U.S. patent application number 13/146612 was filed with the patent office on 2012-02-23 for oligomer-phenothiazine conjugates.
This patent application is currently assigned to Nektar Therapeutics. Invention is credited to Xuyuan Gu, Jennifer Riggs-Sauthier.
Application Number | 20120046279 13/146612 |
Document ID | / |
Family ID | 42101449 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120046279 |
Kind Code |
A1 |
Gu; Xuyuan ; et al. |
February 23, 2012 |
Oligomer-Phenothiazine Conjugates
Abstract
The invention relates to (among other things)
oligomer-phenothiazine conjugates and related compounds. A
conjugate of the invention, when administered by any of a number of
administration routes, exhibits advantages over un-conjugated
phenothiazine compounds.
Inventors: |
Gu; Xuyuan; (Madison,
AL) ; Riggs-Sauthier; Jennifer; (Huntsville,
AL) |
Assignee: |
Nektar Therapeutics
San Francisco
CA
|
Family ID: |
42101449 |
Appl. No.: |
13/146612 |
Filed: |
January 28, 2010 |
PCT Filed: |
January 28, 2010 |
PCT NO: |
PCT/US10/22342 |
371 Date: |
November 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61148016 |
Jan 28, 2009 |
|
|
|
Current U.S.
Class: |
514/226.2 ;
544/41 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 1/08 20180101; A61P 25/04 20180101; A61K 47/60 20170801; A61P
43/00 20180101; A61P 35/00 20180101; A61P 1/12 20180101; A61P 25/22
20180101; A61P 25/20 20180101 |
Class at
Publication: |
514/226.2 ;
544/41 |
International
Class: |
A61K 31/5415 20060101
A61K031/5415; A61P 25/20 20060101 A61P025/20; A61P 1/08 20060101
A61P001/08; C07D 279/26 20060101 C07D279/26 |
Claims
1. A compound comprising a phenothiazine residue covalently
attached via a stable or degradable linkage to a water-soluble,
non-peptidic oligomer.
2. The compound of claim 1, having the following structure:
##STR00016## wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each
independently are selected from the group consisting of hydrogen,
unsubstituted alkyl, and substituted alkyl; or R.sup.3 and R.sup.4
together with the nitrogen form a heterocycle; n is an integer
equal to or greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
3. The compound of claim 1, having the structure: ##STR00017##
wherein: R.sup.1, R.sup.2, and R.sup.3, each independently are
selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; n is an integer equal to or greater
than one; X is a spacer moiety; and POLY is a water-soluble,
non-peptidic oligomer.
4. The compound of claim 1, having the structure: ##STR00018##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; n is an integer equal to or
greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
5. The compound of claim 1, wherein the phenothiazine residue is a
residue of a phenothiazine having the formula: ##STR00019##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; and n is an integer equal to or
greater than one.
6. The compound of claim 1, wherein the phenothiazine residue is a
residue of a phenothiazine having the formula: ##STR00020##
7. The compound of claim 1, wherein the phenothiazine residue is a
residue of a phenothiazine having the formula: ##STR00021##
8. The compound of claim 1, wherein the phenothiazine residue is
selected from the group consisting of mequitazine, promethazine,
promezine, and thiazinamium methylsulfate.
9. The compound of claim 1, wherein the water-soluble, non-peptidic
oligomer is a poly(alkylene oxide).
10. The compound of claim 9, wherein the poly(alkylene oxide) is a
poly(ethylene oxide).
11. The compound of claim 1, wherein the water-soluble,
non-peptidic oligomer is made of from about 1 to about 30
monomers.
12. The compound of claim 11, wherein the water-soluble,
non-peptidic oligomer is made of from about 1 to about 10
monomers.
13. The compound of claim 9, wherein the poly(alkylene oxide)
includes an alkoxy or hydroxy end-capping moiety.
14. The compound of claim 1, wherein a single water-soluble,
non-peptidic oligomer is attached to the phenothiazine residue.
15. The compound of claim 1, wherein more than one water-soluble,
non-peptidic oligomer is attached to the phenothiazine residue.
16. The compound of claim 1, wherein the phenothiazine residue is
covalently attached via a stable linkage.
17. The compound of claim 1, wherein the phenothiazine residue is
covalently attached via a degradable linkage.
18. The compound of claim 1, wherein the linkage is an ether
linkage.
19. The compound of claim 1, wherein the linkage is an ester
linkage.
20. A composition comprising a compound comprising a phenothiazine
residue covalently attached via a stable or degradable linkage to a
water-soluble and non-peptidic oligomer, and optionally, a
pharmaceutically acceptable excipient.
21. A composition of matter comprising a compound comprising a
phenothiazine residue covalently attached via a stable or
degradable linkage to a water-soluble, non-peptidic oligomer,
wherein the compound is present in a dosage form.
22. A method of making a phenothiazine conjugate, the method
comprising covalently attaching a water-soluble, non-peptidic
oligomer to a phenothiazine.
23. A method of treatment comprising administering a compound
comprising a phenothiazine residue covalently attached via a stable
or degradable linkage to a water-soluble, non-peptidic oligomer to
a subject in need thereof.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application Ser. No.
61/148,016, filed 28 Jan. 2009, the disclosure of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention comprises (among other things) chemically
modified phenothiazines that possess certain advantages over
phenothiazines lacking the chemical modification. The chemically
modified phenothiazines described herein relate to and/or have
application(s) in (among others) the fields of drug discovery,
pharmacotherapy, physiology, organic chemistry and polymer
chemistry.
BACKGROUND OF THE INVENTION
[0003] Phenothiazines have been shown to possess antihistaminic,
sedative, anti-motion-sickness, antiemetic, and anticholinergic
effects. They are generally indicated for: 1) amelioration of
allergic reactions to blood or plasma; 2) in anaphylaxis as an
adjunct to epinephrine and other standard measures after the acute
symptoms have been controlled; 3) for other uncomplicated allergic
conditions of the immediate type when oral therapy is impossible or
contraindicated; 4) active treatment of motion sickness; 5)
preoperative, postoperative, and obstetric (during labor) sedation;
6) prevention and control of nausea and vomiting associated with
certain types of anesthesia and surgery; 7) as an adjunct to
analgesics for the control of postoperative pain; 8) for sedation
and relief of apprehension and to produce light sleep from which
the patient can be easily aroused; and 9) intravenously in special
surgical situations, such as repeated bronchoscopy, ophthalmic
surgery, and poor-risk patients, with reduced amounts of meperidine
or other narcotic analgesic as an adjunct to anesthesia and
analgesia. Phenothiazines are also indicated as tranquilizers in
veterinary medicine. Recently, phenothiazines have been shown to be
inhibitors of KSP kinesin which is involved in microtubule-mediated
mitotic spindle-related distribution of replicate copies of the
genome to each daughter cell that result from cell division.
Therefore, phenothiazines may have a role in cancer treatment as
well. However, with the use of phenothiazines some incidents of
venous thrombosis at the injection site have been encountered.
Other clinical case reports involving the use of promethazine HCl
have indicated irritation and other serious adverse reactions at
the local area of injection particularly gangrene at the extremity
of the injection site. Promethazine hydrochloride has also been
reported to raise plasma creatine kinase levels after intramuscular
injection, which is an indication of muscle irritation.
[0004] Therefore, pharmacotherapy with phenothiazines would be
improved if these and/or other side effects associated with their
use could be decreased or if their pharmacology may be improved.
Thus, there is a large unmet need for developing novel
phenothiazine compounds.
[0005] The present invention seeks to address these and other needs
in the art.
SUMMARY OF THE INVENTION
[0006] In one or more embodiments of the invention, a compound is
provided, the compound comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer.
[0007] Exemplary compounds of the invention include those having
the following structure:
##STR00001##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; n is an integer equal to or
greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
[0008] Further exemplary compounds include those having the
following structure:
##STR00002##
wherein: R.sup.1, R.sup.2, and R.sup.3, each independently are
selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; n is an integer equal to or greater
than one; X is a spacer moiety; and POLY is a water-soluble,
non-peptidic oligomer.
[0009] Exemplary compounds include those having the following
structure:
##STR00003##
R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently are
selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; n is an integer equal to or
greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
[0010] The "phenothiazine residue" is a compound having a structure
of a phenothiazine compound that is altered by the presence of one
or more bonds, which bonds serve to attach (either directly or
indirectly) one or more water-soluble, non-peptidic oligomers.
[0011] In this regard, any phenothiazine compound having receptor
binding activity can be used as a phenothiazine moiety. Exemplary
phenothiazine moieties have a structure encompassed by Formula
I:
##STR00004##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; and n is an integer equal to or
greater than one.
[0012] In one or more embodiments of the invention, a composition
is provided, the composition comprising a compound comprising a
phenothiazine residue covalently attached via a stable or
degradable linkage to a water-soluble, non-peptidic oligomer, and
optionally, a pharmaceutically acceptable excipient.
[0013] In one or more embodiments of the invention, a dosage foini
is provided, the dosage form comprising a compound comprising a
phenothiazine residue covalently attached via a stable or
degradable linkage to a water-soluble, non-peptidic oligomer,
wherein the compound is present in a dosage form.
[0014] In one or more embodiments of the invention, a method is
provided, the method comprising covalently attaching a
water-soluble, non-peptidic oligomer to a phenothiazine moiety.
[0015] In one or more embodiments of the invention, a method is
provided, the method comprising administering a compound to a
mammal in need thereof, comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer.
[0016] These and other objects, aspects, embodiments and features
of the invention will become more fully apparent to one of ordinary
skill in the art when read in conjunction with the following
detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0017] This paragraph is intentionally left blank.
DETAILED DESCRIPTION OF THE INVENTION
[0018] As used in this specification, the singular forms "a," "an,"
and "the" include plural referents unless the context clearly
dictates otherwise.
[0019] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0020] "Water soluble, non-peptidic oligomer" indicates an oligomer
that is at least 35% (by weight) soluble, preferably greater than
70% (by weight), and more preferably greater than 95% (by weight)
soluble, in water at room temperature. Typically, an unfiltered
aqueous preparation of a "water-soluble" oligomer transmits at
least 75%, more preferably at least 95%, of the amount of light
transmitted by the same solution after filtering. It is most
preferred, however, that the water-soluble oligomer is at least 95%
(by weight) soluble in water or completely soluble in water. With
respect to being "non-peptidic," an oligomer is non-peptidic when
it has less than 35% (by weight) of amino acid residues.
[0021] The terms "monomer," "monomeric subunit" and "monomeric
unit" are used interchangeably herein and refer to one of the basic
structural units of a polymer or oligomer. In the case of a
homo-oligomer, a single repeating structural unit forms the
oligomer. In the case of a co-oligomer, two or more structural
units are repeated--either in a pattern or randomly--to form the
oligomer. Preferred oligomers used in connection with present the
invention are homo-oligomers. The water-soluble, non-peptidic
oligomer comprises one or more monomers serially attached to form a
chain of monomers. The oligomer can be formed from a single monomer
type (i.e., is homo-oligomeric) or two or three monomer types
(i.e., is co-oligomeric).
[0022] An "oligomer" is a molecule possessing from about 1 to about
30 monomers. Specific oligomers for use in the invention include
those having a variety of geometries such as linear, branched, or
forked, to be described in greater detail below.
[0023] "PEG" or "polyethylene glycol," as used herein, is meant to
encompass any water-soluble poly(ethylene oxide). Unless otherwise
indicated, a "PEG oligomer" or an oligoethylene glycol is one in
which substantially all (preferably all) monomeric subunits are
ethylene oxide subunits, though, the oligomer may contain distinct
end capping moieties or functional groups, e.g., for conjugation.
PEG oligomers for use in the present invention will comprise one of
the two following structures: "-(CH.sub.2CH.sub.2O).sub.n--" or
"--(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2--," depending upon
whether or not the terminal oxygen(s) has been displaced, e.g.,
during a synthetic transformation. As stated above, for the PEG
oligomers, the variable (n) ranges from about 1 to 30, and the
terminal groups and architecture of the overall PEG can vary. When
PEG further comprises a functional group, A, for linking to, e.g.,
a small molecule drug, the functional group when covalently
attached to a PEG oligomer does not result in formation of (i) an
oxygen-oxygen bond (--O--O--, a peroxide linkage), or (ii) a
nitrogen-oxygen bond (N--O, O--N).
[0024] The terms "end-capped" or "terminally capped" are
interchangeably used herein to refer to a terminal or endpoint of a
polymer having an end-capping moiety. Typically, although not
necessarily, the end-capping moiety comprises a hydroxy or
C.sub.1-20 alkoxy group. Thus, examples of end-capping moieties
include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as
aryl, heteroaryl, cyclo, heterocyclo, and the like. In addition,
saturated, unsaturated, substituted and unsubstituted forms of each
of the foregoing are envisioned. Moreover, the end-capping group
can also be a silane. The end-capping group can also advantageously
comprise a detectable label. When the polymer has an end-capping
group comprising a detectable label, the amount or location of the
polymer and/or the moiety (e.g., active agent) of interest to which
the polymer is coupled, can be determined by using a suitable
detector. Such labels include, without limitation, fluorescers,
chemiluminescers, moieties used in enzyme labeling, colorimetric
moieties (e.g., dyes), metal ions, radioactive moieties, and the
like. Suitable detectors include photometers, films, spectrometers,
and the like. In addition, the end-capping group may contain a
targeting moiety.
[0025] The term "targeting moiety" is used herein to refer to a
molecular structure that helps the conjugates of the invention to
localize to a targeting area, e.g., help enter a cell, or bind a
receptor. Preferably, the targeting moiety comprises of vitamin,
antibody, antigen, receptor, DNA, RNA, sialyl Lewis X antigen,
hyaluronic acid, sugars, cell specific lectins, steroid or steroid
derivative, RGD peptide, ligand for a cell surface receptor, serum
component, or combinatorial molecule directed against various
intra- or extracellular receptors. The targeting moiety may also
comprise a lipid or a phospholipid. Exemplary phospholipids
include, without limitation, phosphatidylcholines,
phospatidylserine, phospatidylinositol, phospatidylglycerol, and
phospatidylethanolamine. These lipids may be in the form of
micelles or liposomes and the like. The targeting moiety may
further comprise a detectable label or alternately a detectable
label may serve as a targeting moiety. When the conjugate has a
targeting group comprising a detectable label, the amount and/or
distribution/location of the polymer and/or the moiety (e.g.,
active agent) to which the polymer is coupled can be determined by
using a suitable detector. Such labels include, without limitation,
fluorescers, chemiluminescers, moieties used in enzyme labeling,
colorimetric (e.g., dyes), metal ions, radioactive moieties, gold
particles, quantum dots, and the like.
[0026] "Branched," in reference to the geometry or overall
structure of an oligomer, refers to an oligomer having two or more
polymers "arms" extending from a branch point.
[0027] "Forked," in reference to the geometry or overall structure
of an oligomer, refers to an oligomer having two or more functional
groups (typically through one or more atoms) extending from a
branch point.
[0028] A "branch point" refers to a bifurcation point comprising
one or more atoms at which an oligomer branches or forks from a
linear structure into one or more additional arms.
[0029] The term "reactive" or "activated" refers to a functional
group that reacts readily or at a practical rate under conventional
conditions of organic synthesis. This is in contrast to those
groups that either do not react or require strong catalysts or
impractical reaction conditions in order to react (i.e., a
"nonreactive" or "inert" group).
[0030] "Not readily reactive," with reference to a functional group
present on a molecule in a reaction mixture, indicates that the
group remains largely intact under conditions that are effective to
produce a desired reaction in the reaction mixture.
[0031] A "protecting group" is a moiety that prevents or blocks
reaction of a particular chemically reactive functional group in a
molecule under certain reaction conditions. The protecting group
may vary depending upon the type of chemically reactive group being
protected as well as the reaction conditions to be employed and the
presence of additional reactive or protecting groups in the
molecule. Functional groups which may be protected include, by way
of example, carboxylic acid groups, amino groups, hydroxyl groups,
thiol groups, carbonyl groups and the like. Representative
protecting groups for carboxylic acids include esters (such as a
p-methoxybenzyl ester), amides and hydrazides; for amino groups,
carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl
groups, ethers and esters; for thiol groups, thioethers and
thioesters; for carbonyl groups, acetals and ketals; and the like.
Such protecting groups are well-known to those skilled in the art
and are described, for example, in T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and references cited therein.
[0032] A functional group in "protected form" refers to a
functional group bearing a protecting group. As used herein, the
term "functional group" or any synonym thereof encompasses
protected forms thereof.
[0033] A "physiologically cleavable" or "hydrolyzable" or
"degradable" bond is a relatively labile bond that reacts with
water (i.e., is hydrolyzed) under physiological conditions. The
tendency of a bond to hydrolyze in water may depend not only on the
general type of linkage connecting two central atoms but also on
the substituents attached to these central atoms. Appropriate
hydrolytically unstable or weak linkages include but are not
limited to carboxylate ester, phosphate ester, anhydrides, acetals,
ketals, acyloxyalkyl ether, imines, orthoesters, peptides,
oligonucleotides, thioesters, and carbonates.
[0034] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes.
[0035] A "stable" linkage or bond refers to a chemical bond that is
substantially stable in water, that is to say, does not undergo
hydrolysis under physiological conditions to any appreciable extent
over an extended period of time. Examples of hydrolytically stable
linkages include but are not limited to the following:
carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides,
urethanes, amines, and the like. Generally, a stable linkage is one
that exhibits a rate of hydrolysis of less than about 1-2% per day
under physiological conditions. Hydrolysis rates of representative
chemical bonds can be found in most standard chemistry
textbooks.
[0036] "Substantially" or "essentially" means nearly totally or
completely, for instance, 95% or greater, more preferably 97% or
greater, still more preferably 98% or greater, even more preferably
99% or greater, yet still more preferably 99.9% or greater, with
99.99% or greater being most preferred of some given quantity.
[0037] "Monodisperse" refers to an oligomer composition wherein
substantially all of the oligomers in the composition have a
well-defined, single molecular weight and defined number of
monomers, as determined by chromatography or mass spectrometry.
Monodisperse oligomer compositions are in one sense pure, that is,
substantially having a single and definable number (as a whole
number) of monomers rather than a large distribution. A
monodisperse oligomer composition possesses a MW/Mn value of 1.0005
or less, and more preferably, a MW/Mn value of 1.0000. By
extension, a composition comprised of monodisperse conjugates means
that substantially all oligomers of all conjugates in the
composition have a single and definable number (as a whole number)
of monomers rather than a large distribution and would possess a
MW/Mn value of 1.0005, and more preferably, a MW/Mn value of 1.0000
if the oligomer were not attached to the therapeutic moiety. A
composition comprised of monodisperse conjugates may, however,
include one or more nonconjugate substances such as solvents,
reagents, excipients, and so forth.
[0038] "Bimodal," in reference to an oligomer composition, refers
to an oligomer composition wherein substantially all oligomers in
the composition have one of two definable and different numbers (as
whole numbers) of monomers rather than a large distribution, and
whose distribution of molecular weights, when plotted as a number
fraction versus molecular weight, appears as two separate
identifiable peaks. Preferably, for a bimodal oligomer composition
as described herein, each peak is generally symmetric about its
mean, although the size of the two peaks may differ. Ideally, the
polydispersity index of each peak in the bimodal distribution,
Mw/Mn, is 1.01 or less, more preferably 1.001 or less, and even
more preferably 1.0005 or less, and most preferably a MW/Mn value
of 1.0000. By extension, a composition comprised of bimodal
conjugates means that substantially all oligomers of all conjugates
in the composition have one of two definable and different numbers
(as whole numbers) of monomers rather than a large distribution and
would possess a MW/Mn value of 1.01 or less, more preferably 1.001
or less and even more preferably 1.0005 or less, and most
preferably a MW/Mn value of 1.0000 if the oligomer were not
attached to the therapeutic moiety. A composition comprised of
bimodal conjugates may, however, include one or more nonconjugate
substances such as solvents, reagents, excipients, and so
forth.
[0039] An "phenothiazine" is broadly used herein to refer to an
organic, inorganic, or organometallic compound having a molecular
weight of less than about 1000 Daltons and having some degree of
activity as a phenothiazine therapeutic. Phenothiazine activity of
a compound may be measured by assays known in the art and also as
described herein.
[0040] A "biological membrane" is any membrane made of cells or
tissues that serves as a barrier to at least some foreign entities
or otherwise undesirable materials. As used herein a "biological
membrane" includes those membranes that are associated with
physiological protective barriers including, for example: the
blood-brain barrier (BBB); the blood-cerebrospinal fluid barrier;
the blood-placental barrier; the blood-milk barrier; the
blood-testes barrier; and mucosal barriers including the vaginal
mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual
mucosa, and rectal mucosa. Unless the context clearly dictates
otherwise, the term "biological membrane" does not include those
membranes associated with the middle gastro-intestinal tract (e.g.,
stomach and small intestines).
[0041] A "biological membrane crossing rate," provides a measure of
a compound's ability to cross a biological membrane, such as the
blood-brain barrier ("BBB"). A variety of methods may be used to
assess transport of a molecule across any given biological
membrane. Methods to assess the biological membrane crossing rate
associated with any given biological barrier (e.g., the
blood-cerebrospinal fluid barrier, the blood-placental barrier, the
blood-milk barrier, the intestinal barrier, and so forth), are
known, described herein and/or in the relevant literature, and/or
may be determined by one of ordinary skill in the art.
[0042] A "reduced rate of metabolism" refers to a measurable
reduction in the rate of metabolism of a water-soluble
oligomer-small molecule drug conjugate as compared to the rate of
metabolism of the small molecule drug not attached to the
water-soluble oligomer (i.e., the small molecule drug itself) or a
reference standard material. In the special case of "reduced first
pass rate of metabolism," the same "reduced rate of metabolism" is
required except that the small molecule drug (or reference standard
material) and the corresponding conjugate are administered orally.
Orally administered drugs are absorbed from the gastro-intestinal
tract into the portal circulation and may pass through the liver
prior to reaching the systemic circulation. Because the liver is
the primary site of drug metabolism or biotransformation, a
substantial amount of drug may be metabolized before it ever
reaches the systemic circulation. The degree of first pass
metabolism, and thus, any reduction thereof, may be measured by a
number of different approaches. For instance, animal blood samples
may be collected at timed intervals and the plasma or serum
analyzed by liquid chromatography/mass spectrometry for metabolite
levels. Other techniques for measuring a "reduced rate of
metabolism" associated with the first pass metabolism and other
metabolic processes are known, described herein and/or in the
relevant literature, and/or may be determined by one of ordinary
skill in the art. Preferably, a conjugate of the invention may
provide a reduced rate of metabolism reduction satisfying at least
one of the following values: at least about 30%; at least about
40%; at least about 50%; at least about 60%; at least about 70%; at
least about 80%; and at least about 90%. A compound (such as a
small molecule drug or conjugate thereof) that is "orally
bioavailable" is one that preferably possesses a bioavailability
when administered orally of greater than 25%, and preferably
greater than 70%, where a compound's bioavailability is the
fraction of administered drug that reaches the systemic circulation
in unmetabolized form.
[0043] "Alkyl" refers to a hydrocarbon chain, ranging from about 1
to 20 atoms in length. Such hydrocarbon chains are preferably but
not necessarily saturated and may be branched or straight chain.
Exemplary alkyl groups include methyl, ethyl, propyl, butyl,
pentyl, 2-methylbutyl, 2-ethylpropyl, 3-methylpentyl, and the like.
As used herein, "alkyl" includes cycloalkyl when three or more
carbon atoms are referenced. An "alkenyl" group is an alkyl of 2 to
20 carbon atoms with at least one carbon-carbon double bond.
[0044] The terms "substituted alkyl" or "substituted C.sub.q,
alkyl" where q and r are integers identifying the range of carbon
atoms contained in the alkyl group, denotes the above alkyl groups
that are substituted by one, two or three halo (e.g., F, Cl, Br,
I), trifluoromethyl, hydroxy, C.sub.1-7 alkyl (e.g., methyl, ethyl,
n-propyl, isopropyl, butyl, t-butyl, and so forth), C.sub.1-7
alkoxy, C.sub.1-7 acyloxy, C.sub.3-7 heterocyclic, amino, phenoxy,
nitro, carboxy, acyl, cyano. The substituted alkyl groups may be
substituted once, twice or three times with the same or with
different substituents.
[0045] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, and may be straight chain or branched, as
exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl. "Lower
alkenyl" refers to a lower alkyl group of 2 to 6 carbon atoms
having at least one carbon-carbon double bond.
[0046] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0047] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C.sub.1-C.sub.20 alkyl (e.g.,
methoxy, ethoxy, propyloxy, etc.), preferably C.sub.1-C.sub.7.
[0048] "Pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" refers to component that may be included in the
compositions of the invention causes no significant adverse
toxicological effects to a patient.
[0049] The term "aryl" means an aromatic group having up to 14
carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl,
phenanthrenyl, naphthalenyl, and the like. "Substituted phenyl" and
"substituted aryl" denote a phenyl group and aryl group,
respectively, substituted with one, two, three, four or five (e.g.
1-2, 1-3 or 1-4 substituents) chosen from halo (F, Cl, Br, I),
hydroxy, cyano, nitro, alkyl (e.g., C.sub.1-6 alkyl), alkoxy (e.g.,
C.sub.1-6 alkoxy), benzyloxy, carboxy, aryl, and so forth.
[0050] Chemical moieties are defined and referred to throughout
primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.).
Nevertheless, such terms are also used to convey corresponding
multivalent moieties under the appropriate structural circumstances
clear to those skilled in the art. For example, while an "alkyl"
moiety generally refers to a monovalent radical (e.g.,
CH.sub.3--CH.sub.2--), in certain circumstances a bivalent linking
moiety can be "alkyl," in which case those skilled in the art will
understand the alkyl to be a divalent radical (e.g.,
--CH.sub.2--CH.sub.2--), which is equivalent to the term
"alkylene." (Similarly, in circumstances in which a divalent moiety
is required and is stated as being "aryl," those skilled in the art
will understand that the term "aryl" refers to the corresponding
multivalent moiety, arylene). All atoms are understood to have
their normal number of valences for bond formation (i.e., 1 for H,
4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on
the oxidation state of the S).
[0051] "Pharmacologically effective amount," "physiologically
effective amount," and "therapeutically effective amount" are used
interchangeably herein to mean the amount of a water-soluble
oligomer-small molecule drug conjugate present in a composition
that is needed to provide a desired level of active agent and/or
conjugate in the bloodstream or in the target tissue. The precise
amount may depend upon numerous factors, e.g., the particular
active agent, the components and physical characteristics of the
composition, intended patient population, patient considerations,
and may readily be determined by one skilled in the art, based upon
the information provided herein and available in the relevant
literature.
[0052] A "difunctional" oligomer is an oligomer having two
functional groups contained therein, typically at its termini. When
the functional groups are the same, the oligomer is said to be
homodifunctional. When the functional groups are different, the
oligomer is said to be heterodifunctional.
[0053] A basic reactant or an acidic reactant described herein
include neutral, charged, and any corresponding salt forms
thereof.
[0054] The term "patient," refers to a living organism suffering
from or prone to a condition that can be prevented or treated by
administration of a conjugate as described herein, and includes
both humans and animals.
[0055] "Optional" or "optionally" means that the subsequently
described circumstance may but need not necessarily occur, so that
the description includes instances where the circumstance occurs
and instances where it does not.
[0056] As indicated above, the present invention is directed to
(among other things) a compound comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer.
[0057] The "phenothiazine residue" is a compound having a structure
of a phenothiazine compound that is altered by the presence of one
or more bonds, which bonds serve to attach (either directly or
indirectly) one or more water-soluble, non-peptidic oligomers.
Exemplary phenothiazines have a structure encompassed by at least
one of the structures defined herein as Formula I:
##STR00005##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; and n is an integer equal to or
greater than one.
[0058] In one or more embodiments of the invention, a compound is
provided, the compound comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer, wherein the phenothiazine has
a structure encompassed by the following formula:
##STR00006##
[0059] In one or more embodiments of the invention, a compound is
provided, the compound comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer, wherein the phenothiazine has
a structure encompassed by the following formula:
##STR00007##
[0060] In one or more embodiments of the invention, a compound is
provided, the compound comprising a phenothiazine residue
covalently attached via a stable or degradable linkage to a
water-soluble, non-peptidic oligomer, wherein the phenothiazine is
selected from the group consisting of mequitazine, promethazine,
promezine, and thiazinamium methylsulfate.
[0061] In some instances, phenothiazines can be obtained from
commercial sources. In addition, phenothiazines can be obtained
through chemical synthesis. Examples of phenothiazines as well as
synthetic approaches for preparing phenothiazines are described in
the literature and in, for example, U.S. Pat. Nos. 2,519,886,
2,530,451, 2,607,773, 3,987,042, GB Patent No. 641452. Each of
these (and other) phenothiazines can be covalently attached (either
directly or through one or more atoms) to a water-soluble,
non-peptidic oligomer.
[0062] Exemplary compounds of the invention include those having
the following structure:
##STR00008##
wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently
are selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; n is an integer equal to or
greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
[0063] Further exemplary compounds of the invention include those
having the following structure:
##STR00009##
wherein:
[0064] R.sup.1, R.sup.2, and R.sup.3, each independently are
selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl;
n is an integer equal to or greater than one; X is a spacer moiety;
and POLY is a water-soluble, non-peptidic oligomer.
[0065] Exemplary compounds of the invention include those having
the following structure:
##STR00010##
R.sup.1, R.sup.2, R.sup.3, and R.sup.4, each independently are
selected from the group consisting of hydrogen, unsubstituted
alkyl, and substituted alkyl; or R.sup.3 and R.sup.4 together with
the nitrogen form a heterocycle; n is an integer equal to or
greater than one; X is a spacer moiety; and POLY is a
water-soluble, non-peptidic oligomer.
[0066] Use of discrete oligomers (e.g., from a monodisperse or
bimodal composition of oligomers, in contrast to relatively impure
compositions) to form oligomer-containing compounds may
advantageously alter certain properties associated with the
corresponding small molecule drug. For instance, a compound of the
invention, when administered by any of a number of suitable
administration routes, such as parenteral, oral, transdermal,
buccal, pulmonary, or nasal, exhibits reduced penetration across
the blood-brain barrier. It is preferred that the compounds of the
invention exhibit slowed, minimal or effectively no crossing of the
blood-brain barrier, while still crossing the gastro-intestinal
(GI) walls and into the systemic circulation if oral delivery is
intended. Moreover, the compounds of the invention maintain a
degree of bioactivity as well as bioavailability in comparison to
the bioactivity and bioavailability of the compound free of all
oligomers.
[0067] With respect to the blood-brain barrier ("BBB"), this
barrier restricts the transport of drugs from the blood to the
brain. This barrier consists of a continuous layer of unique
endothelial cells joined by tight junctions. The cerebral
capillaries, which comprise more than 95% of the total surface area
of the BBB, represent the principal route for the entry of most
solutes and drugs into the central nervous system.
[0068] For compounds whose degree of blood-brain barrier crossing
ability is not readily known, such ability may be determined using
a suitable animal model such as an in situ rat brain perfusion
("RBP") model as described herein. Briefly, the RBP technique
involves cannulation of the carotid artery followed by perfusion
with a compound solution under controlled conditions, followed by a
wash out phase to remove compound remaining in the vascular space.
(Such analyses may be conducted, for example, by contract research
organizations such as Absorption Systems, Exton, Pa.). In one
example of the RBP model, a cannula is placed in the left carotid
artery and the side branches are tied off. A physiologic buffer
containing the analyte (typically but not necessarily at a 5
micromolar concentration level) is perfused at a flow rate of about
10 mL/minute in a single pass perfusion experiment. After 30
seconds, the perfusion is stopped and the brain vascular contents
are washed out with compound-free buffer for an additional 30
seconds. The brain tissue is then removed and analyzed for compound
concentrations via liquid chromatograph with tandem mass
spectrometry detection (LC/MS/MS). Alternatively, blood-brain
barrier permeability can be estimated based upon a calculation of
the compound's molecular polar surface area ("PSA"), which is
defined as the sum of surface contributions of polar atoms (usually
oxygens, nitrogens and attached hydrogens) in a molecule. The PSA
has been shown to correlate with compound transport properties such
as blood-brain barrier transport. Methods for determining a
compound's PSA can be found, e.g., in, Ertl, P., et al., J. Med.
Chem. 2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res.
1999, 16, 1514-1519.
[0069] With respect to the blood-brain barrier, the water-soluble,
non-peptidic oligomer-small molecule drug conjugate exhibits a
blood-brain barrier crossing rate that is reduced as compared to
the crossing rate of the small molecule drug not attached to the
water-soluble, non-peptidic oligomer. Exemplary reductions in
blood-brain barrier crossing rates for the compounds described
herein include reductions of: at least about 5%; at least about
10%; at least about 25%; at least about 30%; at least about 40%; at
least about 50%; at least about 60%; at least about 70%; at least
about 80%; or at least about 90%, when compared to the blood-brain
barrier crossing rate of the small molecule drug not attached to
the water-soluble oligomer. A preferred reduction in the
blood-brain barrier crossing rate for a conjugate of the invention
is at least about 20%.
[0070] Assays for determining whether a given compound (regardless
of whether the compound includes a water-soluble, non-peptidic
oligomer or not) can act as a phenothiazine are known and/or may be
prepared by one of ordinary skill in the art and are further
described infra.
[0071] Each of these (and other) phenothiazine moieties can be
covalently attached (either directly or through one or more atoms)
to a water-soluble, non-peptidic oligomer.
[0072] Exemplary molecular weights of small molecule drugs include
molecular weights of: less than about 950; less than about 900;
less than about 850; less than about 800; less than about 750; less
than about 700; less than about 650; less than about 600; less than
about 550; less than about 500; less than about 450; less than
about 400; less than about 350; and less than about 300
Daltons.
[0073] The small molecule drug used in the invention, if chiral,
may be obtained from a racemic mixture, or an optically active
form, for example, a single optically active enantiomer, or any
combination or ratio of enantiomers (i.e., scalemic mixture). In
addition, the small molecule drug may possess one or more geometric
isomers. With respect to geometric isomers, a composition can
comprise a single geometric isomer or a mixture of two or more
geometric isomers. A small molecule drug for use in the present
invention can be in its customary active form, or may possess some
degree of modification. For example, a small molecule drug may have
a targeting agent, tag, or transporter attached thereto, prior to
or after covalent attachment of an oligomer. Alternatively, the
small molecule drug may possess a lipophilic moiety attached
thereto, such as a phospholipid (e.g.,
distearoylphosphatidylethanolamine or "DSPE,"
dipalmitoylphosphatidylethanolamine or "DPPE," and so forth) or a
small fatty acid. In some instances, however, it is preferred that
the small molecule drug moiety does not include attachment to a
lipophilic moiety.
[0074] The phenothiazine moiety for coupling to a water-soluble,
non-peptidic oligomer possesses a free hydroxyl, carboxyl, thio,
amino group, or the like (i.e., "handle") suitable for covalent
attachment to the oligomer. In addition, the phenothiazine moiety
may be modified by introduction of a reactive group, preferably by
conversion of one of its existing functional groups to a functional
group suitable for formation of a stable covalent linkage between
the oligomer and the drug.
[0075] Accordingly, each oligomer is composed of up to three
different monomer types selected from the group consisting of:
alkylene oxide, such as ethylene oxide or propylene oxide; olefinic
alcohol, such as vinyl alcohol, 1-propenol or 2-propenol; vinyl
pyrrolidone; hydroxyalkyl methacrylamide or hydroxyalkyl
methacrylate, where alkyl is preferably methyl; .alpha.-hydroxy
acid, such as lactic acid or glycolic acid; phosphazene, oxazoline,
amino acids, carbohydrates such as monosaccharides, alditol such as
mannitol; and N-acryloylmorpholine. Preferred monomer types include
alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide or
methacrylate, N-acryloylmorpholine, and .alpha.-hydroxy acid.
Preferably, each oligomer is, independently, a co-oligomer of two
monomer types selected from this group, or, more preferably, is a
homo-oligomer of one monomer type selected from this group.
[0076] The two monomer types in a co-oligomer may be of the same
monomer type, for example, two alkylene oxides, such as ethylene
oxide and propylene oxide. Preferably, the oligomer is a
homo-oligomer of ethylene oxide. Usually, although not necessarily,
the terminus (or termini) of the oligomer that is not covalently
attached to a small molecule is capped to render it unreactive.
Alternatively, the terminus may include a reactive group. When the
terminus is a reactive group, the reactive group is either selected
such that it is unreactive under the conditions of formation of the
final oligomer or during covalent attachment of the oligomer to a
small molecule drug, or it is protected as necessary. One common
end-functional group is hydroxyl or --OH, particularly for
oligoethylene oxides.
[0077] The water-soluble, non-peptidic oligomer (e.g., "POLY" in
various structures provided herein) can have any of a number of
different geometries. For example, the water-soluble, non-peptidic
oligomer can be linear, branched, or forked. Most typically, the
water-soluble, non-peptidic oligomer is linear or is branched, for
example, having one branch point. Although much of the discussion
herein is focused upon poly(ethylene oxide) as an illustrative
oligomer, the discussion and structures presented herein can be
readily extended to encompass any water-soluble, non-peptidic
oligomers described above.
[0078] The molecular weight of the water-soluble, non-peptidic
oligomer, excluding the linker portion, is generally relatively
low. Exemplary values of the molecular weight of the water-soluble
polymer include: below about 1500; below about 1450; below about
1400; below about 1350; below about 1300; below about 1250; below
about 1200; below about 1150; below about 1100; below about 1050;
below about 1000; below about 950; below about 900; below about
850; below about 800; below about 750; below about 700; below about
650; below about 600; below about 550; below about 500; below about
450; below about 400; below about 350; below about 300; below about
250; below about 200; and below about 100 Daltons.
[0079] Exemplary ranges of molecular weights of the water-soluble,
non-peptidic oligomer (excluding the linker) include: from about
100 to about 1400 Daltons; from about 100 to about 1200 Daltons;
from about 100 to about 800 Daltons; from about 100 to about 500
Daltons; from about 100 to about 400 Daltons; from about 200 to
about 500 Daltons; from about 200 to about 400 Daltons; from about
75 to 1000 Daltons; and from about 75 to about 750 Daltons.
[0080] Preferably, the number of monomers in the water-soluble,
non-peptidic oligomer falls within one or more of the following
ranges: between about 1 and about 30 (inclusive); between about 1
and about 25; between about 1 and about 20; between about 1 and
about 15; between about 1 and about 12; between about 1 and about
10. In certain instances, the number of monomers in series in the
oligomer (and the corresponding conjugate) is one of 1, 2, 3, 4, 5,
6, 7, or 8. In additional embodiments, the oligomer (and the
corresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 monomers. In yet further embodiments, the
oligomer (and the corresponding conjugate) possesses 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 monomers in series. Thus, for example,
when the water-soluble, non-peptidic polymer includes
CH.sub.3--(OCH.sub.2CH.sub.2).sub.n--, "n" is an integer that can
be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and can fall
within one or more of the following ranges: between about 1 and
about 25; between about 1 and about 20; between about 1 and about
15; between about 1 and about 12; between about 1 and about 10.
[0081] When the water-soluble, non-peptidic oligomer has 1, 2, 3,
4, 5, 6, 7, 8, 9, or monomers, these values correspond to a methoxy
end-capped oligo(ethylene oxide) having a molecular weights of
about 75, 119, 163, 207, 251, 295, 339, 383, 427, and 471 Daltons,
respectively. When the oligomer has 11, 12, 13, 14, or 15 monomers,
these values correspond to methoxy end-capped oligo(ethylene oxide)
having molecular weights corresponding to about 515, 559, 603, 647,
and 691 Daltons, respectively.
[0082] When the water-soluble, non-peptidic oligomer is attached to
the phenothiazine (in contrast to the step-wise addition of one or
more monomers to effectively "grow" the oligomer onto the
phenothiazine), it is preferred that the composition containing an
activated form of the water-soluble, non-peptidic oligomer be
monodisperse. In those instances, however, where a bimodal
composition is employed, the composition will possess a bimodal
distribution centering around any two of the above numbers of
monomers. For instance, a bimodal oligomer may have any one of the
following exemplary combinations of monomer subunits: 1-2, 1-3,
1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5,
2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8,
3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so
forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9, 6-10,
and so forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and so
forth.
[0083] In some instances, the composition containing an activated
form of the water-soluble, non-peptidic oligomer will be trimodal
or even tetramodal, possessing a range of monomers units as
previously described. Oligomer compositions possessing a
well-defined mixture of oligomers (i.e., being bimodal, trimodal,
tetramodal, and so forth) can be prepared by mixing purified
monodisperse oligomers to obtain a desired profile of oligomers (a
mixture of two oligomers differing only in the number of monomers
is bimodal; a mixture of three oligomers differing only in the
number of monomers is trimodal; a mixture of four oligomers
differing only in the number of monomers is tetramodal), or
alternatively, can be obtained from column chromatography of a
polydisperse oligomer by recovering the "center cut", to obtain a
mixture of oligomers in a desired and defined molecular weight
range.
[0084] It is preferred that the water-soluble, non-peptidic
oligomer is obtained from a composition that is preferably
unimolecular or monodisperse. That is, the oligomers in the
composition possess the same discrete molecular weight value rather
than a distribution of molecular weights. Some monodisperse
oligomers can be purchased from commercial sources such as those
available from Sigma-Aldrich, or alternatively, can be prepared
directly from commercially available starting materials such as
Sigma-Aldrich. Water-soluble, non-peptidic oligomers can be
prepared as described in Chen Y., Baker, G. L., J. Org. Chem.,
6870-6873 (1999), WO 02/098949, and U.S. Patent Application
Publication 2005/0136031.
[0085] When present, the spacer moiety (through which the
water-soluble, non-peptidic polymer is attached to the
phenothiazine moiety) may be a single bond, a single atom, such as
an oxygen atom or a sulfur atom, two atoms, or a number of atoms. A
spacer moiety is typically but is not necessarily linear in nature.
The spacer moiety, "X," is hydrolytically stable, and is preferably
also enzymatically stable. Preferably, the spacer moiety "X" is one
having a chain length of less than about 12 atoms, and preferably
less than about 10 atoms, and even more preferably less than about
8 atoms and even more preferably less than about 5 atoms, whereby
length is meant the number of atoms in a single chain, not counting
substituents. For instance, a urea linkage such as this,
R.sub.oligomer--NH--(C.dbd.O)--NH--R'.sub.drug, is considered to
have a chain length of 3 atoms (--NH--C(O)--NH--). In selected
embodiments, the linkage does not comprise further spacer
groups.
[0086] In some instances, the spacer moiety "X" comprises an ether,
amide, urethane, amine, thioether, urea, or a carbon-carbon bond.
Functional groups such as those discussed below, and illustrated in
the examples, are typically used for forming the linkages. The
spacer moiety may less preferably also comprise (or be adjacent to
or flanked by) other atoms, as described further below.
[0087] More specifically, in selected embodiments, a spacer moiety
of the invention, X, may be any of the following: "--" (i.e., a
covalent bond, that may be stable or degradable, between the
phenothiazine residue and the water-soluble, non-peptidic
oligomer), ), --O--, --NH--, --S--, --C(O)--, --C(O)O--, --OC(O)--,
--CH.sub.2--C(O)O--, --CH.sub.2--OC(O)--, --C(O)O--CH.sub.2--,
--OC(O)--CH.sub.2--, C(O)--NH, NH--C(O)--NH, O--C(O)--NH, --C(S)--,
--CH.sub.2--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --O--CH.sub.2--,
--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--NH--C(O)--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--,
--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--CH.sub.2--, --O--C(O)--NH--CH.sub.2--CH.sub.2--,
--NH--CH.sub.2--, --NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--NH--CH.sub.2--,
--C(O)--CH.sub.2--, --C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C-
H.sub.2--, bivalent cycloalkyl group, --N(R.sup.6)--, R.sup.6 is H
or an organic radical selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl and substituted aryl. Additional spacer
moieties include, acylamino, acyl, aryloxy, alkylene bridge
containing between 1 and 5 inclusive carbon atoms, alkylamino,
dialkylamino having about 2 to 4 inclusive carbon atoms,
piperidino, pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino,
1-piperizinyl, 4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-lower
alkyl)-1-piperizinyl, 4-(methoxy-lower alkyl)-1-piperizinyl, and
guanidine. In some instances, a portion or a functional group of
the drug compound may be modified or removed altogether to
facilitate attachment of the oligomer. In some instances, it is
preferred that X is not an amide, i.e., --CONR-- or --RNCO--).
[0088] For purposes of the present invention, however, a group of
atoms is not considered a linkage when it is immediately adjacent
to an oligomer segment, and the group of atoms is the same as a
monomer of the oligomer such that the group would represent a mere
extension of the oligomer chain.
[0089] The linkage "X" between the water-soluble, non-peptidic
oligomer and the small molecule is formed by reaction of a
functional group on a terminus of the oligomer (or nascent oligomer
when it is desired to "grow" the oligomer onto the phenothiazine)
with a corresponding functional group within the phenothiazine.
Illustrative reactions are described briefly below. For example, an
amino group on an oligomer may be reacted with a carboxylic acid or
an activated carboxylic acid derivative on the small molecule, or
vice versa, to produce an amide linkage. Alternatively, reaction of
an amine on an oligomer with an activated carbonate (e.g.
succinimidyl or benzotriazolyl carbonate) on the drug, or vice
versa, forms a carbamate linkage. Reaction of an amine on an
oligomer with an isocyanate (R--N.dbd.C.dbd.O) on a drug, or vice
versa, forms a urea linkage (R--NH--(C.dbd.O)--NH--R'). Further,
reaction of an alcohol (alkoxide) group on an oligomer with an
alkyl halide, or halide group within a drug, or vice versa, forms
an ether linkage. In yet another coupling approach, a small
molecule having an aldehyde function is coupled to an oligomer
amino group by reductive amination, resulting in formation of a
secondary amine linkage between the oligomer and the small
molecule.
[0090] A particularly preferred water-soluble, non-peptidic
oligomer is an oligomer bearing an aldehyde functional group. In
this regard, the oligomer will have the following structure:
CH.sub.3O--(CH.sub.2--CH.sub.2--O).sub.n--(CH.sub.2).sub.p--C(O)H,
wherein (n) is one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is
one of 1, 2, 3, 4, 5, 6 and 7. Preferred (n) values include 3, 5
and 7 and preferred (p) values 2, 3 and 4.
[0091] The termini of the water-soluble, non-peptidic oligomer not
bearing a functional group may be capped to render it unreactive.
When the oligomer includes a further functional group at a terminus
other than that intended for formation of a conjugate, that group
is either selected such that it is unreactive under the conditions
of formation of the linkage "X," or it is protected during the
formation of the linkage "X."
[0092] As stated above, the water-soluble, non-peptidic oligomer
includes at least one functional group prior to conjugation. The
functional group comprises an electrophilic or nucleophilic group
for covalent attachment to a small molecule, depending upon the
reactive group contained within or introduced into the small
molecule. Examples of nucleophilic groups that may be present in
either the oligomer or the small molecule include hydroxyl, amine,
hydrazine (--NHNH.sub.2), hydrazide (--C(O)NHNH.sub.2), and thiol.
Preferred nucleophiles include amine, hydrazine, hydrazide, and
thiol, particularly amine. Most small molecule drugs for covalent
attachment to an oligomer will possess a free hydroxyl, amino,
thio, aldehyde, ketone, or carboxyl group.
[0093] Examples of electrophilic functional groups that may be
present in either the oligomer or the small molecule include
carboxylic acid, carboxylic ester, particularly imide esters,
orthoester, carbonate, isocyanate, isothiocyanate, aldehyde,
ketone, thione, alkenyl, acrylate, methacrylate, acrylamide,
sulfone, maleimide, disulfide, iodo, epoxy, sulfonate,
thiosulfonate, silane, alkoxysilane, and halosilane. More specific
examples of these groups include succinimidyl ester or carbonate,
imidazoyl ester or carbonate, benzotriazole ester or carbonate,
vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyl
disulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, and
tresylate (2,2,2-trifluoroethanesulfonate).
[0094] Also included are sulfur analogs of several of these groups,
such as thione, thione hydrate, thioketal, 2-thiazolidine thione,
etc., as well as hydrates or protected derivatives of any of the
above moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketone
hydrate, hemiketal, ketal, thioketal, thioacetal).
[0095] An "activated derivative" of a carboxylic acid refers to a
carboxylic acid derivative that reacts readily with nucleophiles,
generally much more readily than the underivatized carboxylic acid.
Activated carboxylic acids include, for example, acid halides (such
as acid chlorides), anhydrides, carbonates, and esters. Such esters
include imide esters, of the general form --(CO)O--N[(CO)--].sub.2;
for example, N-hydroxysuccinimidyl (NHS) esters or
N-hydroxyphthalimidyl esters. Also preferred are imidazolyl esters
and benzotriazole esters. Particularly preferred are activated
propionic acid or butanoic acid esters, as described in co-owned
U.S. Pat. No. 5,672,662. These include groups of the form
--(CH.sub.2).sub.2-3C(.dbd.O)O-Q, where Q is preferably selected
from N-succinimide, N-sulfosuccinimide, N-phthalimide,
N-glutarimide, N-tetrahydrophthalimide,
N-norbornene-2,3-dicarboximide, benzotriazole, 7-azabenzotriazole,
and imidazole.
[0096] Other preferred electrophilic groups include succinimidyl
carbonate, maleimide, benzotriazole carbonate, glycidyl ether,
imidazoyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate,
aldehyde, and orthopyridyl disulfide.
[0097] These electrophilic groups are subject to reaction with
nucleophiles, e.g., hydroxy, thio, or amino groups, to produce
various bond types. Preferred for the present invention are
reactions which favor formation of a hydrolytically stable linkage.
For example, carboxylic acids and activated derivatives thereof,
which include orthoesters, succinimidyl esters, imidazolyl esters,
and benzotriazole esters, react with the above types of
nucleophiles to form esters, thioesters, and amides, respectively,
of which amides are the most hydrolytically stable. Carbonates,
including succinimidyl, imidazolyl, and benzotriazole carbonates,
react with amino groups to form carbamates. Isocyanates
(R--N.dbd.C.dbd.O) react with hydroxyl or amino groups to form,
respectively, carbamate (RNH--C(O)--OR') or urea (RNH--C(O)--NHR')
linkages. Aldehydes, ketones, glyoxals, diones and their hydrates
or alcohol adducts (i.e., aldehyde hydrate, hemiacetal, acetal,
ketone hydrate, hemiketal, and ketal) are preferably reacted with
amines, followed by reduction of the resulting imine, if desired,
to provide an amine linkage (reductive amination).
[0098] Several of the electrophilic functional groups include
electrophilic double bonds to which nucleophilic groups, such as
thiols, can be added, to form, for example, thioether bonds. These
groups include maleimides, vinyl sulfones, vinyl pyridine,
acrylates, methacrylates, and acrylamides. Other groups comprise
leaving groups that can be displaced by a nucleophile; these
include chloroethyl sulfone, pyridyl disulfides (which include a
cleavable S--S bond), iodoacetamide, mesylate, tosylate,
thiosulfonate, and tresylate. Epoxides react by ring opening by a
nucleophile, to fowl, for example, an ether or amine bond.
Reactions involving complementary reactive groups such as those
noted above on the oligomer and the small molecule are utilized to
prepare the conjugates of the invention.
[0099] In some instances the phenothiazine may not have a
functional group suited for conjugation. In this instance, it is
possible to modify (or "functionalize") the "original"
phenothiazine so that it does have a functional group suited for
conjugation. For example, if the phenothiazine has an amide group,
but an amine group is desired, it is possible to modify the amide
group to an amine group by way of a Hofmann rearrangement, Curtius
rearrangement (once the amide is converted to an azide) or Lossen
rearrangement (once amide is concerted to hydroxamide followed by
treatment with tolyene-2-sulfonyl chloride/base).
[0100] It is possible to prepare a conjugate of small molecule
phenothiazine bearing a carboxyl group wherein the carboxyl
group-bearing small molecule phenothiazine is coupled to an
amino-terminated oligomeric ethylene glycol, to provide a conjugate
having an amide group covalently linking the small molecule
phenothiazine to the oligomer. This can be performed, for example,
by combining the carboxyl group-bearing small molecule
phenothiazine with the amino-terminated oligomeric ethylene glycol
in the presence of a coupling reagent, (such as
dicyclohexylcarbodiimide or "DCC") in an anhydrous organic
solvent.
[0101] Further, it is possible to prepare a conjugate of a small
molecule phenothiazine bearing a hydroxyl group wherein the
hydroxyl group-bearing small molecule phenothiazine is coupled to
an oligomeric ethylene glycol halide to result in an ether (--O--)
linked small molecule conjugate. This can be performed, for
example, by using sodium hydride to deprotonate the hydroxyl group
followed by reaction with a halide-terminated oligomeric ethylene
glycol.
[0102] Further, it is possible to prepare a conjugate of a small
molecule phenothiazine moiety bearing a hydroxyl group wherein the
hydroxyl group-bearing small molecule phenothiazine moiety is
coupled to an oligomeric ethylene glycol bearing an haloformate
group [e.g., CH.sub.3(OCH.sub.2CH.sub.2).sub.nOC(O)-halo, where
halo is chloro, bromo, iodo] to result in a carbonate
[--O--C(O)--O--] linked small molecule conjugate. This can be
performed, for example, by combining a phenothiazine moiety and an
oligomeric ethylene glycol bearing a haloformate group in the
presence of a nucleophilic catalyst (such as
4-dimethylaminopyridine or "DMAP") to thereby result in the
corresponding carbonate-linked conjugate.
[0103] In another example, it is possible to prepare a conjugate of
a small molecule phenothiazine bearing a ketone group by first
reducing the ketone group to form the corresponding hydroxyl group.
Thereafter, the small molecule phenothiazine now bearing a hydroxyl
group can be coupled as described herein.
[0104] In still another instance, it is possible to prepare a
conjugate of a small molecule phenothiazine bearing an amine group.
In one approach, the amine group-bearing small molecule
phenothiazine and an aldehyde-bearing oligomer are dissolved in a
suitable buffer after which a suitable reducing agent (e.g.,
NaCNBH.sub.3) is added. Following reduction, the result is an amine
linkage formed between the amine group of the amine
group-containing small molecule phenothiazine and the carbonyl
carbon of the aldehyde-bearing oligomer.
[0105] In another approach for preparing a conjugate of a small
molecule phenothiazine bearing an amine group, a carboxylic
acid-bearing oligomer and the amine group-bearing small molecule
phenothiazine are combined, in the presence of a coupling reagent
(e.g., DCC). The result is an amide linkage formed between the
amine group of the amine group-containing small molecule
phenothiazine and the carbonyl of the carboxylic acid-bearing
oligomer.
[0106] While it is believed that the full scope of the conjugates
disclosed herein behave as described, an optimally sized oligomer
can be identified as follows.
[0107] First, an oligomer obtained from a monodisperse or bimodal
water soluble oligomer is conjugated to the small molecule drug.
Preferably, the drug is orally bioavailable, and on its own,
exhibits a non-negligible blood-brain barrier crossing rate. Next,
the ability of the conjugate to cross the blood-brain barrier is
determined using an appropriate model and compared to that of the
unmodified parent drug. If the results are favorable, that is to
say, if, for example, the rate of crossing is significantly
reduced, then the bioactivity of conjugate is further evaluated.
Preferably, the compounds according to the invention maintain a
significant degree of bioactivity relative to the parent drug,
i.e., greater than about 30% of the bioactivity of the parent drug,
or even more preferably, greater than about 50% of the bioactivity
of the parent drug.
[0108] The above steps are repeated one or more times using
oligomers of the same monomer type but having a different number of
subunits and the results compared.
[0109] For each conjugate whose ability to cross the blood-brain
barrier is reduced in comparison to the non-conjugated small
molecule drug, its oral bioavailability is then assessed. Based
upon these results, that is to say, based upon the comparison of
conjugates of oligomers of varying size to a given small molecule
at a given position or location within the small molecule, it is
possible to determine the size of the oligomer most effective in
providing a conjugate having an optimal balance between reduction
in biological membrane crossing, oral bioavailability, and
bioactivity. The small size of the oligomers makes such screenings
feasible and allows one to effectively tailor the properties of the
resulting conjugate. By making small, incremental changes in
oligomer size and utilizing an experimental design approach, one
can effectively identify a conjugate having a favorable balance of
reduction in biological membrane crossing rate, bioactivity, and
oral bioavailability. In some instances, attachment of an oligomer
as described herein is effective to actually increase oral
bioavailability of the drug.
[0110] For example, one of ordinary skill in the art, using routine
experimentation, can determine a best suited molecular size and
linkage for improving oral bioavailability by first preparing a
series of oligomers with different weights and functional groups
and then obtaining the necessary clearance profiles by
administering the conjugates to a patient and taking periodic blood
and/or urine sampling. Once a series of clearance profiles have
been obtained for each tested conjugate, a suitable conjugate can
be identified.
[0111] Animal models (rodents and dogs) can also be used to study
oral drug transport. In addition, non-in vivo methods include
rodent everted gut excised tissue and Caco-2 cell monolayer
tissue-culture models. These models are useful in predicting oral
drug bioavailability.
[0112] To determine whether the phenothiazine or the conjugate of a
phenothiazine and a water-soluble non-peptidic polymer has activity
as a phenothiazine therapeutic, it is possible to test such a
compound. The phenothiazine compounds may be tested using in vitro
binding studies to receptors using various cell lines expressing
these receptors that have become routine in pharmaceutical industry
and described herein.
[0113] The present invention also includes pharmaceutical
preparations comprising a conjugate as provided herein in
combination with a pharmaceutical excipient. Generally, the
conjugate itself will be in a solid form (e.g., a precipitate),
which can be combined with a suitable pharmaceutical excipient that
can be in either solid or liquid form.
[0114] Exemplary excipients include, without limitation, those
selected from the group consisting of carbohydrates, inorganic
salts, antimicrobial agents, antioxidants, surfactants, buffers,
acids, bases, and combinations thereof.
[0115] A carbohydrate such as a sugar, a derivatized sugar such as
an alditol, aldonic acid, an esterified sugar, and/or a sugar
polymer may be present as an excipient. Specific carbohydrate
excipients include, for example: monosaccharides, such as fructose,
maltose, galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, maltitol, lactitol, xylitol, sorbitol, myoinositol,
and the like.
[0116] The excipient can also include an inorganic salt or buffer
such as citric acid, sodium chloride, potassium chloride, sodium
sulfate, potassium nitrate, sodium phosphate monobasic, sodium
phosphate dibasic, and combinations thereof.
[0117] The preparation may also include an antimicrobial agent for
preventing or deterring microbial growth. Nonlimiting examples of
antimicrobial agents suitable for the present invention include
benzalkonium chloride, benzethonium chloride, benzyl alcohol,
cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl
alcohol, phenylmercuric nitrate, thimersol, and combinations
thereof.
[0118] An antioxidant can be present in the preparation as well.
Antioxidants are used to prevent oxidation, thereby preventing the
deterioration of the conjugate or other components of the
preparation. Suitable antioxidants for use in the present invention
include, for example, ascorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorous acid, monothioglycerol,
propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate,
sodium metabisulfite, and combinations thereof.
[0119] A surfactant may be present as an excipient. Exemplary
surfactants include: polysorbates, such as "Tween 20" and "Tween
80," and pluronics such as F68 and F88 (both of which are available
from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as
phospholipids such as lecithin and other phosphatidylcholines,
phosphatidylethanolamines, fatty acids and fatty esters; steroids,
such as cholesterol; and chelating agents, such as EDTA, zinc and
other such suitable cations.
[0120] Pharmaceutically acceptable acids or bases may be present as
an excipient in the preparation. Nonlimiting examples of acids that
can be used include those acids selected from the group consisting
of hydrochloric acid, acetic acid, phosphoric acid, citric acid,
malic acid, lactic acid, formic acid, trichloroacetic acid, nitric
acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric
acid, and combinations thereof. Examples of suitable bases include,
without limitation, bases selected from the group consisting of
sodium hydroxide, sodium acetate, ammonium hydroxide, potassium
hydroxide, ammonium acetate, potassium acetate, sodium phosphate,
potassium phosphate, sodium citrate, sodium formate, sodium
sulfate, potassium sulfate, potassium fumerate, and combinations
thereof.
[0121] The amount of the conjugate in the composition will vary
depending on a number of factors, but will optimally be a
therapeutically effective dose when the composition is stored in a
unit dose container. A therapeutically effective dose can be
determined experimentally by repeated administration of increasing
amounts of the conjugate in order to determine which amount
produces a clinically desired endpoint.
[0122] The amount of any individual excipient in the composition
will vary depending on the activity of the excipient and particular
needs of the composition. The optimal amount of any individual
excipient is determined through routine experimentation, i.e., by
preparing compositions containing varying amounts of the excipient
(ranging from low to high), examining the stability and other
parameters, and then determining the range at which optimal
performance is attained with no significant adverse effects.
[0123] Generally, however, excipients will be present in the
composition in an amount of about 1% to about 99% by weight,
preferably from about 5%-98% by weight, more preferably from about
15-95% by weight of the excipient, with concentrations less than
30% by weight most preferred.
[0124] These foregoing pharmaceutical excipients along with other
excipients and general teachings regarding pharmaceutical
compositions are described in "Remington: The Science &
Practice of Pharmacy", 19.sup.th ed., Williams & Williams,
(1995), the "Physician's Desk Reference", 52.sup.nd ed., Medical
Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook of
Pharmaceutical Excipients, 3.sup.rd Edition, American
Pharmaceutical Association, Washington, D.C., 2000.
[0125] The pharmaceutical compositions can take any number of forms
and the invention is not limited in this regard. Exemplary
preparations are most preferably in a form suitable for oral
administration such as a tablet, caplet, capsule, gel cap, troche,
dispersion, suspension, solution, elixir, syrup, lozenge,
transdermal patch, spray, suppository, and powder.
[0126] Oral dosage forms are preferred for those conjugates that
are orally active, and include tablets, caplets, capsules, gel
caps, suspensions, solutions, elixirs, and syrups, and can also
comprise a plurality of granules, beads, powders or pellets that
are optionally encapsulated. Such dosage forms are prepared using
conventional methods known to those in the field of pharmaceutical
formulation and described in the pertinent texts.
[0127] Tablets and caplets, for example, can be manufactured using
standard tablet processing procedures and equipment. Direct
compression and granulation techniques are preferred when preparing
tablets or caplets containing the conjugates described herein. In
addition to the conjugate, the tablets and caplets will generally
contain inactive, pharmaceutically acceptable carrier materials
such as binders, lubricants, disintegrants, fillers, stabilizers,
surfactants, coloring agents, flow agents, and the like. Binders
are used to impart cohesive qualities to a tablet, and thus ensure
that the tablet remains intact. Suitable binder materials include,
but are not limited to, starch (including corn starch and
pregelatinized starch), gelatin, sugars (including sucrose,
glucose, dextrose and lactose), polyethylene glycol, waxes, and
natural and synthetic gums, e.g., acacia sodium alginate,
polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl
cellulose, hydroxypropyl methylcellulose, methyl cellulose,
microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose,
and the like), and Veegum. Lubricants are used to facilitate tablet
manufacture, promoting powder flow and preventing particle capping
(i.e., particle breakage) when pressure is relieved. Useful
lubricants are magnesium stearate, calcium stearate, and stearic
acid. Disintegrants are used to facilitate disintegration of the
tablet, and are generally starches, clays, celluloses, algins,
gums, or crosslinked polymers. Fillers include, for example,
materials such as silicon dioxide, titanium dioxide, alumina, talc,
kaolin, powdered cellulose, and microcrystalline cellulose, as well
as soluble materials such as mannitol, urea, sucrose, lactose,
dextrose, sodium chloride, and sorbitol. Stabilizers, as well known
in the art, are used to inhibit or retard drug decomposition
reactions that include, by way of example, oxidative reactions.
[0128] Capsules are also preferred oral dosage forms, in which case
the conjugate-containing composition can be encapsulated in the
form of a liquid or gel (e.g., in the case of a gel cap) or solid
(including particulates such as granules, beads, powders or
pellets). Suitable capsules include hard and soft capsules, and are
generally made of gelatin, starch, or a cellulosic material.
Two-piece hard gelatin capsules are preferably sealed, such as with
gelatin bands or the like.
[0129] Included are parenteral formulations in the substantially
dry form (as a lyophilizate or precipitate, which can be in the
form of a powder or cake), as well as formulations prepared for
injection, which are liquid and require the step of reconstituting
the dry form of parenteral formulation. Examples of suitable
diluents for reconstituting solid compositions prior to injection
include bacteriostatic water for injection, dextrose 5% in water,
phosphate-buffered saline, Ringer's solution, saline, sterile
water, deionized water, and combinations thereof.
[0130] In some cases, compositions intended for parenteral
administration can take the form of nonaqueous solutions,
suspensions, or emulsions, normally being sterile. Examples of
nonaqueous solvents or vehicles are propylene glycol, polyethylene
glycol, vegetable oils, such as olive oil and corn oil, gelatin,
and injectable organic esters such as ethyl oleate.
[0131] The parenteral formulations described herein can also
contain adjuvants such as preserving, wetting, emulsifying, and
dispersing agents. The formulations are rendered sterile by
incorporation of a sterilizing agent, filtration through a
bacteria-retaining filter, irradiation, or heat.
[0132] The conjugate can also be administered through the skin
using conventional transdermal patch or other transdermal delivery
system, wherein the conjugate is contained within a laminated
structure that serves as a drug delivery device to be affixed to
the skin. In such a structure, the conjugate is contained in a
layer, or "reservoir," underlying an upper backing layer. The
laminated structure can contain a single reservoir, or it can
contain multiple reservoirs.
[0133] The conjugate can also be formulated into a suppository for
rectal administration. With respect to suppositories, the conjugate
is mixed with a suppository base material which is (e.g., an
excipient that remains solid at room temperature but softens, melts
or dissolves at body temperature) such as coca butter (theobroma
oil), polyethylene glycols, glycerinated gelatin, fatty acids, and
combinations thereof. Suppositories can be prepared by, for
example, performing the following steps (not necessarily in the
order presented): melting the suppository base material to form a
melt; incorporating the conjugate (either before or after melting
of the suppository base material); pouring the melt into a mold;
cooling the melt (e.g., placing the melt-containing mold in a room
temperature environment) to thereby form suppositories; and
removing the suppositories from the mold.
[0134] The invention also provides a method for administering a
conjugate as provided herein to a patient suffering from a
condition that is responsive to treatment with the conjugate. The
method comprises administering, generally orally, a therapeutically
effective amount of the conjugate (preferably provided as part of a
pharmaceutical preparation). Other modes of administration are also
contemplated, such as pulmonary, nasal, buccal, rectal, sublingual,
transdermal, and parenteral. As used herein, the term "parenteral"
includes subcutaneous, intravenous, intra-arterial,
intraperitoneal, intracardiac, intrathecal, and intramuscular
injection, as well as infusion injections.
[0135] In instances where parenteral administration is utilized, it
may be necessary to employ somewhat bigger oligomers than those
described previously, with molecular weights ranging from about 500
to 30K Daltons (e.g., having molecular weights of about 500, 1000,
2000, 2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or
even more).
[0136] The method of administering may be used to treat any
condition that can be remedied or prevented by administration of
the particular conjugate. Those of ordinary skill in the art
appreciate which conditions a specific conjugate can effectively
treat. The actual dose to be administered will vary depend upon the
age, weight, and general condition of the subject as well as the
severity of the condition being treated, the judgment of the health
care professional, and conjugate being administered.
Therapeutically effective amounts are known to those skilled in the
art and/or are described in the pertinent reference texts and
literature. Generally, a therapeutically effective amount will
range from about 0.001 mg to 1000 mg, preferably in doses from 0.01
mg/day to 750 mg/day, and more preferably in doses from 0.10 mg/day
to 500 mg/day.
[0137] The unit dosage of any given conjugate (again, preferably
provided as part of a pharmaceutical preparation) can be
administered in a variety of dosing schedules depending on the
judgment of the clinician, needs of the patient, and so forth. The
specific dosing schedule will be known by those of ordinary skill
in the art or can be determined experimentally using routine
methods. Exemplary dosing schedules include, without limitation,
administration five times a day, four times a day, three times a
day, twice daily, once daily, three times weekly, twice weekly,
once weekly, twice monthly, once monthly, and any combination
thereof. Once the clinical endpoint has been achieved, dosing of
the composition is halted.
[0138] All articles, books, patents, patent publications and other
publications referenced herein are incorporated by reference in
their entireties. In the event of an inconsistency between the
teachings of this specification and the art incorporated by
reference, the meaning of the teachings in this specification shall
prevail.
EXPERIMENTAL
[0139] It is to be understood that while the invention has been
described in conjunction with certain preferred and specific
embodiments, the foregoing description as well as the examples that
follow are intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modifications within the
scope of the invention will be apparent to those skilled in the art
to which the invention pertains.
[0140] All non-PEG chemical reagents referred to in the appended
examples are commercially available unless otherwise indicated. The
preparation of PEG-mers is described in, for example, U.S. Patent
Application Publication No. 2005/0136031.
[0141] .sup.1H NMR (nuclear magnetic resonance) data was generated
by an NMR spectrometer. A list of certain compounds as well as the
source of the compounds is provided below.
Example 1
Synthesis of Small PEG Promethazine Conjugates
[0142] Scheme 1: Synthesis of mPEG.sub.n-N-Promethazine
conjugates.
##STR00011##
[0143] Promethazine, 1-chloroethyl chloroformate, dichloroethane
(DCE), phosphorous oxychloride (POCl.sub.3), sodium borohydride
(NaBH.sub.4), sodium hydride (NaH), and N,N-dimethylformamide (DMF)
were from Sigma-Aldrich (St Louis, Mo.). mPEG.sub.n-OMs and
mPEG.sub.n-Br were from Sai Chemicals (India). Sodium carbonate
(Na.sub.2CO.sub.3), sodium bicarbonate (NaHCO.sub.3), sodium
sulfate (Na.sub.2SO.sub.4), potassium carbonate (K.sub.2CO.sub.3),
sodium chloride (NaCl), and sodium hydroxide (NaOH) were from EM
Science (Gibbstown, N.J.). DCM was distilled from CaH.sub.2.
[0144] Desalting of Promethazine-HCl: The Sigma-Aldrich
promethazine.HCl (10 g, 31.2 mmol) was dissolved in H.sub.2O (250
mL) in a 500-mL flask. Na.sub.2CO.sub.3 (8.2 g, 78 mmol) was added
in one portion. The salt was first dissolved in water and then a
thick oil-like product appeared sticking to the stirring bar. DCM
(50 mL) was added to dissolve the product and the solution became
clear after 2.5 hrs. The solution was diluted by adding DCM (50 mL)
and the organic phase was separated. The aqueous phase was then
extracted with DCM (50 mL.times.2) and the combined organic phases
were dried over Na.sub.2SO.sub.4. After filtration, the solution
was concentrated and a viscous product was obtained.
[0145] Promethazine R.sub.f=0.26 (DCM:MeOH=10:1), RP-HPLC (betasil
C18, 0.5 mL/min, 10-60% ACN in 10 min) 8.45 min, purity >99%,
LC-MS (ESI, MH.sup.+) 285.3, .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 1.06 (3H, d, J=6.5 Hz), 2.35 (6H, s), 3.05-3.12 (1H, m),
3.69 (1H, dd, J=9, 13.5 Hz), 4.07 (1H, dd, J=4.0, 13.0 Hz),
6.92-6.95 (4H, m), 7.15-7.17 (4H, m).
[0146] Demethylation: The promethazine obtained above (2.71 g, 9.51
mmol) was dissolved in DCE (50 mL) in a 250-mL flask. 1-Chloroethyl
chloroformate (12.4 mL, 114 mmol) was added slowly at room
temperature. The mixture was heated in oil-bath to 100.degree. C.
and the reaction was kept overnight (16 hrs) at this temperature.
After cooling down the reaction mixture under the dry N.sub.2
atmosphere, the solvent was evaporated under vacuum. The residue
was dried under high vacuum for 3 hrs before it was re-dissolved in
DCE (25 mL) and ethanol (25 mL). The mixture was heated to
83.degree. C. and reflux was continued at this temperature
overnight (20 hrs). After cooling down to the room temperature, the
solvents were evaporated and the residue was diluted in DCM (100
mL) and saturated NaHCO.sub.3 solution (250 mL). After the organic
phase was separated, the aqueous solution was extracted with DCM
(50 mL.times.2) and the combined organic phases were dried over
Na.sub.2SO.sub.4. After filtration and concentration, the resulting
residue was loaded onto a Biotage 40M column (1-6% of MeOH in DCM
in 16 CV). Only part of the pure product was obtained based on HPLC
fraction analysis. The mixture was purified a second time on a
Biotage 25M column under the same conditions. The slight yellowish
product (1.39 g, 54% yield) was obtained together with a mixture
(560 mg, 20%).
[0147] N-demethyl-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 3.68 min,
LC-MS (ESI, MH.sup.+) 271.2. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 1.16 (3H, d, J=6.5 Hz), 2.38 (3H, s), 3.05-3.12 (1H, m),
3.83 (1H, dd, J=5.0, 13.5 Hz), 3.91 (1H, dd, J=3.0, 13.5 Hz),
6.92-6.96 (4H, m), 7.15-7.19 (4H, m).
[0148] General procedure for N-PEGylation from mPEG.sub.n-OMs: The
above secondary amine starting material (about 200 mg, 1 eq) was
combined with mPEG.sub.n-OMs (1.2 eq) in 12-mL microwave reaction
tube. K.sub.2CO.sub.3 (1.5 eq) and water (1.2 mL) was added. After
sealing the tube, the reaction was performed using a 3-stage
program (65.degree. C., 2 min, 85.degree. C., 2 min, 100.degree.
C., 100 min). After the reaction was complete, the mixture was
diluted with NaHCO.sub.3 (60 mL) and extracted with DCM (20
mL.times.3). The combined DCM solution was dried over
Na.sub.2SO.sub.4. After filtration, the solution was concentrated
and the resulting residue was purified on a Biotage 25M normal
phase column (1-6% MeOH in DCM in 16 CV). The product mixture was
purified one more time on under normal phase conditions to collect
more pure product. The final fractions were combined and loaded on
Biotage reverse phase column (25M, 20% in 5 CV following with
20-65% acetonitrile in water in 20 CV). The product fractions were
again combined and acetonitrile was evaporated until the solution
become cloudy. The aqueous phase was then extracted with DCM (15
mL.times.3), with small amount of solid NaCl was added each time in
extraction. The combined DCM solution was dried over
Na.sub.2SO.sub.4 and filtrated. The solution was then concentrated
and dried under vacuum over 24 hrs before characterization.
[0149] General procedure for N-PEGylation from mPEG.sub.n-Br: The
N-PEGylation can be designed using mPEG.sub.n-Br using a similar
procedure as described above with over 95% conversion. No
differences in the work up and purification. The product contained
a darker color which was a contaminate from the starting
mPEG.sub.n-Br and was subsequently removed during the reverse phase
purification.
[0150] mPEG.sub.3-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.43 min,
purity >99%, LC-MS (ESI, MH.sup.+) 417.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.65-2.74
(2H, m), 3.20-3.24 (1H, m), 3.38 (3H, s), 3.51-3.65 (10H, m), 3.70
(1H, dd, J=9.0, 13.0 Hz), 4.03 (1H, dd, J=4.5, 13.5 Hz), 6.91-6.94
(4H, m), 7.14-7.17 (4H, m).
[0151] mPEG.sub.4-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.80 min,
purity >98%, LC-MS (ESI, MH.sup.+) 461.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.65-2.74
(2H, m), 3.20-3.24 (1H, m), 3.38 (3H, s), 3.51-3.65 (14H, m), 3.70
(1H, dd, J=9.0, 13.5 Hz), 4.03 (1H, dd, J=4.5, 13.0 Hz), 6.91-6.94
(4H, m), 7.14-7.17 (4H, m).
[0152] mPEG.sub.5-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.80 min,
purity >99%, LC-MS (ESI, MH.sup.+) 505.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.68-2.70
(2H, m), 3.21 (1H, bs), 3.38 (3H, s), 3.52-3.65 (18H, m), 3.70 (1H,
dd, J=9.0, 12.5 Hz), 4.02 (1H, dd, J=4.0, 13.0 Hz), 6.91-6.94 (4H,
m), 7.15-7.17 (4H, m).
[0153] mPEG.sub.6-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.43 min,
purity >99%, LC-MS (ESI, MH.sup.+) 549.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.66-2.72
(2H, m), 3.19-3.23 (1H, m), 3.38 (3H, s), 3.51-3.65 (22H, m), 3.70
(1H, dd, J=9.0, 13.0 Hz), 4.02 (1H, dd, J=4.0, 13.5 Hz), 6.91-6.94
(4H, m), 7.14-7.17 (4H, m).
[0154] mPEG.sub.7-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.32 min,
purity >99%, LC-MS (ESI, MH.sup.+) 593.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.64-2.73
(2H, m), 3.20-3.24 (1H, m), 3.38 (3H, s), 3.51-3.66 (26H, m), 3.70
(1H, dd, J=9.0, 13.0 Hz), 4.02 (1H, dd, J=4.0, 13.5 Hz), 6.91-6.94
(4H, m), 7.14-7.17 (4H, m).
[0155] mPEG.sub.9-N-promethazine R.sub.f=0.33 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 30-60% ACN in 10 min) 5.60 min,
purity >99%, LC-MS (ESI, MH.sup.+) 680.4. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.04 (3H, d, J=6.5 Hz), 2.34 (3H, s), 2.64-2.73
(2H, m), 3.20-3.24 (1H, m), 3.38 (3H, s), 3.50-3.66 (34H, m), 3.69
(1H, dd, J=9.0, 13.5 Hz), 4.02 (1H, dd, J=4.0, 13.5 Hz), 6.91-6.94
(4H, m), 7.14-7.17 (4H, m).
[0156] Scheme 2: Synthesis of mPEG.sub.n-O-Promethazine
conjugates.
##STR00012##
[0157] Vilsmeier formylation: Desalted promethazine (1.68 g, 5.92
mmol) was dissolved in DMF (1.92 mL, 24.9 mmol) in a 100-mL flask
with a nitrogen bubble. The solution mixture was cooled down to
0.degree. C. before a mixture of POCl.sub.3 (1.41 mL, 15.4 mmol)
and DMF (0.96 mL, 12.4 mmol) were added dropwise. The reaction
solution was heated slowly to 90.degree. C. for 6 hrs before an
additional mixture of POCl.sub.3 (1.5 mL) and DMF (0.8 mL) was
added. The reaction was kept at this temperature and N.sub.2
bubbled slowly during the next 40 hrs. The progress of the reaction
was monitored by HPLC (SM<5% UV 254 nm, ELS .about.100%
conversion). The reaction was quenched by adding 30 g of ice and
diluting with saturated NaHCO.sub.3 (250 mL). NaOH (1N) was added
until the pH reached 10-12. The resulting solution was then
extracted by DCM (80 mL+50 mL.times.2). The combined organic phases
were dried over Na.sub.2SO.sub.4. After filtration, the solution
was concentrated and the DMF residue solution was loaded onto a
Biotage 40S column and purified (2-7% MeOH in DCM in 16 CV). The
product fractions were combined and concentrated to give a brownish
product (1.63 g, 88% yield). HPLC analysis showed the starting
material contamination is less than 3%.
[0158] (4-Aldhyde)-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10 min) 7.75 min,
LC-MS (ESI, MH.sup.+) 313.2. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 1.08 (3H, d, J=5.5 Hz), 2.35 (6H, s), 3.10 (1H, bs), 3.76
(1H, dd, J=4.0, 13.0 Hz), 4.14 (1H, bs), 6.96-7.02 (3H, m),
7.16-7.22 (2H, m), 7.64-7.69 (2H, m), 9.82 (1H, s).
[0159] Aldehyde reduction: The above aldehyde (1.63 g, 5.22 mmol)
was dissolved in methanol (30 mL). NaBH.sub.4 (433 mg, 12 mmol) was
added at room temperature in small portions. The reaction was kept
at room temperature for 15 min. The methanol solvent was evaporated
and the residue was dissolved in NaHCO.sub.3 (100 mL) and extracted
with DCM (60 mL.times.3). The organic phase was combined and loaded
onto a Biotage 40S column (2-20% MeOH in DCM in 20 CV). The two
product peaks (same products) were combined and concentrated to
give a colorless foam product (1.27 g, 77% yield).
[0160] (4-methylene hydroxyl)-promethazine R.sub.f=0.19
(DCM:MeOH=10:1), RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10
min) 6.58+6.75 min, LC-MS (ESI, MH.sup.+) 315.3. .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 1.07 (3H, d, J=6.5 Hz), 2.35 (6H, s), 3.09
(1H, bs), 3.68 (1H, dd, J=4.0, 13.5 Hz), 4.09 (1H, dd, J=3.5, 13.0
Hz), 4.59 (2H, s), 6.90-6.95 (3H, m), 7.15-7.18 (4H, m).
[0161] General procedure for O-PEGylation from mPEGn-OMs: The above
reductive product (about 200 mg, 1 eq) was combined with
mPEG.sub.n-OMs (1.2-1.5 eq) in 100-mL flask. The mixture was
dissolved in toluene (30 mL) and azeotropic evaporated to about 2
mL. DMF (3 mL) and NaH (10 eq) was added. The reaction mixture was
warmed up to 45.degree. C. in an oil bath and the reaction was kept
at this temperature for 6 hrs (or overnight). After removing the
oil bath, the mixture was quenched with 30 g ice, diluted with
saturated NaHCO.sub.3 (60 mL), and extracted with DCM (20
mL.times.3). The combined DCM solution was dried over
Na.sub.2SO.sub.4. After filtration, the solution was concentrated
and the residue was purified on a Biotage 25M normal phase column
(1-6% MeOH in DCM in 16 CV). The fractions (UV: >98% pure) were
combined and loaded onto a Biotage reverse phase column (25M, 20%
in 5 CV following with 20-65% acetonitrile in water in 20 CV). The
product fractions were combined and acetonitrile was evaporated
until the solution become cloudy. The aqueous phase was then
extracted with DCM (15 mL.times.3), each time a small amount of
solid NaCl was added in extraction. The combined DCM solution was
dried over Na.sub.2SO.sub.4 and filtered. The solution was then
concentrated and dried under vacuum over 24 hrs before
characterization.
[0162] mPEG.sub.3-O-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10 min) 8.27 min,
purity >95%, LC-MS (ESI, MH.sup.+) 461.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.05 (3H, d, J=6.0 Hz), 2.34 (6H, s), 3.07 (1H,
bs), 3.38 (3H, s), 3.54-3.56 (2H, m), 3.59-3.61 (2H, m), 3.64-3.70
(9H, m), 4.06 (1H, d, J=12.5 Hz), 4.52 (2H, s), 6.87-6.95 (3H, m),
7.12-7.16 (4H, m).
[0163] mPEG.sub.5-O-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10 min) 8.31 min,
purity >97%, LC-MS (ESI, MH.sup.+) 549.3. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.07 (3H, d, J=5.5 Hz), 2.36 (6H, s), 3.10 (1H,
bs), 3.37 (3H, s), 3.53-3.55 (2H, m), 3.59-3.61 (2H, m), 3.63-3.70
(17H, m), 4.09 (1H, d, J=9.0 Hz), 4.45 (2H, s), 6.88-6.95 (3H, m),
7.13-7.18 (4H, m).
[0164] mPEG.sub.7-O-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10 min) 8.39 min,
purity >98%, LC-MS (ESI, MH.sup.+) 637.4. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.06 (3H, bs), 2.35 (6H, s), 3.08 (1H, bs),
3.38 (3H, s), 3.53-3.56 (2H, m), 3.59-3.61 (2H, m), 3.63-3.70 (25H,
m), 4.06 (1H, bs), 4.45 (2H, s), 6.88-6.95 (3H, m), 7.13-7.18 (4H,
m).
[0165] mPEG.sub.9-O-promethazine R.sub.f=0.23 (DCM:MeOH=10:1),
RP-HPLC (betasil C18, 0.5 mL/min, 10-60% ACN in 10 min) 8.35 min,
purity >99%, LC-MS (ESI, MH.sup.+) 725.5. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.05 (3H, d, J=3.5 Hz), 2.34 (6H, s), 3.07 (1H,
bs), 3.38 (3H, s), 3.54-3.56 (2H, m), 3.59-3.61 (2H, m), 3.63-3.70
(33H, m), 4.06 (1H, dd, J=4, 13.0 Hz), 4.45 (2H, s), 6.87-6.94 (3H,
m), 7.12-7.18 (4H, m).
Example 2
In Vitro Receptor Binding
[0166] Binding to Histamine Receptors
[0167] The receptor binding affinity of promethazine (parent) and
N- and O-PEG derivatives are evaluated using radioligand binding
assays in membranes prepared from CHO cells that express the
recombinant human H1, H2, H3 or H4 histamine receptors. Competition
binding experiments are conducted by incubating membranes with a
fixed concentration of radioligand in the presence of variable
concentrations of test compounds. The radioligands used are
specific for each receptor subtype and the assay conditions are
described in Table 2. Following incubations, the membranes are
washed, and the bound radioactivity is measured. Non-specific
binding is measured in the presence of excess cold ligand and
subtraction of this value from the total binding yields the
specific binding at each test compound concentration. IC.sub.50
values are obtained from non-linear regression analysis of
dose-response curves and are calculated only for those compounds
that show >50% inhibition of binding at the highest
concentrations. Ki is obtained using the Cheng Prusoff correction
using experimental Kd vales that are determined under these assay
conditions.
[0168] The binding affinities of Promethazine, mPEG-N-promethazine
and mPEG-O -Promethazine conjugates to Histamine receptors is shown
in Table 1. Promethazine and PEG-promethazine conjugates displayed
high affinity binding to the H1 receptor. PEG conjugation results
in a reduction in binding affinity and this effect is PEG size
dependent.
[0169] Binding at the H2, H3 and H4 receptors is significantly
lower for all molecules tested. Ki values could not be calculated
at these receptors since <50% inhibition of binding is obtained
at the highest concentrations tested.
TABLE-US-00001 TABLE 1 Binding affinities of Promethazine
conjugates to Histamine receptors. Ki (nM) Fold change in binding
Molecule H1 H2 H3 H4 affinity at H1 Promethazine 0.278 NS NS NS 1
mPEG.sub.3-N-Promethazine 2.28 NS NS NS 8.20
mPEG.sub.4-N-Promethazine 1.95 NS NS NS 7.01 mPEG.sub.5-N
Promethazine 8.53 NS NS NS 30.68 mPEG.sub.6-N Promethazine 13.2 NS
NS NS 47.48 mPEG.sub.7-N Promethazine 16.9 NS NS NS 60.79
mPEG.sub.9-N Promethazine 44 NS NS NS 158.27
mPEG.sub.3-O-Promethazine 7.62 NS NS NS 27.41
mPEG.sub.5-O-Promethazine 7.16 NS NS NS 25.76
mPEG.sub.7-O-Promethazine 33.2 NS NS NS 119.42
mPEG.sub.9-O-Promethazine 45.1 NS NS NS 162.23 NS: No significant
binding
TABLE-US-00002 TABLE 2 Assay Conditions for histamine receptor
binding assays Non- Receptor specific Receptor Source Radioligand
binding Methods Test Conc. Histamine Human [.sup.3H]- Pyrilamine
Reaction in 50 mM 0.01, 0.1, H1 rCHO Pyrilamine (1 .mu.M) Tris-HCl
(pH 7.4), 2 mM 0.3, 1, 3, cells (1.2 nM) MgCl.sub.2, 100 mM 10, 30,
100 nM NaCl and 250 mM 3, 30 .mu.M Sucrose at 25.degree. C. for 3
h. Histamine Human [.sup.125I]- Tiotidine Reaction in 50 mM 0.01,
0.1, H2 rCHO- Aminopotentidine (3 .mu.M) phosphate (pH 7.4) 0.3, 1,
3, K1 cells (0.1 nM) at 25.degree. C. for 2 h. 10, 30, 100 nM, 3,
30 .mu.M Histamine Human [.sup.3H]R(-)-.alpha.- R(-)-.alpha.-
Reaction in 50 mM 0.01, 0.1, H3 rCHO- Methylhistamine
Methylhistamine Tris-HCl (pH 7.4), 5 mM 0.3, 1, 3, K1 cells (3 nM)
(1 .mu.M) MgCl.sub.2, 0.04% 10, 30, 100 nM, BSA at 25.degree. C.
for 1.5 h. 3, 30 .mu.M Histamine Human [.sup.3H]Histamine Histamine
Reaction in 50 mM 0.01, 0.1, H4 rCHO- (8.2 nM) (1 .mu.M) Tris-HCl
(pH 7.4), 0.3, 1, 3, K1 cells 1.25 mM EDTA at 10, 30, 100 nM,
25.degree. C. for 1.5 h. 3, 30 .mu.M rCHO--recombinant CHO
cells
[0170] Binding to Muscarinic Receptors
[0171] The receptor binding affinities of Promethazine (parent) and
PEG-Promethazine conjugates are evaluated using radioligand binding
assays in membranes prepared from CHO cells that express the
recombinant human M1, M2, M3, M4 or M5 muscarinic acetylcholine
receptors. Competition binding experiments are conducted by
incubating membranes with a fixed concentration of radioligand in
the presence of variable concentrations of test compounds.
.sup.3H--N-Methylscopolamine at 0.8 nM is used as the radioligand
for all receptor subtypes. Incubations are carried out for 2 hours
at 25.degree. C. in buffer containing 50 mM Tris HCl, 10 mM
MgCl.sub.2 and 1 mM EDTA. Following incubations, the membranes are
washed, and the bound radioactivity is measured. Non-specific
binding is measured in the presence of excess Atropine as the cold
ligand and subtraction of this value from the total binding yields
the specific binding at each test compound concentration. IC.sub.50
values are obtained from non-linear regression analysis of
dose-response curves and are calculated only for those compounds
that show >50% inhibition of binding at the highest
concentration tested. Ki is obtained using the Cheng Prusoff
correction using Kd values that are experimentally determined under
these assay conditions.
[0172] The binding affinities of Promethazine, mPEG-N-promethazine
and mPEG-.beta.-Promethazine conjugates at the five muscarinic
receptor subtypes are shown in Table 3. Promethazine displays high
binding affinity to all muscarinic receptor subtypes, with Ki
values ranging from .about.7-26 nM and displays little selectivity
for any muscarinic receptor subtype. In contrast, the PEG
conjugates display a reduction in binding affinity at all
subtypes--the Ki at any particular receptor subtype was reduced
about 10-100-fold. In several cases, an inhibition of radioligand
binding could not be obtained at the highest concentration tested
and hence data are shown as "no significant binding (NS)". These
data suggest that PEG conjugation reduces the binding affinity of
promethazine conjugates to muscarinic acetylcholine receptors.
TABLE-US-00003 TABLE 3 Binding affinities of Promethazine
conjugates to muscarinic receptors. Ki (nM) Molecule M1 M2 M3 M4 M5
Promethazine 8.18 26.4 16.2 7.74 9.34 mPEG.sub.3-N-Promethazine NS
NS NS NS NS mPEG.sub.4-N-Promethazine NS NS NS NS NS
mPEG.sub.5-N-Promethazine NS 817 NS NS NS mPEG.sub.6-N-Promethazine
NS 337 NS NS 1740 mPEG.sub.7-N-Promethazine NS 90 NS 435 905
mPEG.sub.9-N-Promethazine NS 308 NS 455 2050
mPEG.sub.3-O-Promethazine NS NS NS NS NS mPEG.sub.5-O-Promethazine
NS NS NS NS NS mPEG.sub.7-O-Promethazine NS NS NS NS NS
mPEG.sub.9-O-Promethazine NS NS NS NS NS NS: No significant
binding
Example 3
Scale up of mPEG.sub.4-N-promethazine
[0173] Materials: Promethazine hydrochloride, 1-chloroethyl
chloroformate were purchased from Sigma-Aldrich (St Louis, Mo.).
mPEG.sub.4-Br was received from India Sai CRO. Sodium bicarbonate
(NaHCO.sub.3), sodium sulfate (Na.sub.2SO.sub.4), sodium chloride
(NaCl), Potassium carbonate (K.sub.2CO.sub.3), Sodium hydroxide
(NaOH), and hydrochloride acid (HCl) were purchased from EM Science
(Gibbstown, N.J.). Toluene, dichloroethylene (DCE), dichloromethane
(DCM), and other organic solvents were used as they purchased.
Desalting Promethazine
[0174] The reaction was carried out in NaHCO.sub.3/DCM. The product
was solidified in high vacuo drying.
Demethylation and Product Precipitation:
[0175] The desalted promethazine (13.16 g, 46.3 mmol) was added to
a 1000-mL flask. Toluene (110 mL) and DCE (100 mL) were added and
azeotropically distilled while under 45.degree. C. then followed
with distillation under high vacuum for an additional 15 min. DCE
(100 mL) was added and after a homogenous solution was obtained,
1-chloroethyl chloroformate (25 g.times.6, 1.06 mol) was added. The
reaction mixture was heated to 105.degree. C. and allowed to reflux
for 18 hours. The reaction was allowed to cool to approximately
50.degree. C. at which point the solvent was removed by rotary
evaporation and then subsequently under high vacuum for 3 hrs
before additional DCE (100 mL) and ethanol (100 mL) were added. The
solution was kept under 83.degree. C. and refluxed for another 18
hrs. The solvent was removed while keeping the temperature under
55.degree. C. and the resulting residue was dissolved in 0.2N HCl.
The aqueous phase was washed with ether (200 mL.times.5). A
salt-like precipitate was generated while extracting the aqueous
phase with DCM. The precipitate was collected by filtration and
washed with DCM. The DCM solution was evaporated and heated to
50.degree. C. The precipitation process was repeated. The salt-like
product was basified in NaHCO.sub.3 (3 piece of NaOH) and extracted
with DCM (100 mL.times.3). The combined DCM was dried over
Na.sub.2SO.sub.4. Filtration, evaporation, and drying under high
vacuum give an oily-like product (-10.0 grams, 79% yield).
[0176] N-demethyl-promethazine: RP-HPLC (betasil C18, 0.5 mL/min,
30-60% ACN in 10 min) 4.31 min, LC-MS (ESI, MH.sup.+) 271.1.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.14 (3H, d, J=6.5 Hz),
2.37 (3H, s), 3.03-3.09 (1H, m), 3.81 (1H, dd, J=5.0, 13.5 Hz),
3.88 (1H, dd, J=8.0, 13.5 Hz), 6.92-6.96 (4H, m), 7.15-7.19 (4H,
m).
N-alkylation and Purification.
[0177] The demethylation product (2.06 g, 7.62 mmol) was mixed with
mPEG.sub.4-Br (2.53, 9.33 mmol). Water (10 mL) was premixed with
K.sub.2CO.sub.3 (5.37 g, 38.9 mmol) and the resulting solution was
added to the above mixture. The water solution was heated in oil
bath while the temperature remained under 100.degree. C. The
reaction was kept at this temperature for 20 hrs and HPLC analysis
indicated the reaction was complete. The product residue was
dissolved in DCM and the aqueous phase separated. The DCM was
removed under reduced pressure and the resulting residue was
dissolved in 0.3N HCl (240 mL) and washed with ether (100
mL.times.3). Then the aqueous solution was washed with a mixture of
EtOAc and ether (2 times). The aqueous phase was basified with
solid NaOH to pH>10 and extracted with DCM (50 mL.times.3). The
combined DCM phase was washed with a saturated solution of
NaHCO.sub.3 (50 mL.times.5+1 piece of NaOH.about.100 mg each time).
The DCM phase was separated, dried over Na.sub.2SO.sub.4, and the
solvent removed under reduced pressure. A slight yellowish product
(2.9 g) was obtained after the high vacuo drying with >99%
purity.
[0178] mPEG.sub.4-N-Promethazine RP-HPLC (betasil C18, 0.5 mL/min,
30-60% ACN in 10 min) 6.23 min, purity >99%, LC-MS (ESI,
MH.sup.+) 461.1. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.04
(3H, d, J=6.5 Hz), 2.34 (3H, s), 2.67-2.71 (2H, m), 3.20-3.23 (1H,
m), 3.38 (3H, s), 3.50-3.67 (14H, m), 3.70 (1H, dd, J=9.0, 13.5
Hz), 4.03 (1H, dd, J=4.5, 13.0 Hz), 6.91-6.94 (4H, m), 7.14-7.18
(4H, m).
Example 4
Scale Up Synthesis of mPEG.sub.3-Promethazine
[0179] Materials: Promethazine hydrochloride, 1-chloroethyl
chloroformate were purchased from Sigma-Aldrich (St Louis, Mo.).
mPEG.sub.3-Br was received from CRO-Sai Advandium in india. The
other inorganic salts, base, acid and organic solvents were used as
purchased.
Desalting of Promethazine HCl
##STR00013##
[0181] Promethazine HCl (20.2723 g, 63.2 mmol) was dissolved in DCM
(250 mL), washed with aq. 10% Na.sub.2HCO.sub.3 solution
(2.times.150 mL). The aq. solution was extracted with DCM (50 mL).
The combined organic solution was dried over Na.sub.2SO.sub.4,
concentrated to dryness. The residue was mixed with toluene (150
mL), concentrated to dryness and dried under high vacuum to afford
18.6892 g of promethazine in 96% yield.
Demethylation of Promethazine
##STR00014##
[0183] First run: Promethazine (10.743 g, 36.3 mmol) was dissolved
in dichloroethane (150 mL) at room temperature, cooled to 0.degree.
C. 1-Chloroethyl chloroformate (100 g, 4 bottles of 25 g per
bottle, 685 mmol) was added slowly. The reaction mixture became
green in color after a few minutes and then became more yellow. The
mixture was stirred at room temperature for 2 h, refluxed for 19 h
(oil bath temp. 100.degree. C.). HPLC analysis showed low
conversion and some side products. The reaction mixture was
concentrated to remove some DCE (.about.120 mL). More of
1-chloroethyl chloroformate (25 g, 171 mmol) was added. The mixture
was stirred at 115.degree. C. for 22 h. HPLC analysis didn't show
starting material. The mixture was concentrated to remove all of
solvents under reduced pressure and dried under high vacuum.
[0184] The crude mixture was dissolved in MeOH (100 mL) at room
temperature and stirred at room temperature for 15 min (open flask)
then refluxed for 18 h (oil bath temperature: 75 degree), cooled to
room temperature. The mixture was concentrated to remove all of
solvents, the residue was dissolved in DCM (250 mL), washed with
10% aq. NaHCO.sub.3 solution (2.times.100 mL), concentrated to
dryness. The residue was dissolved in 750 mL of 0.2 N HCl solution,
washed with ether (3.times.150 mL). The aqueous solution was
extracted with DCM, and adjusted the mixture to basic with aq. KOH
solution, extracted again with DCM (3 times) until no product was
found in DCM solution by TLC. All of organic solutions were
combined and dried over Na.sub.2SO.sub.4, concentrated to afford
8.5066 g of the product as oil. The yield was 92%.
[0185] Second run: Promethazine (7.9456 g, 26.8 mmol) was mixed
with 1-chloroethyl chloroformate (20 g, 137 mmol). The mixture was
stirred at 120.degree. C. (oil temperature) for 17.5 h. HPLC showed
the reaction was complete. The reaction was cooled and
concentrated. The residue was dried under high vacuum.
[0186] The resulting residue was dissolved in MeOH (100 mL),
refluxed for 2 h, cooled to Lt. The mixture was concentrated to
remove all of solvent to afford a solid. The solid was mixed with
0.2 N HCl solution. Some of solid didn't dissolve in the acidic
solution. The mixture was filtered and the solid was washed with
ether and then dissolved in DCM. The aq. solution was washed with
ether (2.times.150 mL). The aq. solution was adjusted pH to 10-11
with aq. KOH solution, and then extracted with DCM (4.times.150
mL). The combined DCM solution was dried over Na.sub.2SO.sub.4,
concentrated to yield an oil. The oil product was dissolved in 0.2
N HCl (500 mL). The solution was washed with ether (2.times.150
mL), and adjusted the pH to 11.32 with aq. KOH solution, extracted
with DCM (4.times.150 mL), dried over Na.sub.2SO.sub.4,
concentrated to afford 4.9763 g of product in 69% yield.
[0187] .sup.1H-NMR (CDCl.sub.3, 500 MHz): 7.181-7.138 (m, 4H,
Ar--H), 6.951-6.915 (m, 4 H, Ar--H), 3.886-3.785 (m, 2H, CH.sub.2),
3.067-3.028 (m, 1H, CH), 2.358 (s, 3H, CH.sub.3), 1.127 (d, J=6.0
Hz, CH.sub.3).
Synthesis of mPEG.sub.3-N-Promethazine
##STR00015##
[0189] First run: Demethylpromethazine (1.7301 g, 6.4 mmol) and
mPEG.sub.3-Br (1.7877 g, 7.87 mmol) were placed in a 25-mL vial. A
solution of K.sub.2CO.sub.3 (5.47 g, 39.6 mmol) in water (10 mL)
was added. The resulting mixture was stirred at room temperature
for 68.5 h. HPLC showed only about 30% starting material remained.
The mixture was heated at 120.degree. C. for 50 min under microwave
conditions. HLPC showed the reaction was complete. When the
reaction mixture was cooled to room temperature, EtOAc (50 mL) was
added. The mixture was washed with 0.2 N HCl (100 ml). The aq.
solution only contained impurities based on HPLC; therefore, the
organic EtOAc solution was extracted with 0.2N HCl solution
(2.times.150 mL). The combined aq. extraction solution was washed
once again with EtOAc (100 mL) and adjusted the pH to 11.84 with
aq. KOH solution, extracted with DCM (3.times.60 mL). The combined
DCM solution was dried over Na.sub.2SO.sub.4, and concentrated to
afford 2.3094 g of product in 87% yield.
[0190] Second run: Demethylpromethazine (6.4773 g, 23.96 mmol) and
mPEG.sub.3-Br (5.983 g, 16.30 mmol) were placed in a 100-mL round
bottomed flask. A solution of K.sub.2CO.sub.3 (17.34 g, 125 mmol)
in water (35 mL) was added. The resulting mixture was stirred at
110.degree. C. (oil temperature) for a few hours. The mixture was
stirred at 100.degree. C. for 1.5 h at which point more
mPEG.sub.3-Br (0.643 g, 2.83 mmol) was added. The mixture was
stirred at 105.degree. C. for 2.5 h. The reaction mixture was
cooled and EtOAc (150 mL) was added. The aq. solution was separated
and the organic solution was washed once with 5% aq. NaHCO.sub.3.
The EtOAc solution was extracted with 0.2 N HCl (3.times.150 mL).
The combined acidic solution was washed with EtOAc (2.times.100
mL), and then adjusted pH to 10-11 with aq. KOH solution, extracted
with DCM (4.times.200 mL). The combined DCM solution was dried over
Na.sub.2SO.sub.4, concentrated to afford 9.2668 g of final product
as oil in 93% yield.
[0191] .sup.1H-NMR (CDCl.sub.3, 500 MHz): 7.170-7.139 (m, 4H,
Ar--H), 6.935-6.902 (m, 4 H, Ar--H), 4.021 (dd, J=4.5 and 13.5 Hz,
1H), 3.690 (dd, J=9.0 and 13.5 Hz, 1H), 3.648-3.576 (m, 6H),
3.560-3.486 (m, 4H), 3.273 (s, 3H, CH.sub.3), 3.246-3.181 (m, 1H),
2.732-2.643 (m, 2H, CH.sub.2), 2.332 (s, 3H, CH.sub.3), 1.034 (d,
J=6.5 Hz, 3H, CH.sub.3). LC-MS: 417.3 (MH.sup.+).
Example 5
Analgesic Assay
[0192] An analgesic assay was used to determine whether a given
compound can reduce and/or prevent visceral pain in mice.
[0193] The assay utilized CD-1 male mice (5-8 mice per group), each
mouse being approximately 0.015-0.030 kg on the study day. Mice
were treated according to standard protocols.
[0194] Mice were given a single "pretreatment" dose of a compound
lacking covalent attachment of a water-soluble, non-peptidic
oligomer, a corresponding version comprising the compound
covalently attached to a water-soluble, non-peptidic oligomer, or
control solution (IV, SC, IP or orally) thirty minutes prior to the
administration of the acetic acid solution. The animal was given an
IP injection of an irritant (acetic acid) that induces "writhing"
which may include: contractions of the abdomen, twisting and
turning of the trunk, arching of the back and the extension of the
hindlimbs. Mice were given a single IP injection (0.1 mL/10 g
bodyweight) of a 0.5% acetic acid solution. After the injection the
animals were returned to their observation enclosure and their
behavior was observed. Contractions were counted between 0 and 20
minutes after the injection. The animals were used once. Each
tested article was dosed at different doses, when possible. The
results are shown in the table below.
TABLE-US-00004 TABLE Acetic Acid Writhing Data PEG.sub.3-
PEG.sub.4- PEG.sub.5- PEG.sub.6- PEG.sub.7- PEGS.sub.9- PEG.sub.3-
PEG.sub.5- PEG.sub.7- PEG.sub.9- TA Promethazine Parent N-Pro N-Pro
N-Pro N-Pro N-Pro N-Pro O-Pro O-Pro O-Pro O-Pro Dose (mg/kg) 1 3 5
10 10 10 10 10 10 10 10 10 10 10 Mean 0.5814 147.1 119.0 119.0
100.6 49.14 88.00 68.00 64.57 -59.69 13.12 90.82 28.42 44.32 (%
Morphine) SD 132.5 63.69 0.0 0.0 41.42 57.26 50.04 34.79 78.00
62.30 50.11 43.45 81.70 62.87 SEM 59.27 28.48 0.0 0.0 18.53 25.61
22.38 15.56 34.88 27.86 22.41 19.43 36.54 28.11
Example 6
Brain:Plasma Ratio for PEG-Promethazine Conjugates
[0195] The ability of the PEG-promethazine conjugates to cross the
blood brain barrier (BBB) and enter the CNS was determined by
measuring the ratio of their relative concentrations in brain and
plasma in rats. Briefly, rats were injected intravenously with 5
mg/kg of promethazine, PEG-promethazine conjugates or atenolol. An
hour following injection, the animals were sacrificed and plasma
and brain were collected and frozen immediately. Following tissue
and plasma extractions, concentrations of the compounds in brain
and plasma were measured using LC-MS/MS. The brain:plasma ratio was
calculated as the ratio of measured concentrations in the brain
(ng/g) and plasma (ng/mL). Atenolol, which does not cross the blood
brain barrier is used as a measure of vascular contamination of the
brain tissue.
[0196] The following table shows the ratios of brain to plasma
concentrations of PEG-Promethazine conjugates. The brain:plasma
ratio of promethazine is 39.93:1, indicating a nearly 40 fold
greater concentration of promethazine in the brain compared to the
plasma compartment. PEG conjugation reduced the entry of
promethazine into the CNS as evidenced by 20-500-fold lower
brain:plasma ratios of the PEG-promethazine conjugates.
TABLE-US-00005 Brain/plasma ratio Compound Mean Std. Dev. N
Promethazine 39.926 13.286 3 mPEG.sub.4-N-Promethazine 1.863 0.470
3 mPEG.sub.5-N-Promethazine 0.243 0.058 3 mPEG.sub.6-N-Promethazine
0.098 0.027 3 mPEG.sub.7-N-Promethazine 0.112 0.009 3
mPEG.sub.9-N-Promethazine 0.080 0.023 3 Atanolol 0.024 0.006 3
Example 7
In Vivo Efficacy of PEG-Promethazine Conjugates
[0197] The antihistamine efficacy of PEG-Promethazine conjugates is
evaluated in vivo by their ability to antagonize
bronchoconstriction induced by histamine in guinea pigs. Groups of
3-4 guinea pigs are anesthetized with urethane, demobilized by
succinylcholine, and placed on a heated plate to keep body
temperature at 37.degree. C. The left carotid artery is cannulated
with a catheter (PE50, inner diameter 0.58 mm) connected to a
pressure transducer for measurements of blood pressure (BP). A
cannula (20 nun in length and with a 2 mm inner diameter) is
inserted into the upper trachea through a tracheotomy and connected
to a constant-volume mechanical ventilator. A respiratory volume of
10 ml/kg and a frequency of 50 breaths per minute are used with
animals placed in a supine position. Respiratory air flow is
measured using a flow transducer connected to a pneumotachograph.
Pleural pressure is measured using a differential pressure
transducer connected to two catheters, one through a 3-way stopcock
connecting the pneumotachograph and trachea for obtaining
intrapulmonary pressure, another from a catheter which is directly
inserted into the pleural cavity for obtaining intrapulmonary
pressure. Tidal volume (VT) is calculated by integration of the
flow signal. Lung resistance (R.sub.L) is calculated from pleural
pressure (.DELTA.P) and flow signals (.DELTA.F) measured at
isovolumetric points (50%) during inspiration and expiration, i.e.,
R.sup.L=.DELTA.P/.DELTA.F. Dynamic Compliance (C.sub.dyn) is the
amount of lung expansion (volume change) per unit of pleural
pressure change when the pressure and volume changes are measured
at times of zero flow. Briefly, dividing tidal volume (VT) with
transpulmonary pressure (.DELTA.PTP) between points of zero flow
yields C.sub.dyn=VT/.DELTA.PTP. All signals are captured by a data
acquisition and analysis system (PO-NE-MAH Inc., USA).
[0198] PEG-Promethazine conjugates are administered
intra-tracheally, subcutaneously or orally at various times prior
to an IV histamine challenge. The various parameters (R.sub.L,
C.sub.dyn, BP, HR) are recorded immediately before administration
of test substance and vehicle as well as immediately before (0 min)
and at 0.33, 0.67, 1, 2, 3, 4 and 5 minutes following challenge
with histamine phosphate (10 .mu.g/kg, 1 ml/kg, intravenously).
Histamine produces a pronounced bronchoconstriction in guinea pigs
measured as a decrease in pulmonary compliance and increase in
pulmonary resistance that is dose-dependently antagonized by
molecules with antihistamine activity.
Example 8
pKa and Log P Determination for Promethazine and PEG Promethazine
Conjugates
[0199] The Sirius GLpKa instrument is used to determine the pKa and
Log P for Promethazine and Peg Promethazine conjugates. A blank
standardization has to be completed first and passed in order to
conduct the compound experiments. The solutions used by the
instrument for the blank titration include water, a pH solution,
and a surfactant (TRITON X-100). The TRITON X-100 was purchased
commercially from Sirus. The 0.5% solution of TRITON X-100 was used
in the experiment. In a 1 L volumetric flask 5 mls of TRITON X-100
is added and MILLI Q water to volume. The vessels used by the
instrument were filled to 1/3 of their volume with each solution.
Therefore the assay tray contains one vessel for water, pH
solution, and surfactant for the blank run. The temperature of the
water bath for the instrument is 25.degree. C. The blank is
calculated as an average of three "good" experiments. The "good"
was denoted by a green check mark next to the assay number in the
computer software. Once the blank was run successfully the
instrument was ready to conduct the experiments.
[0200] The pKa had to be determined first in order to calculate the
Log P. The API is used to determine the pKa needed for the Log P
experiment followed by the conjugates. Promethazine was first
weighed into a vessel. The amount weighed for each sample was 10%
of the molecular weight of the compound (i.e. 2.84 g for
Promethazine). The same procedure was followed for each
PEG-Promethazine conjugate n=3-9. The vessels were placed in the
assay tray of the instrument. A method had to be set up for the pKa
run. The experimental parameters are entered into the computer
software. These included sample weight, molecular weight, assay
type (aqueous pKa), number of assays (three), and titration range
(generally 3.5-12). The run was started. There are three
measurements for each run that are averaged to obtain the final
results from the data points collected in the calculation software.
All runs were denoted as "good" by the green check mark next to the
assay number. The pKa values were obtained for all compounds as
listed in the table below.
[0201] An additional method has to be set up for the Log P
determinations for each compound. The Promethazine and
PEG-Promethazine conjugates (n=3-9) are weighed into vessels in the
same manner as for the previous experiment, i.e., 10% of the
molecular weight. The vessels are placed in the assay tray of the
instrument. The experimental parameters are entered into the
computer. These included sample weight, molecular weight, assay
type (Partition Log P), number of assays (three), and pKa value of
the parent drug from the previous experiment. The run was started.
There are three measurements for each run that are averaged to
obtain the final results from the data points collected in the
calculation software. All runs were denoted as "good" by the green
check mark next to the assay number. The Log P values were obtained
for all compounds as listed in the table below.
TABLE-US-00006 Compound Description MW pKa LogP Promethazine 284.4
8.988 3.431 mPEG.sub.3-N-Promethazine 416.58 8.405 3.654
mPEG.sub.4-N-Promethazine 460.6 8.448 3.511
mPEG.sub.5-N-Promethazine 504 8.674 3.598 mPEG.sub.6-N-Promethazine
548.7 8.529 3.250 mPEG.sub.7-N-Promethazine 592.8 8.486 2.995
mPEG.sub.8-N-Promethazine 636 8.238 2.648 mPEG.sub.9-N-Promethazine
680.9 8.296 2.540
* * * * *