may
also be structurally arranged in the oligomer in any suitable
manner involving direct or indirect covalent bonding, relative to
one another. A wide variety of peptide species may be accommodated
in the broad practice of the present invention, as necessary or
desirable in a given end use therapeutic application.
[0150] While the description is primarily and illustratively
directed to the use of enkephalin as a peptide component in various
compositions and formulations of the invention, it will be
appreciated that the utility of the invention is not thus limited,
but rather extends to any peptide species which capable of
conjugation to the oligomers herein described, or which are capable
of being modified, as for example by the incorporation of a proline
residue, so as to enable the peptide to be conjugated to the
oligomers described herein.
[0151] Accordingly, appropriate peptides include, but or not
limited to: adrenocorticotropic hormone, adenosine deaminase
ribonuclease, alkaline phosphatase, angiotensin, antibodies,
arginase, arginine deaminease, asparaginase, caerulein, calcitonin,
chemotrypsin, cholecystokinin, clotting factors, dynorphins,
endorphins, endorphins, enkephalins, enkephalins, erythropoietin,
gastrin-releasing peptide, glucagon, hemoglobin, hypothalmic
releasing factors, interferon, katacalcin, motilin, neuropeptide Y,
neurotensin, non-naturally occurring opioids, oxytosin, papain,
parathyroid hormone, peptides prolactin, soluble CD-4, somatomedin,
somatostatin, somatostatin, somatotropin, superoxide dismutase,
thyroid stimulating hormone, tissue plasminogen activator, trypsin,
vasopressin, and analogues of such peptides, as well as other
suitable enzymes, hormones, proteins, polypeptides, enzyme-protein
conjugates, antibody-hapten conjugates, viral epitopes, etc.
[0152] In other aspect, the therapeutic peptide of the amphiphilic
drug-oligomer conjugates are as described in United States Patent
5,641,861, which is incorporated herein by reference, so long as
any of such peptides contains a lysine residue. Exemplary peptides
described therein include: Ac-Phe-Arg-Trp-Trp-Tyr-Lys--NH.sub.2;
Ac-Arg-Trp-Ile-Gly-Trp-Lys--NH.sub.2;
Trp-Pro-Lys-His-Xaa--NH.sub.2, where Xaa can be any one of the
twenty naturally occurring amino acids, or
Trp-Trp-Pro-Xaa--NH.sub.2 , where Xaa is Lys or Arg;
Tyr-Pro-Phe-Gly-Phe-Xaa--NH.sub.2, wherein Xaa can be any one of
the twenty naturally occurring amino acids;
(D)lle-(D)Met-(D)Ser-(D)Trp-(D)Tr- p-Gly.sub.n-Xaa--NH.sub.2,
wherein Xaa is Gly or the D-form of a naturally-occurring amino
acid and n is 0 or 1, peptides of this formula can be hexapeptides
when Gly is absent (n is 0) and heptapeptides when Gly is present
(n is 1); (D)Ile-(D)Met-(D)Thr-(D)Trp-Gly-Xaa--NH.sub.2, wherein
Xaa is Gly or the D-form of a naturally-occurring amino acid;
Tyr-Al-B2-C3--NH.sub.2, wherein Al is (D)Nve or (D)Nle, B2 is Gly,
Phe, or Trp, and C3 is Trp or Nap; Pm and red
{MeXHYN-Tyr-(NMe).sub.Z-Tyr-Xaa.- sub.Z--NH.sub.2}, wherein x and y
independently are 0, 1, or 2 and z is 0 or 1, and wherein Xaa is
Phe D-Phe, or NHBzl.
[0153] In other aspect, the therapeutic peptide of the amphiphilic
drug-oligomer conjugates are as described in U.S. Pat. No.
5,602,099, which is incorporated herein by reference. with the
proviso that the conjugation can occur only where there is a free
carboxyl or free N-terminal. Exemplary peptides include:
H-Tyr-Tic-Phe-Phe-OH; H-Tyr- Tic-Phe-Phe-NH.sub.2;
Tyr(N.alpha.Me)-Tic-Phe-Phe-OH; Tyr(Na Cpm)-Tic-Phe-Phe-OH;
Tyr(N.alpha.Hex)-Tic-Phe-Phe-OH;
Tyr(N.alpha.Et.sub.2)-Tic-Phe-Phe-OH; H-Dmt-Tic-Phe-Phe-OH;
H-Dmt-Tic-Phe-Phe-NH.sub.2; H-Tyr(3-F)-Tic-Phe-Phe-OH;
H-Tyr(3-Cl)-Tic-Phe-Phe-OH; H-Tyr(3-Br)-Tic-Phe-Phe-OH;
H-Dmt-Tic.PSI.[CH.sub.2-NH]Phe-Phe-OH; H-Dmt-Tic.PSI.[CH.sub.2--NH
]Phe-Phe-NH.sub.2; H-Tyr-Tic.PSI.[CH.sub.2--NCH.sub.3]Phe-Phe-OH;
H-Tyr-Tic-.PSI.[CH.sub.2--NH]Hfe-Phe-OH;
Tyr(NMe)-Tic.PSI.[CH.sub.2-NH]Hf- e-Phe-OH); H-Tyr-Tic-Phg-Phe-OH;
H-Tyr-Tic-Trp-Phe-OH; H-Tyr-Tic-Trp-Phe-NH.sub.2;
H-Tyr-Tic-His-Phe-OH; H-Tyr-Tic-2-Nal-Phe-OH; H-Tyr-Tic-Atc-Phe-OH;
H-Tyr-Tic-Phe-Phe(pNO.sub.2)-OH; H-Tyr-Tic-Trp-Phe(pNO.sub.2)-OH;
H-Tyr-Tic-Phe-Trp-NH.sub.2; H-Tyr-Tic-Phe-Phe-Val-Val-Gly-NH.sub.2;
H-Tyr-Tic-Phe-Phe-Tyr-Pro-Ser-NH.- sub.2;
H-Tyr-Tic-Trp-Phe-Tyr-Pro-Ser-NH.sub.2; H-Tyr-Tic-Trp-Phe
(pNO.sub.2) -Tyr-Pro-Ser-NH.sub.2 and
H-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH.su- b.2.
[0154] Abbreviations in the aforementioned peptides of U.S. Pat.
No. 5,602,099 may be interpreted as follows:
Aib=.alpha.-aminoisobutyric acid; Atc=2-aminotetralin-2-carboxylic
acid ; Boc=tert-butoxycarbonyl; Cpm=cyclopropylmethyl;
DCC=dicyclohexyl-carbodiimide; DI EA=diisopropylethylamine;
Dmt=2,6-dimethyltyrosine; Et=ethyl; Hex=hexyl;
Hfe=homophenylalanine; HOBt=1-hydroxybenzotriazole; MVD=mouse vas
deferens; 1-Nal=3-(1 -naphthyl)alanine;
2-Nal=3-(2'-naphthyl)alanine; Phe(pNO.sub.2)=4-nitrophenylalanine;
Phg=phenylglycine; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid; TIP=H-Tyr-Tic-Phe-OH; TIP-NH.sub.2=H-Tyr-Tic-Phe-NH.sub.2;
TIP(.PSI.)=H-Tyr-Tic.PSI.[CH.sub.2-NH]Phe-OH;
TIPP=H-Tyr-Tic-Phe-Phe-OH; TIPP-NH.sub.2=H
-Tyr-Tic-Phe-Phe-NH.sub.2; TIPP(.PSI.)=H-Tyr-Tic.PSI.[CH.-
sub.2-NH]Phe-Phe-OH; Tyr(3-Br)=3-bromotyrosine;
Tyr(3-Cl)=3-chlorotyrosine- ; Tyr(3-F)=3-fluorotyrosine; and Tyr(Na
Me)=Na -methyltyrosine.
[0155] In another aspect, the peptides are as described in U.S.
Pat. No. 5,545,719, which is incorporated herein by reference.
[0156] Other exemplary peptides include, for example, ACTH-related
peptides for inducing neural regeneration, cyclosporin for treating
infection, enkephalin analogs for treating pain and drug addiction,
MIF-1 for treating depression, neurotensin for relieving pain, and
peptide T for treating AIDS-associated dementia.
Adrenocorticotropic hormone (ACTH) and its analogue peptides are
also known to restore the avoidance learning caused by removal of
the pituitary gland and can also be used to treat passive avoidance
conditions.
[0157] Particularly preferred peptides are the endogenous and
synthetic Opioid peptides such as the enkephalins. A particularly
preferred Opioid is [Met.sup.5]Enkephalin
(Tyr-Gly-Gly-Phe-Met).
[0158] Peptides according to the present invention may be
synthesized according to any method of sysnthesis known in the art.
Such methods include, but are not limited to chemical synthesis
techniques and recombinant DNA expression techniques.
[0159] The therapeutic compounds of the present invention can be
modified in order to facilitate coupling to the amphiphilic
oligomer. A functional group may be added to the C-terminus or the
N-terminus of the peptide or to a side chain of the peptide in
order to provide a point of attachment for the oligomer.
[0160] Alternatively, specific amino acids may be inserted within
the amino acid chain of the peptide therapeutic, or may replace an
amino acid of the therapeutic or may be added to the C-terminus or
N-terminus of the peptide in order to facilitate attachment of the
oligomer where such modification does not eliminate the activity of
the peptide. For example, a proline or alanine residue can be added
to the N-terminus of a therapeutic peptide, such as an enkephalin,
such as [met.sup.5]enkephalin, in order to facilitate attachment of
the amphiphilic oligomer.
[0161] One skilled in the art would know that one or more amino
acids within the exemplified peptides could be modified or
substituted, as for example, by a conservative amino acid
substitution of one or more of the specific amino acids shown in
the exemplified peptides. A conservative amino acid substitution
change can include, for example, the substitution of one acidic
amino acid for another acidic amino acid, of one hydrophobic amino
acid for another hydrophobic amino acid or other conservative
substitutions known in the art, including the use of non-naturally
occurring amino acids, such as NIe for Leu or ornithine (Orn) or
homoArginine (homoArg) for Arg.
[0162] In addition to the above types of modifications or
substitutions, a mimic of one or more amino acids, otherwise known
as a peptide mimetic or peptidominetic, can also be used. As used
herein, the term "mimic" means an amino acid or an amino acid
analog that has the same or similar functional characteristic of an
amino acid. Thus, for example, a (D)arginine analog can be a mimic
of (D)arginine if the analog contains a side chain having a
positive charge at physiological pH, as is characteristic of the
guinidinium side chain reactive group of arginine. A peptide
mimetic or peptidomimetic is an organic molecule that retains
similar peptide chain pharmacophore groups as are present in the
corresponding peptide.
[0163] The substitution of amino acids by non-naturally occurring
amino acids and peptidomimetics as described above can enhance the
overall activity or properties of an individual peptide based on
the modifications to the side chain functionalities. For example,
these types of alterations can be employed along with the
amphiphilic oligomers of the present invention to further enhance
the peptide's stability to enzymatic breakdown and increase
biological activity.
[0164] One skilled in the art can easily synthesize the peptides
for use as therapeutics in this invention. Standard procedures for
preparing synthetic peptides are well known in the art. The
peptides can be synthesized using the solid phase peptide synthesis
(SPPS) method of Merrifield (J. Am. Chem. Soc., 85:2149 (1964),
which is incorporated herein by reference) or using standard
solution methods well known in the art (see, for example,
Bodanzsky, M., Principles of Peptide Synthesis 2nd revised ed.
(Springer-Verlag, 1988 and 1993), which is incorporated herein by
reference). Alternatively, simultaneous multiple peptide synthesis
(SMPS) techniques well known in the art can be used. Peptides
prepared by the method of Merrifield can be synthesized using an
automated peptide synthesizer such as the Applied Biosystems 431
A-01 Peptide Synthesizer (Mountain View, Calif.) or using the
manual peptide synthesis technique described by Houghten, Proc.
Natl. Acad. Sci., USA 82:5131 (1985), which is incorporated herein
by reference.
[0165] Peptides can be synthesized using amino acids or amino acid
analogs, the active groups of which are protected as necessary
using, for example, a t-butyidicarbonate (t-BOC) group or a
fluorenylmethoxy carbonyl (FMOC) group. Amino acids and amino acid
analogs can be purchased commercially (Sigma Chemical Co.; Advanced
Chemtec) or synthesized using methods known in the art. Peptides
synthesized using the solid phase method can be attached to resins
including 4-methylbenzhydrylamine (MBHA),
4-(oxymethyl)-phenylacetamidomethyl and
4-(hydroxymethyl)phenoxymethyl- copoly(styrene-1% divinylbenzene)
(Wang resin), all of which are commercially available, or to
p-nitrobenzophenone oxime polymer (oxime resin), which can be
synthesized as described by De Grado and Kaiser, J. Org. Chem.
47:3258 (1982), which is incorporated herein by reference.
[0166] A newly synthesized peptide can be purified using a method
such as reverse phase high performance liquid chromatography
(RP-HPLC) or other methods of separation based on the size or
charge of the peptide. Furthermore, the purified peptide can be
characterized using these and other well known methods such as
amino acid analysis and mass spectrometry.
[0167] 4.2 SYNTHESIS
[0168] A general synthesis scheme for the oligomers of the present
invention is provided in FIG. 9, and a general synthesis scheme for
attaching such oligomer to the therapeutic peptides of the instant
invention is provided in FIG. 10.
[0169] Several methods of modifying fatty acid to achieve the
desired oligomer will be discussed in further detail with
structural illustrations.
[0170] In the synthesis of oligomers containing fatty acids and
polyethylene glycols, where the ethylene glycol is connected to the
fatty acid in a hydrolysable ester bond, it is desirable to start
with the acid chloride of the fatty acid or its acid anhydride. A
desired polyethylene glycol having two free hydroxyls at the
termini is then treated in inert solvent with equal molar
equivalent of acid chloride or acid anhydride. The glycol unit is
first dissolved in inert solvent and treated with organic base
before the addition of the acid chloride or acid anhydride. The
product is extracted from the reaction medium and further purified
using column chromatograph: 4
[0171] In some instances it is desired to create oligomers that
have stronger hydrolysable bond such as amide. The acid chloride or
the acid anhydride of the selected fatty acid is treated with amino
derivative of polyethylene glycol in a controlled reaction
condition to effect only the amino residue and not the hydroxyl
portion. Other conditions that ensure selectivity is by converting
the fatty acid into N-hydroxysuccinimide ester and reacting with
the amino residue of the polyethylene glycol. 5
[0172] Coupling of the oligomer to the peptide drug is effected by
converting the free hydroxyl moiety of the oligomer to
N-hydroxysuccinimide ester (NSU). N-hydroxysuccinimide group reacts
readily with the nucleophilic amino residue of the peptide. 6
[0173] In the synthesis of oligomers in which the lipophilic
portion of the oligmers is connected to the hydrophilic portion by
ether linkage, the desired polyethylene glycol (hydrophile) is
first protected. One of the two free hydroxyls at the termini is
protected with a trityl group in pyridine using one mole of trityl
chloride. The protected polyethylene glycol is dissolved in a
suitable inert solvent and treated with sodium hydride. Bromo or
tosylate derivative of the lipophilic portion is dissolved in inert
solvent and added to the solution of the protected polyethylene
glycol. The product is treated with a solution of
para-toluenesulfonic acid in anhydrous inert solvent at room
temperature. The desired product is extracted in inert solvent and
purified by column chromatography. The structures of the
transformation are depicted below: 7
[0174] The lipophilic portion can be alkyl, cholesteryl, adamantyl
moieties.
[0175] In the synthesis of oligomers where the lipophilic portion
of the oligomer is connected to the hydrophilic portion in ether
bond and the terminal ends in carboxylic acid moiety, it is
desirable to protect the carboxylic group. Polyethylene glycol
having free hydroxyl group at one end and carboxylic group at the
other end is selected. The carboxylic group is protected by
esterification. The protected polyethylene glycol is dissolved in a
suitable inert solvent and treated with sodium hydride. Bromo or
tosylate derivatives of the lipophilic portion is dissolved in
inert solvent and added to the solution of the protected
polyethylene glycol. The product is treated with solution of sodium
hydroxide to liberate free acid. The desired product is extracted
in inert solvent and purified by column chromatography. The
structures of the transformation are depicted below. 8
[0176] The lipophilic portion can be alkyl, cholesteryl or
adamantyl moieties.
[0177] This group of acidic oligomers can be coupled to peptide
drugs by first reacting the carboxylic group with
N-hydroxysuccinimide (NSU) to from easily leavable group. A
solution of the activated oligomers in inert solvent is treated
with the desired peptide drug dissolved in a suitable solvent.
Inverse addition may be selected. 9
[0178] Sometimes it is desirable to replace the lipophilic moiety
with lipophilic sugars. The sugar moiety is first esterified with
desired fatty acid chloride to obtain selective or partial
acylation. The product is treated in inert solvent with diacid
chloride of desired dicarboxylic acid derivative of polyethylene
glycol.
[0179] Reaction is conducted with one molar equivalent of each
reacting moiety. This reaction leaves one end of the hydrophile
bearing acid chloride, which is further converted to N-
hydroxysuccinimide ester. The activated ester is reacted with
peptide drug in suitable inert solvent. 10
[0180] Where R=fatty acid, alkyl.sub.1-26, cholesterol or
adamantane.
[0181] 4.3 THERAPEUTIC METHODS
[0182] The invention provides methods of treatment and prevention
by administration to a subject of an effective amount of an
amphiphilic drug-oligomer conjugate of the invention.
[0183] One embodiment of the invention provides for methods of
administering a pharmaceutical composition which is comprised of a
therapeutically effective amount of an amphiphilic drug-oligomer
conjugate according to the present invention.
[0184] Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The conjugates
may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local.
[0185] In certain circumstances, it may be desirable to introduce
the pharmaceutical compositions of the invention directly into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
[0186] Pulmonary or nasal administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with an
aerosolizing agent.
[0187] In another embodiment, the conjugates can be delivered in a
controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201
(1987); Buchwald et al., Surgery88:507 (1980); Saudek et al., N.
Engl. J. Med. 321:574 (1989)). In yet another embodiment, a
controlled release system can be placed in proximity of the
therapeutic target, i.e., the brain, thus requiring only a fraction
of the systemic dose (see, e.g., Goodson, in Medical Applications
of Controlled Release, supra, vol. 2, pp.115-138 (1984)).
[0188] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0189] The subject is preferably an animal, including, but not
limited to, animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0190] 4.4 PHARMACEUTICAL COMPOSITIONS
[0191] Exemplary means of administration include oral, parenteral,
rectal, topical, sublingual, mucosal, nasal, opthalmic,
subcutaneous, intramuscular, intravenous, transdermal, spinal,
intrathecal, intra-articular, intra-arterial, sub-arachnoid,
bronchial, lymphatic, and intrauterine administration.
[0192] The present invention contemplates the use of pharmaceutical
formulations for human medical use which comprise the vector
structures of the present invention as therapeutic ingredients.
Such pharmaceutical formulations may include pharmaceutically
effective carriers, and optionally, may include other therapeutic
ingredients. The carrier or carriers must be pharmaceutically
acceptable in the sense that they are compatible with the
therapeutic ingredients and are not unduly deleterious to the
recipient thereof. The therapeutic ingredient or ingredients are
provided in an amount necessary to achieve the desired therapeutic
effect, described below.
[0193] In another aspect, a pharmaceutical composition is provided
to comprising (1) a mixture of an enkephalin conjugate according to
the present invention wherein the enkephalin peptide has proline or
alanine added to its N-terminus and an enkephalin conjugate
according to the present invention which does not have a proline or
alanine added to the N-terminus, and (2) a pharmaceutical
carrier.
[0194] Various delivery systems are known and can be used to
administer a conjugate of the invention, e.g., encapsulation
microcapsules.
[0195] As used herein, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans.
[0196] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the conjugate is administered.
[0197] Such pharmaceutical carriers can be sterile liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable
solutions.
[0198] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene glycol, water, ethanol and the
like.
[0199] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents.
[0200] The compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides.
[0201] Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0202] Such compositions will contain a therapeutically effective
amount of the drug-oligomer conjugate, preferably in purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the patient. The formulation
should suit the mode of administration.
[0203] The mode of administration and dosage forms will of course
affect the therapeutic amounts of the compounds which are desirable
and efficacious for the given treatment application. A
therapeutically effective amount is an amount necessary to prevent,
delay or reduce the severity of the onset of disease, or an amount
necessary to arrest or reduce the severity of an ongoing disease.
It will be readily apparent to one of skill in the art that this
amount will vary based on factors such as the weight and health of
the recipient, the type of cells being transformed, the mode of
administration of the present compositions and the type of medical
disorder being treated.
[0204] The dosage can be presented in the form of tablets, syrups,
losenges, elixirs, suspensions, and/or emulsions.
[0205] Accessory ingredients may, without limitation, include
diluents, buffers, flavoring agents, disintegrants, surfactants,
thickeners, lubricants, preservatives, and/or antioxidants.
[0206] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings.
[0207] Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent.
[0208] Where the composition is to be administered by infusion, it
can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water or saline. Where the composition is
administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients may be
mixed prior to administration.
[0209] The conjugates of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0210] The amount of the Conjugate of the invention which will be
therapeutically effective in the treatment of a particular disorder
or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vivo and/or in vitro assays may optionally be
employed to help identify optimal dosage ranges.
[0211] For example, suitable doses of a an enkephalin conjugate for
analgesia may genarally be in the range of from 1 mg/kg to 20
mg/kg, preferably 3 mg/kg to 15 mg/kg, more preferably 5 mg/kg to 7
mg/kg.
[0212] Effective doses may be extrapolated from dose-response
curves derived from in vitro or animal model test systems.
[0213] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0214] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0215] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
5. EXAMPLES
[0216] 5.1 SYNTHESIS
[0217] 5.1.1 TRIETHYLENE GLYCOL MONOHEXADECYL ESTER
[0218] Palmitic anhydride (5.00 g; 10.104 mmol) was dissolved in
dry THF (20 mL) and 3 mol excess of dry pyridine and the solution
was stirred at room temperature. To the stirring solution,
triethylene glycol (1.5 g; 10.104 mmol) was added slowly. After
stirring for 1 h, THF was removed under reduced pressure at room
temperature and the reaction mixture was poured into ice cold 10%
sulfuric acid. The aqueous layer was extracted with ethyl acetate
(30 ml.times.3). Combined organic layer was sequentially washed
with water, brine and dried over MgSO.sub.4 and filtered. After
evaporation gave pure product, single spot on TLC.
[0219] 5.1.2 SUCCINIMIDYL TRIETHYLENE GLYCOL MONOHEXADECYL
ESTER:
[0220] To a stirring solution of teg-palmitate (1 g; 2.57 mmol),
dimethylaminopyridine (0.313 g; 2.57 mmol) in dry THF was added
N,N'-disuccinimidyl carbonate (0.691 g) in one portion. The
reaction mixture was stirred overnight at room temperature. The
organic solvent was removed under reduced pressure and reaction
mixture was diluted with ethyl acetate, washed with 1N hydrochloric
acid (1OmL x 2), water and brine. The solvent was dried over
MgSO.sub.4, filtered and evaporated to leave white solid.
[0221] 5.1.2.1 Determination of Activity
[0222] The succinimidyl reactivity was determined by conjugating it
with insulin and it was found to be 67%.
[0223] 5.1.3 SUCCINIMIDYL TRIETHYLENE GLYCOL MONOHEXADECYL
ETHER
[0224] To a cold stirring solution of phosgene (10.0 mL; 20%
solution in toluene) under nitrogen, a solution of triethylene
glycol monohexadecyl ether (1.5 g; 4.00 mmol) in dry
dichloromethane (4mL) was added. The reaction mixture was stirred
at 0.degree. C. for 2 h at room temperature. Excess of phosgene was
distilled off using water aspirator, passing through cold solution
of dilute NaOH.
[0225] The reaction flask was cooled in ice bath and equimolar
quantity of triethyl amine and a solution of hydroxysuccinimide,
dissolved in minimum quantity of THF was added slowly. The reaction
mixture was stirred at room temperature for 12 h. The solvent was
removed completely at 25.degree. C. and residue was redisolved in
ethyl acetate, washed with water, brine, dried over MgSO.sub.4 and
evaporated to give pure succinimidyl derivative
[0226] 5.1.3.1 DETERMINATION OF ACTIVITY
[0227] The succinimidyl reactivity was determined by conjugating it
with insulin and it was found to be 62.5%.
[0228] 5.2 CONJUGATION OF COMPOUND 2 & 4 WITH MET-ENKEPHLIN
[0229] 5.2.1 GENERAL PROCEDURE FOR CONJUGATION
[0230] 5.2.1.1 11
[0231] 5.2.1.2 GENERAL PROCEDURE
[0232] To a stirring solution of met-enkephalin (0.130 g; 0.1854
mmol) in 5mL of DMF-DCM (2:1) was added TEA (25 .mu.L). The
reaction mixture was cooled to 10.degree. C. and a solution of
palmityl-teg-nsu or cetyl-teg-nsu dissolved in 1 mL of DCM was
added in one portion. The reaction mixture was stirred for 2 h at
10.degree. C. The solvent was removed under reduced pressure and
the residue was redissolved in dry ethyl acetate. After evaporation
of the solvent 0.310 g conjugated enkephalin was obtained. HPLC
showed mono & diconjugate in the ratio of 3.1.
[0233] 5.3 SYNTHESIS OF CETYL-PEG.sub.2; IT'S ACTIVATION &
CONJUGATION WITH PROTECTED (BOC) LEUENK
[0234] To a suspension of NaH (4.00 g: 0.1 mol) in dry THF (300 mL)
at 10.degree. C. was added diethylene glycol in one portion. The
cooling bath was removed and reaction mixture was stirred at room
temperature for 2 h. At the end the reaction mixture was cooled to
10.degree. C. and bromohexadecane (29 g).095 mol) was added in one
portion. The cooling bath was removed and the reaction was stirred
at room temperature for 4 h. The solvent was removed under reduced
pressure and crude was admixed with water and extracted with ethyl
acetate (30 mL.times.3). The combined organic extract was
sequentially washed with water, brine, dried over MgSO.sub.4 and
evaporated to leave white solid powder, single spot on TLC and
single molecular ion peak.
[0235] 5.3.1 CETYL-PEG.sub.2-NSU
[0236] To a cold stirring solution of phosgene (10.0 mL; 20%
solution in toluene) under nitrogen, a solution of
cetyl-PEG.sub.2-OH (1.3 g; 4.00 mmol) in dry dichloromethane (5 mL)
was added. The reaction mixture was stirred at 0.degree. C. for 1
hr and 2 h at room temperature. Excess of phosgene was distilled
off using water aspirator, passing through cold solution of dilute
NaOH.
[0237] The reaction flask was cooled in ice bath and equimolar
quantity of triethyl amine and a solution of hydroxy succinimide,
dissolved in minimum quantity of THF was added slowly. The reaction
mixture was stirred at room temperature for 12 h. The solvent was
removed completely at 252 C. and residue was redissolved in ethyl
acetate, washed with water, brine, dried over MgSO.sub.4 and
evaporated to give pure succinimidyl derivative.
[0238] 5.3.2 DETERMINATION OF ACTIVITY
[0239] The succinimidyl reactivity was determined by conjugating it
with insulin and it was found to be 83.5%.
[0240] 5.4 CONJUGATION OF SUCCINIMIDYL CETYL-PEG2 WITH
BOC-LEU...ENK... LYS-OH Boc-Leu...enk...Lys-OH (100 mg; 0.125 mmol)
was dissolved in 5 mL of DMF:DCM(1:1) and stirred at 10.degree. C.
under nitrogen. To this clear solution TEA (17.5 .mu.L) and a
solution of succinimidyl cetyl-PEG.sub.2, dissolved in 1 mL of DCM
were added.
[0241] After 1.5 h (TLC showed single product) the solvent was
removed under reduced pressure at room temperature and reaction
mixture was admixed with water and extracted with ethyl acetate (10
mL.times.3). The organic extract was sequentially washed with
water, brine, dried and evaporated to a solid.
[0242] 5.4.1 PURIFICATION OF DERIVATIZED BOC-LEU...ENK...LYS-OH ON
SILICA GEL COLUMN
[0243] The derivatized blocked enkephalin was purified on silica
gel column using methanol-chloroform (5% methnol-chloroform)
mixture as an eluting solvent. After evaporation of desired
fraction 100 mg pure compound was obtained. A product yield of 100
mg was obtained after removal of the solvent.
[0244] 5.4.2 DEBLOCKING OF BUTYLOXYCARBONYL GROUP FROM DERIVATIZED
LEU...ENK
[0245] Derivatized Boc-Leu...enk. (100 mg: 0.0866 mmol) was treated
with 0.4 ml of TFA-DCM (1:1) for 30 min. at room temperature. The
solvent was removed under reduced pressure. The solid was
redissolved in 2ml of methanol, filtered and evaporated; 80 mg of
pure product was obtained.
[0246] 5.5 SYNTHESIS OF AMPHIPHILIC OLIGOMER-ENKEPHALIN
CONJUGATES
[0247] 5.5.1 A GENERAL SCHEME FOR SYNTHESIS OF NON-HYDROLYZABLE AND
HYDROLYZABLE CONJUGATES
[0248] One-hundred milligrams of enkephalin (100 mg; 0.142 mmol)
was dissolved in dry dimethylformamide (5 mL) at room temperature.
P-nitrophenol or N-hydroxysuccinimide activated (carbonate or
ester) of amphiphilic oligomer (1.1 mole equivalent) was dissolved
in 1 mL tetrahydrofuran and added to above solution and stirred at
room temperature over 1.5 hours. The extent of the reaction was
monitored by a reverse phase (C-18) HPLC using isopropanol/water
(0.1% trifluoracetic acid) gradient system. Reaction mixture was
evaporated under reduced pressure and the contents were dissolved
in an isopropanol-water mixture. This mixture was purified on a 22
mm preparative HPLC column (C-8) with a solvent gradient system
made of either isopropanol/water (0.1% trifluoroacetic acid) or
acetonitrile/water (0.1% trifluoroacetic acid to give pure
monoconjugated and diconjugated enkephalins. The solvent was
evaporated at low temperature (<20.degree. C.) to give dry
produce. The purity of the product was analyzed by reverse phase
analytical HPLC, and the MW information was obtained by MALDI
(TOF)-mass spectral technique.
[0249] 5.5.2 SYNTHESIS OF CHOLESTEROL-PEG.sub.2 HYDROLYZABLE
AMPHIPHILIC OLIGOMER
[0250] PEG.sub.2 diacid (3,6,9-trioxaundecanoic diacid, 10 g) was
dissolved in dry chloroform (50 mL) and added dropwise to
oxalychloride at room temperature under dry condition in the
presence of catalytic amount of dimethylformamide. The reaction was
stirred or 6 hours and the solvent and excess of reagent was
stripped off to give an oily residue.
[0251] Above residue was dissolved in chloroform (50 mL) and to
this was added cholesterol (1.05 mole equivalent) in chloroform
(5OmL) and triethylamine (1 mole equivalent) over 30 minutes at
5.degree. C. The reaction was stirred at 15.degree. C. over 2
hours. To this was added N-hydroxysuccinimide (1 mole equivalent)
in chloroform (5OmL) and followed by triethylamine (1 equivalent)
at 5.degree. C. and allowed to stir overnight. Solvent was stripped
off and the product was extracted with ethylacetate. Crude product
was purified on a silica gel column with 1:10 methanol/chloroform
solvent system to obtain activated amphiphilic oligomer in 80%
yield.
[0252] 5.6 MOLECULAR WEIGHT INFORMATION OF ENKEPHALIN CONJUGATES
OBTAINED BY MALDI (TOF)-MS
1 Enkephalin Conjugate Expected M. W. Observed M. W.
Cholesterol-PEG.sub.2 1274 1275 DHA- PEG.sub.2 1144.4 1144.3
Linolenic- PEG.sub.2 1093.4 1093.3 Cetyl- PEG.sub.2 Avg. 1059 Avg.
1032 Palmitate- PEG.sub.3 1116 1115.6 Cetyl- PEG.sub.3 1101
1101.12
[0253] These results demonstrate that the reactions resulted in
monoconjugates, i.e., each peptide was coupled to only one
oligomer. It is significant to note that a single conjugate is
sufficient to impart amphiphilic properties.
[0254] 5.7 STABILITY OF MET ENKEPHALIN-LYS (ENKEPHALIN) AND ITS
AMPHIPHILIC OLIGOMER CONJUGATES IN RAT BRAIN HOMOGENATE
[0255] Met enkephalin-lys and its conjugates (Cetyl-PEG.sub.2,
Cetyl-PEG.sub.3 and Palmitate-PEG.sub.3) were incubated in 2% rat
brain homogenate. Samples were drawn over time intervals and the
amount of the substance remaining was measured by a HPLC method.
Following experimental procedure was used for the study.
[0256] Procedure: A 2% rat brain homogenate was prepared by
homogenizing freshly perfused (PBS buffer) rat brain in PBS buffer
(pH 7.4). Two 3-mL aliquots of the homogenate were equilibrated at
37.degree. C. in a water bath. To one unmodified enkephalin was
added and to other modified (conjugate) was added, resulting in a
final concentration of 60ug/mL of peptide. At time 0, 1, 2, 3, 5,
15, 30, and 60 minutes, 200 uL of aliquot was withdrawn and
quenched with 200 uL of the quenching agent (1% trifluoroacetic
acid in acetonitrile/isopropanol or 1% trichloroacetic acid in
water). The sample solutions were vortexed and centrifuged at
700ORPM. The supernatant was analyzed by a HPLC method using a
gradient of 10 to 100% isopropanol/water (0.1% trifluoroacetic
acid) on a C-18 column.
[0257] FIG. 2 shows the stability of the cetyl-PEG.sub.2-enkephalin
conjugate as compared to free met-enkephalin-lys. FIG. 3 shows the
stability of the cetyl-PEG.sub.3-enkephalin as compared to
met-enkephalin-lysine. FIG. 4 shows palmitate-PEG.sub.3-enk
(hydrolyzable) conjugate as compared to met-enkephalin-enk.
[0258] 5.7.1 EXTRACTION AND DETECTION OF ENKEPHALIN CONJUGATES FROM
THE BRAIN OF DOSED RATS
[0259] 5.7.1.1 PROCEDURE
[0260] The following procedure was used to identify the presence of
conjugate from the brain specimen of animals dosed with 5 mg/kg
cetyl-PEG.sub.2-enkephalin.
[0261] After 10 monutes of dosing, the brain of the animal was
perfused with 1.5% triflouroacetic acid in PBS solution, and the
brain was removed and frosen at -70.degree. C. The brain was
homogenized with 1mL of 1.5% triflouroacetic acid in PBS solution
and the homogenate was extracted with acetonitrile/isopropanol
solution. The extract was treated with saturated sodium chloride
solution and frozen at -20.degree. C. for 2 hours. The organic
layer was isolated and centrifuged at 4000 RPM. The supernatant was
evaporated and the resulting residue was reconstituted in
acetonitrile/isopropanol/water mixture. The reconstituted solution
was analyzed by HPLC using a gradient of 10 to 100%
isopropanol/water (0.1% trifloiroacetic acid) on a C-18 column. The
presence and the concentration of cetyl-PEG.sub.2-enkephalin
conjugate in the extract were measured by comparing the retention
time and the peak area of standard solution under the same
analytical condition. The results are presented in FIGS. 5A to
5D.
[0262] 5.7.1.2 RESULTS
[0263] The results demonstrate that monoconjugates were isolated
from brain tissue. FIG. 5A shows a peak produced by cetyl
enkephalin standard, while 5B shows a corresponding peak
demonstrating that cetyl enkephalin was actually present in the
brain extract. In contrast, neither the vehicle (FIG. 5C) nor the
unconjugated enkephalin (FIG. 5D) showed a corresponding peak.
[0264] 5.8 RAT PAW-HOT PLATE TEST
[0265] 5.8.1 ANIMALS
[0266] Adult, male Sprague-Dawley rats weighing 150-175 g were
obtained from Charles River Breeding Laboratories (Raleigh, N.C.)
and used for all animal studies. Rats were housed in hanging
wire-bottomed cages in a vivarium equipped with a 12:12 light:dark
cycle and humidity was maintained between 45-65% with a room
temperature of 72.+-.2.degree. C. Rats were provided Purina Rodent
Chow and tap water ad libitum.
[0267] 5.8.2 METHODS
[0268] Met-enkephalin-lys and met-enkephalin-lys derivatives were
assessed for analgesic activity by rat paw-hot plate assay. Rats
were given an injection of naloxone at 0.5 mg/kg (s.c.) then
administered a single administration of cetyl-enkephalin by the
tail vein 10 minutes later at a dose of 5.0 mg/kg. The results as
graphically displayed in FIG. 6 demonstrate that Naloxone, an
p-receptor antagonist prevents competitively inhibits binding of
cetyl-PEG.sub.2-enkephalin, thus demonstrating that at least part
of the activity of cetyl-PEG.sub.2-enkephalin is attributable to
binding at the Opioid p-receptor.
[0269] In a separate study, rats were administered cetyl-enkephalin
(5.0 mg/kg, i.v.) or clonidine (0.125 mg/kg, i.v.).
[0270] The latency to rat paw withdrawal from the hot plate was
measured by a Hot Plate Analgesia Meter (Harvard Apparatus Ltd.,
Kent, England). The temperature of the hot plate was set and
calibrated at 52.degree. C. and rats were removed from the heat
stimulus by 36 seconds after placement. Latency trials were
terminated when the animal was either licking a hind paw or
initiating a jump from the plate. Baseline measurements were
collected 1 hour prior to drug administration and at various times
post-injection, dependent upon the study conducted. All hot plate
testing was terminated by 1 hour after drug dosing.
[0271] 5.8.3 RESULTS
[0272] The results are displayed in the following tables and in the
Graph of FIG. 6. The results demonstrate that while 20 mg/kg
enkephalin alone has 0% analgesic effect as compared to morphine as
a baseline, the enkephalin conjugates of the present invention had
strong analgesic effects and one conjugate, DHA-PEG-ENK had 130% of
the analgesic effect of morphine. The graph of FIG. 7 shows that
CETYL-PEG-ENK produces a response and duration comparable to that
of clonidine, an (.alpha.-adrenergic receptor agonist.
2 ANALGESIC EFFECT OF ENKEPHALIN CONJUGATES IN RATS Mean Analgesia
as Compared with Dose Number Morphine at 3 mg/Kg* Drug or Conjugate
(mg/kg) of Rats @ 5 min @ 30 min Morphine 3 8 100% 100% Enkephalin
20 7 0% 0% Cetyl-PEG-ENK 5 8 84% 75% DHA-PEG-ENK 20 8 130% 67%
Cholesterol-PEG- 5 8 80% 68% ENK Linolenic-PEG-ENK 10 8 77% 73%
[0273] 5.9 AGONIST-STIMULATED [.sup.35S] GTP.gamma.S BINDING IN
BRAIN SECTIONS.
[0274] 5.9.1 MATERIALS
[0275] Male Sprague-Dawley rats (200 g) were purchased from
Zivic-Miller (Zelienople, Pa.). [.sup.35S]GTPTS (1250 Ci/mmol) was
purchased from New England Nuclear Corp. (Boston, Mass.).
[D-Ala.sup.2, N-Me-Phe.sup.4,Gly.sup.5-ol]-enkephalin (DAMGO),
adenosine deaminase, and GDP were obtained from Sigma Chemical Co.
(St. Louis, Mo.). Reflections.RTM. autoradiography film was
purchased from New England Nuclear Corp. (Boston, Mass.). All other
reagent grade chemicals were obtained from Sigma Chemical Co. or
Fisher.
[0276] 5.9.2 AGONIST-STIMULATED [.sup.35S] GTP.gamma.S BINDING IN
BRAIN SECTIONS.
[0277] Agonist-stimulated [.sup.35S] GTP.gamma.S autoradiography
was performed as described by Sim et al. Proc. Nat'l. Acad. Sci.
USA 1992 Pg. 7242 - 7246. Animals were sacrificed by decapitation
and brains were removed and frozen in isopentane at -30.degree. C.
Coronal and horizontal brain sections were cut on a cryostat
maintained at -20.degree. C. Sections were incubated in assay
buffer (50 mM Tris-HCl, 3 mM MgCl.sub.2, 0.2 mM EGTA, 100 mM NaCl,
pH 7.4) at 25.degree. C. for 10 min. Sections were then incubated
in assay buffer containing 2 mM GDP, protease inhibitor cocktail
(10 .mu.l/ml of a solution containing 0.2 mg/ml each of bestatin,
leupeptin, pepstatin A and aprotinin), and adenosine deaminase (9.5
mU/ml) at 25.degree. C. for 15 min. Sections were then incubated in
assay buffer with GDP, 0.04 nM [.sup.35S]GTP.gamma.S and
appropriate agonist at 25.degree. C. for 2 hours. The agonists
were: 10 .mu.M DAMGO, 10 .mu.M cetyl-enkephalin and 10 ,.mu.M
cetyl-TEG-enkephalin. Basal binding was assessed in the absence of
agonist. Slides were rinsed twice for 2 min each in cold Tris
buffer (50 mM Tris-HCl, pH 7.4) and once in deionized H.sub.2O.
Slides were dried overnight and exposed to film for 72 hours. Films
were digitized with a Sony XC-77 video camera and analyzed using
the NIH IMAGE program for Macintosh computers.
[0278] 5.9.3 RESULTS
[0279] Results show that cetyl-TEG-enkephalin stimulates of
[.sup.35S]GTP.gamma.S binding. The anatomical distribution of the
binding is consistent with that of .mu. Opioid receptors. These
results demonstrate that cetyl-TEG-enkephalin does not simply bind
the receptor but also activates the receptor, causing the receptor
to bind to G-protein. This activation provides further
corroborative evidence that cetyl-TEG-enkephalin directly
stimulates analgesia.
* * * * *