U.S. patent application number 10/756774 was filed with the patent office on 2004-08-12 for pulmonary delivery for bioconjugation.
This patent application is currently assigned to CONJUCHEM, INC.. Invention is credited to Bridon, Dominique P., Ezrin, Alan M., Fleser, Angelica, Milner, Peter G., Robitaille, Martin.
Application Number | 20040156859 10/756774 |
Document ID | / |
Family ID | 31949739 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040156859 |
Kind Code |
A1 |
Ezrin, Alan M. ; et
al. |
August 12, 2004 |
Pulmonary delivery for bioconjugation
Abstract
Methods of and compositions for pulmonary delivery of
therapeutic agents which are capable of forming covalent bonds with
a site of interest or which have formed a covalent bond with a
pulmonary solution protein are disclosed. Therapeutic agents useful
in the invention include wound healing agents, antibiotics,
anti-inflammatories, anti-oxidants, anti-proliferatives,
immunosupressants, anti-infective and anti-cancer agents.
Inventors: |
Ezrin, Alan M.; (Moraga,
CA) ; Fleser, Angelica; (Montreal, CA) ;
Robitaille, Martin; (Granby, CA) ; Milner, Peter
G.; (Los Altos Hills, CA) ; Bridon, Dominique P.;
(Ville Mont-Royal, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
425 MARKET STREET
SAN FRANCISCO
CA
94105-2482
US
|
Assignee: |
CONJUCHEM, INC.
Montreal
CA
|
Family ID: |
31949739 |
Appl. No.: |
10/756774 |
Filed: |
January 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10756774 |
Jan 12, 2004 |
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09656121 |
Sep 6, 2000 |
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6706892 |
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60152681 |
Sep 7, 1999 |
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Current U.S.
Class: |
424/185.1 ;
514/397; 514/422; 530/409; 548/314.7; 548/517 |
Current CPC
Class: |
A61P 9/06 20180101; A61K
47/50 20170801; A61P 7/06 20180101; A61K 9/0075 20130101; A61P
11/08 20180101; A61K 47/643 20170801 |
Class at
Publication: |
424/185.1 ;
514/397; 514/422; 530/409; 548/314.7; 548/517 |
International
Class: |
A61K 039/00; C07D
403/02; A61K 031/4178; A61K 031/4025; A61K 031/4015 |
Claims
We claim:
1. A modified therapeutic agent comprising: a therapeutic agent and
a reactive group which reacts in vivo with amino groups, hydroxyl
groups or thiol groups on pulmonary components or blood components
to form a stable covalent bond, the therapeutic agent being
selected from the group consisting of GP-41 peptides, BBB peptides,
anti-cancer agents, antihistamines, bronchodilators,
anti-hypertension agents, anti-angina agents, opioids, analgesics,
anti-depressants, and hypothyroid agents.
2. The modified therapeutic agent of claim 1 wherein said reactive
group is a succinimidyl or a maleimido group.
3. The modified therapeutic agent of claim 1 wherein said reactive
group is a maleimido group which is reactive with a thiol group on
a mobile pulmonary component.
4. The modified therapeutic agent of claim 1 wherein said reactive
group is a maleimido group which is reactive with a thiol group on
a fixed pulmonary component.
5. The modified therapeutic agent of claim 1 wherein said reactive
group is a maleimido group which is reactive with a thiol group on
a mobile blood component.
6. The modified therapeutic agent of claim 1 wherein said reactive
group is a maleimido group which is reactive with a thiol group on
albumin.
7. The modified therapeutic agent of claim 1 wherein said reactive
group is a maleimido group which is reactive with a thiol group on
a fixed blood component.
8. The modified therapeutic agent of claim 1 wherein said
therapeutic agent is an anti-histamine.
9. The modified therapeutic agent of claim 1 wherein said
therapeutic agent is a hypothyroid agent.
10. The modified therapeutic agent of claim 9 wherein said
therapeutic agent is loratidine.
11. The modified therapeutic agent of claim 9 wherein said
therapeutic agent is cetirizine.
12. An aerosol composition for delivery of a therapeutic agent to
the pulmonary system of a host comprising: an aerosolized aqueous
solution containing a modified therapeutic agent, the modified
therapeutic agent comprising a therapeutic agent and a reactive
group which reacts with amino groups, hydroxyl groups or thiol
groups on pulmonary or blood components to form a stable covalent
bond.
13. The aerosol of claim 12 further comprising a pharmaceutically
acceptable carrier.
14. The aerosol of claim 12 wherein said modified therapeutic agent
is 2.5-10% by weight.
15. The aerosol of claim 12 wherein said therapeutic agent an
anti-histamine.
16. The aerosol of claim 15 wherein said therapeutic agent is
loratidine.
17. The aerosol of claim 15 wherein said therapeutic agent is
cetirizine.
18. A particulate formulation for delivery of a therapeutic agent
to the pulmonary system of a host comprising: a dispersable dry
powder containing a modified therapeutic agent, the modified
therapeutic agent comprising a therapeutic agent and a reactive
group which reacts with amino groups, hydroxyl groups or thiol
groups on pulmonary components to form a stable covalent bond.
19. The particulate formulation of claim 18 wherein at least 50% of
the dry powder is in the form of particles having a diameter of 10
um or less.
20. The particulate formulation of claim 18 wherein said
therapeutic agent is an anti-histamine.
21. The particulate formulation of claim 20 wherein said
therapeutic agent is loratidine.
22. The particulate formulation of claim 20 wherein said
therapeutic agent is cetirizine.
23. A method of delivering a therapeutic agent to a host comprising
the steps of: obtaining a modified therapeutic agent, the modified
therapeutic agent comprising a therapeutic agent and a reactive
group which reacts in vivo with amino groups, hydroxyl groups or
thiol groups on pulmonary or blood components to form a stable
covalent bond; and administering the modified therapeutic agent to
the pulmonary system of the host.
24. The method of claim 23 wherein said administering step further
comprises the steps of aerosolizing the modified therapeutic agent
for inhalation by the host.
25. The method of claim 23 wherein said administering step further
comprises the steps of dispersing a dry formulation of the modified
therapeutic agent for inhalation by the host.
26. The method of claim 23 wherein said administering step further
comprises the steps of instilling the modified therapeutic agent
into the pulmonary system of the host.
27. The method of claim 23 wherein said reactive group is a
succinimidyl or a maleimido group.
28. The method of claim 23 wherein said reactive group is a
maleimido group which is reactive with a thiol group on a mobile
pulmonary component.
29. The method of claim 23 wherein said reactive group is a
maleimido group which is reactive with a thiol group on a fixed
pulmonary component.
30. The method of claim 23 wherein said reactive group is a
maleimido group which is reactive with a thiol group on a mobile
blood component.
31. The method of claim 23 wherein said reactive group is a
maleimido group which is reactive with a thiol group on a fixed
blood component.
32. The method of claim 23 wherein said reactive group is a
maleimido group which is reactive with a thiol group on human serum
albumin.
33. The method of claim 23 wherein said therapeutic agent is an
anti-histamine.
34. The method of claim 33 wherein said therapeutic agent is
loratidine.
35. The method of claim 33 wherein said therapeutic agent is
cetirizine.
36. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an antihistamine and analogs
thereof wherein the derivative includes a reactive functional group
which reacts with amino groups, hydroxyl groups, or thiol groups on
blood components to form stable covalent bonds, said reactive
functional group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide an anhistamine effect.
37. Use of a composition according to claim 36 wherein the
antihistamine is selected from cetirizine, loratidine and analogs
thereof.
38. Use of a composition according to claim 36 wherein the
antihistamine is selected from cetirizine and analogs thereof.
39. Use of a composition according to claim 36 wherein the
antihistamine is selected from loratidine and analogs thereof.
40. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-angina agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-angina effect.
41. Use of a composition according to claim 40 wherein the
anti-angina agent is tirofiban.
42. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-hypertensive agent
and analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimid- e and a maleimide
group for use in the treatment of the human body to provide an
anti-hypertensive effect.
43. Use of a composition according to claim 42 wherein the
anti-hypetensive agent is enalapril.
44. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-arrhythmic agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimid- e and a maleimide
group for use in the treatment of the human body to provide an
anti-arrhythmic effect.
45. Use of a composition according to claim 44 wherein the
anti-arrhythmic agent is capobenic acid.
46. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-depressant agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimid- e and a maleimide
group for use in the treatment of the human body to provide an
anti-depressan effect.
47. Use of a composition according to claim 46 wherein the
anti-depressant agent is fluoxetine.
48. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of a bronchodilator and analogs
thereof wherein the derivative includes a reactive functional group
which reacts with amino groups, hydroxyl groups, or thiol groups on
blood components to form stable covalent bonds, said reactive
functional group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide a bronchodilation
effect.
49. Use of a composition according to claim 48 wherein the
bronchodilator is theobromineacetamine and analogs thereof.
50. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-inflammatory agent
and analogs thereof, wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimid- e and a maleimide
group for use in the treatment of the human body to provide an
anti-inflammatory effect.
51. Use of a composition according to claim 50 wherein the
anti-inflammatory agent is loxoprofen and analogs thereof.
52. Use of a composition for the manufacture of a medicament said
composition comprising a derivative of an anti-thyroid deficiency
agent and analogs thereof, wherein the derivative includes a
reactive functional group which reacts with amino groups, hydroxyl
groups, or thiol groups on blood components to form stable covalent
bonds, said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-thyroid deficiency effect.
53. Use of a composition according to claim 52 wherein the
anti-thyroid deficiency agent is thyroxin and analogs thereof.
54. A composition comprising a compound selected from the group
consisting of:
2-[2-[4-[(4-choloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimid-
opropionylacetamide;
11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6-
,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine;
N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropio-
nilamide;
Maleimidopropynamyl-.epsilon.-(3,4,5-trimethoxybenz-amido)-capro-
icamide; Maleimidopropionamyl-1-theobromineacetamide;
Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide
N-maleimidopropionyl-N-methyl-3-(p-trifluoromethylphenoxy)-3-phenylpropyl-
amine; 4-anilino-1-(2-phenethyl)piperdine and
Maleimidopropionamyl-3,5-3',- 5' tetraiodothyroninamide.
55. The composition of claim 54, wherein the compound is
Maleimidopropionamyl-3,5-3',5' tetraiodothyroninamide.
56. An aerosol composition for delivery of a therapeutic agent to
the pulmonary system of a host comprising an aerosolized aqueous
solution containing a modified therapeutic agent conjugated to a
blood protein.
57. The composition of claim 56 wherein said protein is
albumin.
58. The aerosol of claim 56 wherein said therapeutic agent an
anti-histamine.
59. The aerosol of claim 56 wherein said therapeutic agent is
loratidine.
60. The aerosol of claim 56 wherein said therapeutic agent is
cetirizine.
61. A particulate formulation for delivery of a therapeutic agent
to the pulmonary system of a host comprising: a dispersable dry
powder containing a modified therapeutic agent, the modified
therapeutic agent comprising a therapeutic agent and a reactive
group which reacts with amino groups, hydroxyl groups or thiol
groups on pulmonary components to form a stable covalent bond
wherein said therapeutic agent is covalently bonded to a blood
protein.
62. The formulation of claim 61 wherein said protein is
albumin.
63. The formulation of claim 61 wherein said therapeutic agent is
an anti-histamine.
64. The formulation of claim 61 wherein said therapeutic agent is
loratidine.
65. The particulate formulation of claim 61 wherein said
therapeutic agent is cetirizine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of therapeutic and
diagnostic agents in medicine. In particular, this invention
relates to the field of delivery, in particular, pulmonary
delivery, of therapeutic and diagnostic agents wherein the agents
are capable of covalently bonding to a site of interest in vivo, to
provide increased bioavailability and pharmacodynamic duration of
therapeutic and diagnostic benefit for the given agent.
BACKGROUND OF THE INVENTION
[0002] Peptide and protein drugs are being used increasingly in
major research and development programs in the pharmaceutical
industry and are also an important class of therapeutic agents due
to advances in genetic engineering and biotechnology. Systemic
delivery of these macromolecular drugs and other therapeutic and
diagnostic agents, however, has been limited to the parenteral
route largely because of their extensive presystemic elimination
when taken orally. Faced with this dilemma concerning the systemic
delivery of these macromolecules with their unique conformational
complexity for therapeutic activity, pharmaceutical scientists are
continually evaluating the potential of various non-oral routes of
administration as alternatives.
[0003] Despite the tremendous efforts that have been devoted to
this problem, only limited success has been achieved--mostly with
small peptides. An alternative, non-invasive means for systemic
delivery of therapeutic and diagnostic agents is via the pulmonary
system. Delivery via the pulmonary system is advantageous because
the lungs provide a large but extremely thin absorptive mucosal
membrane for increased absorption and delivery to the blood stream.
However, pulmonary delivery of therapeutic and diagnostic agents is
complicated by the complexity of the anatomic structure of the
human respiratory system; the effect of respiration on drug
deposition and an instability of the drugs resulting from
degradation in either the lungs or plasma.
[0004] There is thus a need to improve and enhance delivery of
therapeutic and diagnostic agents, especially pulmonary delivery of
of therapeutic and diagnostic agents through increasing the
stability and blood absorption of the agents.
SUMMARY OF THE INVENTION
[0005] In order to meet these needs, the present invention is
directed to therapeutic and diagnostic agents capable of forming
covalent bonds to blood and pulmonary fluid proteins or other
components ex vivo or in vivo. The therapeutic agents of this
invention have a long duration of action for the management of
disease. The invention relates to ex vivo and in vivo.
bioconjugation of therapeutic agents to protein (e.g. albumin), as
well as an intrapulmonary in vivo bioconjugation of therapeutic
agents to endogenous pulmonary fluid proteins or other components
to dramatically increase the half life of the therapeutic agents
and avoid the need for parenteral administration.
[0006] The present invention reflects the ability to bioconjugate
selected therapeutic agents to blood and pulmonary pulmonary fluid
proteins, including albumin, for processing as a particulate for
intrapulmonary drug delivery. The pulmonary fluid protein conjugate
is targeted to provide a stable drug substance that retains
biological activity for prolonged periods of time. This invention
provides prolonged local retention of therapeutic agent activity in
the airways for use with selected therapeutic agents in managing
pulmonary disease.
[0007] The invention is further directed to methods of facilitating
systemic drug delivery of protein-therapeutic agent bioconjugates
and to agents capable of forming bioconjugates to protein in vivo
via pulmonary delivery with subsequent transcytosis across the
alveolar and pulmonary mucosa. The invention is further directed to
methods of facilitating systemic drug delivery of
protein-therapeutic agent bioconjugates via pulmonary delivery of
agents capable of forming bioconjugates in vivo, the agents
crossing the epithelium of the alveolar or pulmonary mucosa, either
through diffusion or receptor-mediated transport, to conjugate with
blood proteins. The methods of this invention result in long
acting, systemic therapeutics that are stabilized by ex vivo or in
vivo conjugation to pulmonary fluid proteins and/or blood
proteins.
[0008] This invention is further directed to site-specific and
protein-specific bioconjugation of a therapeutic agent to albumin.
Albumin possesses a unique nucleophilic moiety, specifically, the
thiol functionality on cysteine 34 that is capable of undergoing a
nucleophilic attack on electrophile present on a therapeutic agent
modified according to the invention. This selective covalent
bonding enables bioconjugation to extracellular as well as
intracellular albumin for prolonged retention and bioavailabilty of
the therapeutic agent.
[0009] This invention is further directed to the use of reactive
chemistries including: N-hydroxy sulfosuccinimide,
maleimide-benzoyl-succinimide, gamma-maleimido-butyryloxy
succinimide ester, maleimidopropionic acid, isocyanate, thiolester,
thionocarboxylic acid ester, imino ester, and carbodiimide
anhydride. Maleimidopropionic acid is the preferred reactive
chemistry, but the invention also contemplates the selection of the
above and like reactive chemistries as an electrophilic moeity for
bioconjugations with albumin or other proteins.
[0010] This invention is further directed to the use of a
composition for the manufacture of a medicament where the
composition comprises a derivative of an antihistamine and analogs
thereof wherein the derivative includes a reactive functional group
which reacts with amino groups, hydroxyl groups, or thiol groups on
blood components to form stable covalent bonds, said reactive
functional group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide an anhistamine effect.
[0011] The modified antihistamine may be cetirizine, loratidine and
analogs thereof.
[0012] This invention is further directed to the use of a
composition for for the manufacture of a medicament where the
composition comprises a derivative of an anti-angina agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-angina effect.
[0013] The modifed anti-angina agent may be tirofiban or analogs
thereof.
[0014] This invention is further directed to the use of a
composition for the manufacture of a medicament where the
composition comprises a derivative of an anti-hypertensive agent
and analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-hypertensive effect.
[0015] The anti-hypetensive agent may be enalapril or analogs
thereof.
[0016] This invention is further directed to the use of a
composition for the manufacture of a medicament where the
composition comprising a derivative of an anti-arrhythmic agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-arrhythmic effect.
[0017] The anti-arrhythmic agent may be capobenic acid or analogs
thereof.
[0018] This invention is further directed to the use of a
composition for the manufacture of a medicament where the
composition comprising a derivative of an anti-depressant agent and
analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-depressan effect.
[0019] The anti-depressant agent may be fluoxetine or analogs
thereof.
[0020] This invention is further directed to the use of a
composition for the manufacture of a medicament said composition
comprising a derivative of a bronchodilator and analogs thereof
wherein the derivative includes a reactive functional group which
reacts with amino groups, hydroxyl groups, or thiol groups on blood
components to form stable covalent bonds, said reactive functional
group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide a bronchodilation
effect.
[0021] The bronchodilator may be theobromineacetamine or analogs
thereof.
[0022] This invention is further directed to the use of a
composition for the manufacture of a medicament said composition
comprising a derivative of an opioid and analogs thereof, wherein
the derivative includes a reactive functional group which reacts
with amino groups, hydroxyl groups, or thiol groups on blood
components to form stable covalent bonds, said reactive functional
group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide an analgesic effect.
[0023] The opioid may be fentanyl or analogs thereof.
[0024] This invention is further directed to the use of a
composition for the manufacture of a medicament said composition
comprising a derivative of an anti-inflammatory agent and analogs
thereof, wherein the derivative includes a reactive functional
group which reacts with amino groups, hydroxyl groups, or thiol
groups on blood components to form stable covalent bonds, said
reactive functional group being selected from N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide and a maleimide group for use in the
treatment of the human body to provide an anti-inflammatory
effect.
[0025] The anti-inflammatory agent may be loxoprofen or analogs
thereof.
[0026] This invention is further directed to the use of a
composition for the manufacture of a medicament where the
composition comprising a derivative of an anti-thyroid deficiency
agent and analogs thereof, wherein the derivative includes a
reactive functional group which reacts with amino groups, hydroxyl
groups, or thiol groups on blood components to form stable covalent
bonds, said reactive functional group being selected from
N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide
group for use in the treatment of the human body to provide an
anti-thyroid deficiency effect.
[0027] the anti-thyroid deficiency agent may be thyroxin or analogs
thereof.
[0028] This invention is further directed to composition comprising
one or more compounds selected from the group consisting of
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidoprop-
ionylacetamide;
11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6,11-d-
ihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine;
N-(1(S)-Ethoxycarbonyl--
3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropionilamide;
Maleimidopropynamyl-.epsilon.-(3,4,5-trimethoxybenz-amido)-caproicamide;
Maleimidopropionamyl-1-theobromineacetamide;
Maleimidopropamyl2-[4-(2-oxo-
cyclopentan-1-ylmethyl)phenyl]propionamide
N-maleimidopropionyl-N-methyl-3-
-(p-trifluoromethylphenoxy)-3-phenylpropylamine;
4-anilino-1-(2-phenethyl)- piperdine and
Maleimidopropionamyl-3,5-3',5' tetraiodothyroninamide.
[0029] This invention is further directed to the an aerosol
composition for delivery of a therapeutic agent to the pulmonary
system of a host comprising an aerosolized aqueous solution
containing a modified therapeutic agent conjugated to a blood
protein.
[0030] This invention is further directed to the use of a
particulate formulation for delivery of a therapeutic agent to the
pulmonary system of a host comprising:
[0031] a dispersable dry powder containing a modified therapeutic
agent, the modified therapeutic agent comprising a therapeutic
agent and a reactive group which reacts with amino groups, hydroxyl
groups or thiol groups on pulmonary components to form a stable
covalent bond wherein said therapeutic agent is covalently bonded
to a blood protein.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Definitions
[0033] To ensure a complete understanding of the invention the
following definitions are provided:
[0034] Therapeutic Agents: Therapeutic agents are agents that have
a therapeutic effect and inlcude peptides and non-peptide organic
molecules. Therapeutic agents include but are not limited to wound
healing agents, antibiotics, anti-infectives, anti-oxidants,
chemotherapeutic agents, anti-cancer agents, anti-inflammatory
agents, and antiproliferative drugs. Therapeutic agents also
include abortifacients, ace-inhibitor, .alpha.-adrenergic agonists,
.beta.-adrenergic agonists, .alpha.-adrenergic blockers,
.beta.-adrenergic blockers, adrenocortical steroids, adrenocortical
supressants, adrenocorticotrophic hormones, alcohol deterrents,
aldose reductase inhibitors, aldosterone antagonists, 5-alpha
reductase inhibitors, anabolics, analgesics, analgesics,
analgesics, androgens, anesthetics, anesthetics, angiotensin
coverting enzyme inhibitors, anorexics, antacids, anthelmintics,
antiacne agents, antiallergic agents, antialopecia agents,
antiamebic agents, antiandrogen agents, antianginal agents,
antiarrhythmic agents, antiarterioscierotic agents,
antiarthritic/antirheumatic agents, antiasthmatic agents,
antibacterial agents, aminoglycosides, amphenicols, ansamycins,
.beta.-lactams, lincosamides, macrolides, polypeptides,
tetracyclines, antibacterial agents, 2,4-diaminopyrimidines,
nitrofurans, quinolones and analogs, sulfonamides, sulfones,
antibiotics, anticholelithogenic agents, anticholesteremic agents,
anticholinergic agents, anticoagulant agents, anticonvulsant
agents, antidepressant agents, hydrazides/hydrazines, pyrrolidones,
tetracyclics, antidiabetic agents, biguanides, hormones,
sulfonylurea derivatives, antidiarrheal agents, antiduretic agents,
antidotes, antidote, antidote, antidote, antidote, antidyskinetic,
antieczematic, antiemetic agents, antiepileptic agents,
antiestrogen agents, antifibrotic agents, antiflatulent agents,
antifungal agents, polyenes, allylamines, imidazoles, triazoles and
antiglaucoma agents.
[0035] Other therapetic agents include anti-viral agents,
anti-fusogenic agents, blood brain barrier peptides (BBB peptides),
RGD peptides, glucagon-like peptides, antigonadotropin, antigout,
antihemorrhagic and antihistaminic agents; alkylmaine derivatives,
aminoalkyl ethers, ethylenediamine derivatives, piperazines and
tricyclics, antihypercholesterolemic, antihyperlipidemic,
anthyperlipidemic and antihyperlipoproteinemic agents,
aryloxyalkanoic acid derivatives, bile acid sequesterants, hmg coa
reductase inhibitors, nicotine acid derivatives, thyroid
hormones/analogs, antihyperphosphatemic, antihypertensive agents,
arlethanolamine derivatives, arloxypropanolamine derivatives,
benzothiadiazine derivatives, n-carboxyalkyl derivatives,
dihydropyridine derivatives, guanidine derivatives,
hydrazines/phthalazines, imidazole derivatives, quaternary ammonium
compounds, quinazolinyl piperazine derivatives, reserpine
derivatives, sulfonamide derivatives, antihyperthyroid agents,
antihypotensive agents, antihypothyroid agents, anti-infective
agents, anti-inflammatory agents, anti-inflammatory agents,
aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,
arylbutyric acid derivatives and arylcarboxylic acids.
[0036] Therapeutic agents also include arylpropionic acid
derivatives, pyrazoles, pyrazolones, salicylic acid derivatives,
thiazinecarboxamides, antileprotic, antileukemic, antilipemic,
antilipidemic, antimalarial, antimanic, antimethemoglobinemic,
antimigraine, antimycotic, antinauseant, antineoplastic and
alkylating agents, antimetabolites, enzymes, androgens,
antiadrenals, antiandrogens, antiestrogens, lh-rh analogs,
progestogens, adjunct, folic acid replenisher, uroprotective and
antiosteporotic agents
[0037] Therapeutic agents also include antipagetic,
antiparkinsonian, antiperistaltic, antipheochromocytoma,
antipneumocystis, antiprostatic hypertrophy, antiprotozoal,
antiprozoal, antipruritic, antipsoriatic and antipsychotic agents,
butyrophenes, phenothiazines, thioxanthenes, antipyretic,
antirheumatic, antirickettsial, antiseborreheic and
antiseptic/disinfectant agetns, alcohols, aldehydes, dyes,
guanidines, halogens/halogen compounds, mercurial compounds,
nitrofurans, peroxides/permanganates, phenols, quinolines, silver
compounds, others, antispasmodic,antisyphilitic, antithrombotic,
antitubercular, antitumor, antitussive, antiulcerative,
antiurolithic, antivenin, antivertigo and antiviral agents,
purines/pyrimidinomes, anxiolytic, arylpiperazines, benzodiazepine
derivatives, carbamates, astringent, benzodiazepine antagonist,
beta-blocker, bronchodilator, ephedrine derivatives, calcium
channel blockers, arylalkylamines, dihydropyridine derivatives,
piperazine derivatives, calcium regulators, calcium supplements,
cancer chemotherapy agents, capillary protectants, carbonic
anhydrase inhibitors, cardiac depressants, cardiotonic, cathartic,
cation-exchange resin, cck antagonists, central stimulants,cerebral
vasodilators, chelating agents, cholecystokinn antagonists,
choleitholytic agents, choleretic agents, cholinergic agents,
cholinesterase inhibitors, cholinesterase reactivators, cns
stimulants, cognition activators, contraceptives, agents to control
intraocular pressure, converting-enzyme inhibitors, coronary
vasodilators, cytoprotectants, debriging agenta, decongestants,
depigmentora, dermatitis herpretiformis suppresanta, diagnostic
aids, digestive aids, diuretics, benthothiadiazine derivatives,
organomercurials, pteridines, purines, steroids, sulfanamide
derivatives, uracils, others, dopamine and receptor agonists.
[0038] Therapeutic agents also include dopamine receptor
antagonists, ectoparasiticides, electrolyte replenishers, emetics,
enzymes, digestive agents, mucolytic agents, penicillin
inactivating agents, proteolytic agents, enzyme inducers, estrogen
antagonists, expectorant gastric and pancreatic secreation
stimulantd, gastric proton pump inhibitor, gastric secretion
inhibitord, glucocorticoidd, .alpha.-glucosidase inhibitord,
gonad-stimulating principled, gonadotrophic hormoned, gout
suppressant, growth hormone inhibitor, growth hormone releasing
factor, growth stimulant, hematinic, hemolytic, demostatic, heparin
antagonist, hepatoprotectant, histamine h.sub.1-receptor
antagonists, histamine h.sub.2-receptor antagonists, hmg coa
reductase inhibitor, hypnotic, hypocholesteremic and hypolipidemic
agents.
[0039] Therapeutic agents also include hypotensive,
immunomodulators, immunosuppressants, inotrophic agents,
keratolytic agents, lactation stimulating hormone,
laxative/cathargic, lh-rh agonists, lipotrophic agents, local
anesthetics, lupus erythematosus suppressants, major tranquilizers,
mineralocorticoids, minor tranquilizers, miotic agents, monoamine
oxidase ihibitors, mucolytic agents, muscle relaxants, mydriatic
agents, narcotic agents; analgesics, narcotic antagonists, nasal
decongestants, neuroleptic agents, neuromuscular blocking agents,
neuroprotective agents, nmda antagonists, nootropic agents, nsaid
agents, opioid analgesics, oral contraceptives and ovarian
hormones.
[0040] Therapeutic agents also include oxytocic agents, blood brain
barrier protiens, GP41 peptides, insulinotropic peptides
parasympathomimetic agents, pediculicides, pepsin inhibitors,
peripheral vasodilators, peristaltic stimulants, pigmentation
agents, plasma volume expanders, potassium channel
activators/openers, pressor agents, progestogen, prolactin
inhibitors, prostaglandin/prostaglandin analogs, protease
inhibitors, proton pump inhibitors, 5.alpha.-reductase inhibitors,
replenishers/supplements, respiratory stimulants, reverse
transcriptase inhibitors, scabicides, sclerosing agents,
sedative/hypnotic agents, acyclic ureides, alcohols, amides,
barbituric acid derivatives, benzodiazepine derivatives, bromides,
carbamates, chloral derivatives, quinazolone derivatives and
piperidinediones.
[0041] Therapeutic agents also include serotonin receptor agonists,
serotonin receptor antagonists, serotonin uptake inhibitors,
skeletal muscle relaxants, somatostatin analogs, spasmolytic
agents, stool softeners, succinylcholine synergists,
sympathomimetics, thrombolytics, thyroid hormone, thyroid
inhibitors, thyrotrophic hormone, tocolytic, topical protectants,
uricosurics, vasodilators, vasopressors, vasoprotectants,
vitamin/vitamin sources, antichitic, antiscorbutic and
antixerophthalmic agents, enzyme co-factors, hematopoietic,
prombogenic agents and xanthene oxidase inhibitors.
[0042] Diagnostic Imaging Agents: Diagnostic imaging agents are
agents useful in imaging the mammalian vascular system and include
such agents as position emission tomography (PET) agents,
computerized tomography (CT) agents, magnetic resonance imaging
(MRI) agents, nuclear magnetic imaging agents (NMI), fluroscopy
agents and ultrasound contrast agents. Diagnostic agents of
interest include radioisotopes of such elements as iodine (I),
including .sup.123I, .sup.125I, .sup.131I, etc., barium (Ba),
gadolinium (Gd), technetium (Tc), including .sup.99Tc, phosphorus
(P), including .sup.31P, iron (Fe), manganese (Mn), thallium (TV),
chromium (Cr), including .sup.51Cr, carbon (C), including .sup.14C,
or the like, fluorescently labeled compounds, etc.
[0043] Wound Healing Agents: Wound healing agents are agents that
promote wound healing. Wound healing agents include integrins, cell
adhesion molecules such as ICAM, ECAM, ELAM and the like,
antibiotics, growth factors such as EGF, PDGF, IGF, bFGF, aFGF and
KGF, fibrin, thrombin, RGD peptides and the like.
[0044] Antiproliferatives: Antiproliferatives include
antimetabolites, topoisomerase inhibitors, folic acid antagonists
like methotrexate, purine antagonists like mercaptopurine,
azathioprine, and pyrimidine antagonists like fluorouracil,
cytarabine and the like.
[0045] Antioxidants: Antioxidants are agents that prevents
oxidative damage to tissue and include aspartate, orotate,
tacophenol derivative (vitamin E), and free radical scavengers such
as SOD, glutathione and the like.
[0046] Mammalian cells are continuously exposed to activated oxygen
species such as superoxide, hydrogen peroxide, hydroxyl radical,
and singlet oxygen. These reactive oxygen intermediates are
generated in vivo by cells in response to aerobic metabolism,
catabolism of drugs and other xenobiotics, ultraviolet and x-ray
radiation, and the respiratory burst of phagocytic cells (such as
white blood cells) to kill invading bacteria such as those
introduced through wounds. Hydrogen peroxide, for example, is
produced during respiration of most living organisms especially by
stressed and injured cells.
[0047] Active oxygen species can injure cells. An important example
of such damage is lipid peroxidation which involves the oxidative
degradation of unsaturated lipids. Lipid peroxidation is highly
detrimental to membrane structure and function and can cause
numerous cytopathological effects. Cells defend against lipid
peroxidation by producing radical scavengers such as superoxide
dismutase, catalase, and peroxidase. Injured cells have a decreased
ability to produce radical scavengers. Excess hydrogen peroxide can
react with DNA to cause backbone breakage, produce mutations, and
alter and liberate bases. Hydrogen peroxide can also react with
pyrimidines to open the 5,6-double bond, which reaction inhibits
the ability of pyrimidines to hydrogen bond to complementary bases,
Hallaender et al. (1971). Such oxidative biochemical injury can
result in the loss of cellular membrane integrity, reduced enzyme
activity, changes in transport kinetics, changes in membrane lipid
content, and leakage of potassium ions, amino acids, and other
cellular material.
[0048] Antioxidants have been shown to inhibit damage associated
with active oxygen species. For example, pyruvate and other
alpha-ketoacids have been reported to react rapidly and
stoichiometrically with hydrogen peroxide to protect cells from
cytolytic effects, O'Donnell-Tormey et al., J. Exp. Med., 165, pp.
500-514 (1987).
[0049] Anti-infective Agents: Anti-infective agents are agents that
inhibit infection and include anti-viral agents, anti-fungal agents
and antibiotics.
[0050] Anti-Viral Agents: Anti-viral agents are agents that inhibit
virus and include vidarabine, acyclovir and trifluorothymidine.
[0051] Anti-Fungal Agents: Anti-fungal agents are agents that
inhibit fungal growth. Anti-fungal agents include anphoterecin B,
myconazole, terconazole, econazole, isoconazole, thioconazole,
biphonazole, clotrimazole, ketoconazole, butaconazole,
itraconazole, oxiconazole, phenticonazole, nystatin, naphthyphene,
zinoconazole, cyclopyroxolamine and fluconazole.
[0052] Antibiotics: Antibiotics are natural chemical substances of
relatively low molecular weight produced by various species of
microorganisms, such as bacteria (including Bacillus species),
actinomycetes (including Streptomyces) and fungi, that inhibit
growth of or destroy other microorganisms. Substances of similar
structure and mode of action may be synthesized chemically, or
natural compounds may be modified to produce semi-synthetic
antibiotics. These biosynthetic and semi-synthetic derivatives are
also effective as antibiotics. The major classes of antibiotics are
(1) the beta-lactams, including the penicillins, cephalosporins and
monobactams; (2) the aminoglycosides, e.g. gentamicin, tobramycin,
netilmycin, and amikacin; (3) the tetracyclines; (4) the
sulfonamides and trimethoprim; (5) the fluoroquinolones, e.g.
ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) the
macrolides, which include for example, erythromycin, azithromycin,
and clarithromycin; and (8) other antibiotics, e.g., the
polymyxins, chloramphenicol and the lincosamides.
[0053] Antibiotics accomplish their anti-bacterial effect through
several mechanisms of action which can be generally grouped as
follows: (1) agents acting on the bacterial cell wall such as
bacitracin, the cephalosporins, cycloserine, fosfomycin, the
penicillins, ristocetin, and vancomycin; (2) agents affecting the
cell membrane or exerting a detergent effect, such as colistin,
novobiocin and polymyxins; (3) agents affecting cellular mechanisms
of replication, information transfer, and protein synthesis by
their effects on ribosomes, e.g., the aminoglycosides, the
tetracyclines, chloramphenicol, clindamycin, cycloheximide,
fucidin, lincomycin, puromycin, rifampicin, other streptomycins,
and the macrolide antibiotics such as erythromycin and
oleandomycin; (4) agents affecting nucleic acid metabolism, e.g.,
the fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine,
griseofulvin, rifamycins; and (5) drugs affecting intermediary
metabolism, such as the sulfonamides, trimethoprim, and the
tuberculostatic agents isoniazid and para-aminosalicylic acid. Some
agents may have more than one primary mechanism of action,
especially at high concentrations. In addition, secondary changes
in the structure or metabolism of the bacterial cell often occur
after the primary effect of the antimicrobial drug.
[0054] Anti-Cancer Agents: Anti-cancer agents (chemotherapeutic
agents) are natural or synthetic molecules which are effective
against one or more forms of cancer. This definition includes
molecules which by their mechanism of action are cytotoxic
(anti-cancer chemotherapeutic agents), those which stimulate the
immune system (immune stimulators) and modulators of angiogenesis.
The outcome in either case is the slowing of the growth of cancer
cells.
[0055] Anti-cancer therapy include radioactive isotopes such as
.sup.32P used in the treatment of polycythemia vera and in chronic
leukemia. Radioactive phosphorus has a biological half-life of
about 8 days in humans. It emits beta rays that exert a destructive
effect on the rapidly multiplying cells. .sup.32P is usually
administered in doses of about 1 mc daily for 5 days. Either the
oral or intravenous route may be used and the doses are not greatly
different. Radioactive iodine .sup.131I, radioactive gold
.sup.198Au, and other isotopes are not as useful as .sup.32P.
Nevertheless, .sup.131I has some limited applications in metastatic
thyroid carcinoma. Other radioactive isotopes can be used with our
technology either as complexes of radioactive metal such as
.sup.51Cr, .sup.52Mn, .sup.52Mg, .sup.57Ni, .sup.55Co and .sup.56P,
.sup.55Fe, .sup.103Pd, .sup.192Ir, .sup.84Cu and .sup.67Cu or as
chelates of these metals using bifunctional chelating agents like
(BFCs),
6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-t-
etraacetic acid (BAT),
6-[p-(isothiocyanato)benzyl]-1,4,8,11-tetraazacyclo-
tetradecane-1,4,8,11-tetraacetic acid (SCN-TETA),
4-[(1,4,8,11-tetraazacyc- lotetradec-1-yl)methyl]benzoic acid
(CPTA), and 1-[(1,4,7,10,13-pentaazacy-
clopentadec-1-yl)methyl]benzoic acid (PCBA).
[0056] Numerous drugs fall into the category of chemotherapeutic
agents useful in the treatment of neoplastic disease that are
amenable to the embodiment of this application. Such agents
derivitized with this technology can include anti-metabolites such
as metotrexate (folic acid derivatives), fluoroaucil, cytarabine,
mercaptopurine, thioguanine, petostatin (pyrimidine and purine
analogs or inhibitors), a variety of natural products such as
vincristine and vinblastine (vinca alkaloid), etoposide and
teniposide, various antibiotics such as miotomycin, plicamycin,
bleomycin, doxorubicin, danorubicin, dactomycin; a variety of
biological response modifiers including interferon-alpha; a variety
of miscellaneous agents and hormonal modulators including
cisplatin, hydroxyurea, mitoxantorne, procarbozine,
aminogultethimide, prednisone, progestins, estrogens, antiestorgens
such as tamoxifen, androgenic steroids, antiadrogenic agents such
as flutamide, gonadotropin releasing hormones analogs such as
leuprolide, the matrix metalloprotease inhibitors (MMPIs) as well
as anti-cancer agents including Taxol (paclitaxel) and related
molecules collectively termed taxoids, taxines or taxanes.
[0057] Included within the definition of "taxoids" are various
modifications and attachments to the basic ring structure (taxoid
nucleus) as may be shown to be efficacious for reducing cancer cell
growth and which can be constructed by organic chemical techniques
known to those skilled in the art.
[0058] Chemotherapeutics include podophyllotoxins and their
derivatives and analogues. Another important class of
chemotherapeutics useful in this invention are camptothecins.
[0059] Another preferred class of chemotherapeutics useful in this
invention are the anthracyclines (adriamycin and daunorubicin).
[0060] Another important class of chemotherapeutics are compounds
which are drawn from the following list: Taxotere, Amonafide,
Illudin S, 6-hydroxymethylacylfulvene Bryostatin
1,26-succinylbryostatin 1, Palmitoyl Rhizoxin, DUP 941, Mitomycin
B, Mitomycin C, Penclomedine, angiogenesis inhibitor compounds,
Cisplatin hydrophobic complexes such as
2-hydrazino-4,5-dihydro-1H-imidazole with platinum chloride and
5-hydrazino-3,4-dihydro-2H-pyrrole with platinum chloride, vitamin
A, vitamin E and its derivatives, particularly tocopherol
succinate.
[0061] Other compounds useful in the invention include:
1,3-bis(2-chloroethyl)-1-nitrosurea ("carmustine" or "BCNU"),
5-fluorouracil, doxorubicin ("adriamycin"), epirubicin,
aclarubicin, Bisantrene
(bis(2-imidazolen-2-ylhydrazone)-9,10-anthracenedicarboxaldehy- de,
mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyl
dipeptide, lipopolysaccharides, vidarabine and its 2-fluoro
derivative, resveratrol, retinoic acid and retinol, carotenoids,
and tamoxifen.
[0062] Other chemotherapeutic agents useful in the application of
this invention include: Decarbazine, Lonidamine, Piroxantrone,
Anthrapyrazoles, Etoposide, Camptothecin, 9-aminocamptothecin,
9-nitrocamptothecin, camptothecin-11 ("Irinotecan"), Topotecan,
Bleomycin, the Vinca alkaloids and their analogs [Vincristine,
Vinorelbine, Vindesine, Vintripol, Vinxaltine, Ancitabine],
6-aminochrysene, and Navelbine.
[0063] Other compounds useful in the application of the invention
are mimetics of taxol, eleutherobins, sarcodictyins,
discodermolides and epothiolones.
[0064] Antineoplastic Agents--Antineoplastic agents are anti-cancer
agents such as fluoropyrimidines, pyrimidine nucleosides, purines,
platinum analogs, anthracyclines/anthracenediones,
podophyllotoxins, camptothecins, hormones and hormonal analogs,
enzymes, proteins and antibodies, vinca alkaloids, taxanes,
atihormonal agents, antifolates, antimicrotubule agents, alkylating
agents (classical and non-classical), antimetabolites, antibiotics,
topoisomerase inhibitors, antivirals, and miscellaneous cytotoxic
agents, for example hydroxyurea, mitotane, fusion toxins, PZA,
bryostatin, retinoids, butyric acid and derivatives, pentosan,
fumagillin, and others. The objective of all antineoplastic drugs
is to eliminate (cure) or to retard the growth and spread
(remission) of cancer cells. The majority of the above listed
antineoplastic agents pursue this objective by possessing primary
cytotoxic activity, effecting a direct kill on the cancer cells.
Other antineoplastic drugs stimulate the body's natural immunity to
effect cancer cell death.
[0065] Matrix metalloprotease inhibitors (MMPIs)--Also known as
matrix metalloproteinase inhibitors, MMPIs are inhibitors of the
matrix metalloproteases. The metalloproteases are a family of
enzymes containing zinc at the active site, which facilitate the
catalytic hydrolysis of various protein substrates. A subfamily of
the metalloprotease family is known as the matrix metalloproteases
(MMPs) because these enzymes are capable of degrading the major
components of articular cartilage and basement membranes. The
matrix metalloproteases include stromelysin, collagenase,
matrylisin and gelatinase, among other. The action of matrix
metalloptoreases is inhibited by MMPIs used in the preparation of
the derivatized MMPIs of the present invention. Some characterized
MMPs and their preferred substrates are illustrated in the
following table.
[0066] The nomenclature used to describe the interaction of
proteases and their substrates is widely used in the protease
literature. In this system, the binding site for a polypeptide
substrate on a protease is envisioned as a series of subsites; each
subsite interacts with one amino acid reside of the substrate. By
convention, the substrate amino acid residues are called P (for
peptide); the subsites on the protease that interact with the
substrate are called S (for subsite). The subsites are in the
catalytic or active site of the protease. The amino acid residues
on the amino-terminal side of the scissile bond (bond that is
cleaved on the substrate) are numbered P.sub.1, P.sub.2, P.sub.3,
etc., and the residues on the carboxy-terminal side of the scissile
bond are numbered P.sub.1', P.sub.2', P.sub.3', etc. The residues
can be numbered up to P.sub.6 on each side of the scissile bond.
The subsites on the protease are termed S.sub.3, S.sub.2, S.sub.1,
S.sub.1', S.sub.2', S.sub.3', etc. to complement the substrate
residues that interact with the enzyme.
[0067] Characterized MMPs and their preferred substrates.
1 MATRIX MMP PREFERRED METALLOPROTEINASE NUMBER SUBSTRATE CLASS I
Interstitial collagenase 1 Fibrillar collagens, type I, II, III
Neutrophil (PMN) 8 Fibrillar collagens, type I, II, III collagenase
Collagenase-3 13 Fibrillar collagens, type I, II, III Collagenase-4
18 CLASS II Gelatinase A (72 kDa) 2 Collagen types IV, V, gelatin
Gelatinase B (92 kDa) 9 Collagen types IV, V, gelatin
Metalloelastase 12 Elastin CLASS III Stromelysin-1 3 Laminin,
fibronectin, proteoglycans Stromelysin-2 10 Laminin, fibronectin,
proteoglycans Matrylisin (pump) 7 Laminin, fibronectin,
proteoglycans NON-CLASSIFIED Stromelysin-3 11 1-antitrypsin
Membrane-type MMP 14-17 Pro-gelatinase A
[0068] In this application, the term MMPI should be understood to
include matrix metalloprotease inhibitors as well as analogs
thereof. In addition, the term MMPI includes optical isomers and
diastereomers; as well as the racemic and resolved,
enantiomerically pure R and S stereoisomers; as well as other
mixtures of the R and S stereoisomers and pharmaceutically
acceptable salts thereof.
[0069] Oxytocin--Oxytocin is a hormone involved in the enhancement
of lactation, contraction of the uterus, and relaxation of the
pelvis prior to childbirth. Oxytocin secretion in nursing women is
stimulated by direct neural feedback obtained by stimulation of the
nipple during suckling. Its physiological effects include the
contraction of mammary gland myoepithelial cells, which induces the
ejection of milk from mammary glands, and the stimulation of
uterine smooth muscle contraction leading to childbirth. Oxytocin
causes myoepithelial cells surrounding secretory acini of mammary
glands to contract, pushing milk through ducts. In addition, it
stimulates the release of prolactin, and prolactin is trophic on
the breast and stimulates acinar formation of milk.
[0070] Cholecystokinin (CCK)--CCK is a polypeptide of 33 amino
acids originally isolated from pig small intestine that stimulates
gallbladder contraction and bile flow and increases secretion of
digestive enzymes from pancreas. It exists in multiple forms,
including CCK4 and CCK-8, with the octapeptide representing the
dominant molecular species showing the greatest activity. It
belongs to the CCK/gastrin peptide family and is distributed
centrally in the nervous system and peripherally in the
gastrointestinal system. It has many biological roles, including
stimulation of pancreatic secretion, gall bladder contraction and
intestinal mobility in the GI tract as well as the possible
mediation of satiety and painful stimuli.
[0071] Antihypertensive Agents--Antihypertensive agents are various
agents that can be used to treat hypertension, including but not
limited to enalapril, acebutolol, and doxazosin. Enarlapril is a
pro-drug that is activated to the angiotensin-converting enzyme
(ACE) inhibitor, enalaprilat. This pro-drug inhibits the conversion
of angiotensin I to angiotensin II and exerts an antihypertensive
effect by suppressing the renin-angiotensin-aldosterone system.
Acebutolol is in a class of drugs called beta-blockers, which
affect the heart and circulatory system. Acebutolol is used to
lower blood pressure, lower heart rate, and reduce angina (chest
pain). Doxazosin is a member of the alpha blocker family of drugs
used to lower blood pressure in people with hypertension. Doxazosin
is also used to treat symptoms of benign prostatic hyperplasia
(BPH). Doxazosin works by relaxing blood vessels so that blood
passes through them more easily, which helps to lower blood
pressure.
[0072] Methylprednisolone--Methylprednisolone is a synthetic
steroid that suppresses acute and chronic inflammation. In
addition, it stimulates gluconeogenesis, increases catabolism of
proteins and mobilization of free fatty acids. In addition, it
potentiates vascular smooth muscle relaxation by beta adrenergic
agonists, and may alter airway hyperactivity. It is also a potent
inhibitor of the inflammatory response.
[0073] GP-41 Peptides--GP-41 is an HIV transmembrane protein which
has been shown to be essential for the virus to fuse with and
infect healthy cells.
[0074] Anti-viral and antifusogenic peptides: Anti-viral peptides
refers to peptides that inhibit viral infection of cells, by, for
example, inhibiting cell-cell fusion or free virus infection. The
route of infection may involve membrane fusion, as occurs in the
case of enveloped viruses, or some other fusion event involving
viral and cellular structures. Peptides that inhibit viral
infection by a particular virus may be referenced with respect to
that particular virus, e.g., anti-HIV peptide, anti-RSV peptide,
etc. Antifusogenic peptides are peptides demonstrating an ability
to inhibit or reduce the level of membrane fusion events between
two or more entities, e.g., virus-cell or cell-cell, relative to
the level of membrane fusion that occurs in the absence of the
peptide.
[0075] In particular, anti-viral and antifusogenic peptides include
the DP107 and DP178 peptides and analogs thereof, as well as
peptides comprised of amino acid sequences from other (non-HIV)
viruses that correspond to the gp41 region of HIV from which DP107
and DP178 are derived, and that exhibit anti-viral or
anti-fusogenic activity. Thhese peptides can exhibit anti-viral
activity against not only HIV, but other viruses including human
respiratory syncytial virus (RSV), human parainfluenza virus (HPV),
measles virus (MeV) and simian immunodeficiency virus (SIV).
[0076] In particular, anti-HIV peptides refer to peptides that
exhibit anti-viral activity against HIV, including inhibiting CD-4+
cell infection by free virus and/or inhibiting HIV-induced syncytia
formation between infected and uninfected CD-4+ cells. Anti-SIV
peptides are peptides that exhibit anti-viral activity against SIV,
including inhibiting of infection of cells by the SIV virus and
inhibiting syncytia formation between infected and uninfected
cells. Anti-RSV peptides are peptides that exhibit anti-viral
activity against RSV, including inhibiting mucous membrane cell
infection by free RSV virus and syncytia formation between
infection and uninfected cells. Anti-HPV peptides are peptides that
exhibit anti-viral activity against HPV, including inhibiting
infection by free HPV virus and syncytia formation between infected
and uninfected cells. Anti-MeV peptides are peptides that exhibit
anti-viral activity against MeV, including inhibiting infection by
free MeV virus and syncytia formation between infected and
uninfected cells.
[0077] Blood Brain Barrier (BBB) Peptides--The "blood-brain
barrier" is a layer of cells that controls which substances may
penetrate from the general circulation into the brain. BBB proteins
can traverse this barrier through protein transduction. Small
sections of these proteins (10-16 residues long), i.e. BBB
peptides, are responsible for this transduction.
[0078] RGD Peptides: The RGD peptide for conjugation to tissues or
fixed endogenous proteins in accordance with the present invention
includes a sequence of amino acids, preferably naturally occurring
L-amino acids and glycine, having the following formula:
R.sub.1-Arg-Gly-Asp-R.sub.2
[0079] In this formula, R.sub.1 and R.sub.2 represent an amino acid
or a sequence of more than one amino acid or a derivatized or
chemically modified amino acid or more than one derivatized or
chemically modified amino acids.
[0080] Insulinotropic Peptides: Insulinotropic peptides (ITPs) are
peptides with insulinotropic activity. Insulinotropic peptides
stimulate, or cause the stimulation of, the synthesis or expression
of the hormone insulin. Such peptides include precursors,
analogues, fragments of peptides such as Glucagon-like peptide,
exendin 3 and exendin 4 and other peptides with insulinotropic
activity.
[0081] Glucagon-Like Peptide: Glucagon-Like Peptide (GLP) and GLP
derivatives are intestinal hormones which generally simulate
insulin secretion during hyperglycemia, suppresses glucagon
secretion, stimulates (pro) insulin biosynthesis and decelerates
gastric emptying and acid secretion. Some GLPs and GLP derivatives
promote glucose uptake by cells but do not simulate insulin
expression as disclosed in U.S. Pat. No. 5,574,008 which is hereby
incorporated by reference.
[0082] Pulmonary Condition: A pulmonary condition is a disease
which affects lung function. Such conditions may result from a
defect in a gene or genes associated with lung function (e.g.,
cystic fibrosis), asthma, allergies, an immune or autoimmune
disorder, a microbial infection (e.g. bacterial, viral, fungal or
parasitic infection), or a mechanical injury to the lungs.
[0083] Exemplary pulmonary conditions contemplated by the subject
invention include cystic fibrosis, asthmatic bronchitis,
tuberculosis, bronchitis, bronchiectasis, laryngotracheobronchitis,
bronchiolitis, emphysema, bronchial pneumonia, allergic
bronchopneumonia, viral pneumonia, pertussis, diphtheria, spasmodic
croup, pulmonary phthisis, encephalitis with retained secretions,
and pulmonary edema. Other pulmonary conditions, such as those
which develop as a result of injury or surgery (e.g., after
tracheotomy), as well as those associated with insufficient
surfactant secretion in the lungs of premature infants, are also
contemplated by the subject invention. Pulmonary conditions
amenable to treatment by the subject method may also develop as a
result of activity associated with inhalation of particulate matter
e.g. smoking, exposure to construction areas or other high dust
areas, occupational hazards associated with inhalation of
particulates, exposure to environmental particulates (e.g. smog,
pollen, asbestos, siliconis), pulmonary delivery of pharmaceutical
agents (e.g. bronchodilators) or inhalation of cocaine.
[0084] Other pulmonary conditions include diffuse parenchymal lung
disease from infectious cases, such as cytomegaloviral pneumonia or
miliary tuberculosis, drug-induced lung disease (after
administration of penicillin, nitrofurantoin), neoplastic lung
disease having lymphangitic spread pattern or bronchoalveolar cell
carcinoma, granulomatous disease (infectious or noninfectious),
hypersensitivity pneumonitis, histoplasmosis, tuberculosis,
idiophatic pulmonary fibrosis (aka cryptogenic fibrosing
alveolitis), hereditary pulmonary disorders, such as alveolar
microlithiasis and bronchiectasis, eosinophilic granuloma,
lympphangioleimyomatosis, and plumonary alveolar proteinosis
disorder.
[0085] Symptoms of a Pulmonary Condition: Symptoms of a pulmonary
condition are symptoms associated with any of the pulmonary
conditions described above. The classic symptoms associated with
such pulmonary conditions may include coughing, exertional dyspnea,
wheezing, chest pain and purulent sputum production. Other
components of the syndrome which may accompany a pulmonary
condition include hypoxia, CO.sub.2 narcosis, hyperventilation,
decreased expiration volume, and decreased lung capacity.
[0086] Pulmonary Fluid: Pulmonary fluid is the fluid which bathes
the apical surface of the lung epithelium, particularly the
alveolar epithelium and contains fixed and mobile pulmonary fluid
components.
[0087] Pulmonary Delivery Agent: Pulmonary delivery agents are
agents that may be delivered to the lungs. Such agents include
therapeutic agents.
[0088] Fixed Pulmonary Components: Fixed pulmonary components are
non-mobile pulmonary components and include tissues, membrane
receptors, interstitial proteins, fibrin proteins, collagens,
platelets, endothelial cells, epithelial cells and their associated
membrane and membraneous receptors, somatic body cells, skeletal
and smooth muscle cells, neuronal components, osteocytes and
osteoclasts.
[0089] Mobile Pulmonary Components: Mobile pulmonary components are
pulmonary components that do not have a fixed situs for any
extended period of time, generally not exceeding 5, more usually
one minute. Mobile pulmonary components are components of the
pulmonary or lung fluid and include such soluble proteins such as
immunoglobulins, serum albumin, ferritin, transferrin and the
like.
[0090] Blood Components: Blood components may be either fixed or
mobile. Fixed blood components are non-mobile blood components and
include tissues, membrane receptors, interstitial proteins, fibrin
proteins, collagens, platelets, endothelial cells, epithelial cells
and their associated membrane and membraneous receptors, somatic
body cells, skeletal and smooth muscle cells, neuronal components,
osteocytes and osteoclasts and all body tissues especially those
associated with the circulatory and lymphatic systems. Mobile blood
components are blood components that do not have a fixed situs for
any extended period of time, generally not exceeding 5, more
usually one minute. These blood components are not
membrane-associated and are present in the blood for extended
periods of time and are present in a minimum concentration of at
least 0.1 .mu.g/ml. Mobile blood components include serum albumin,
transferrin, ferritin and immunoglobulins such as IgM and IgG. The
half-life of mobile blood components is at least about 12
hours.
[0091] Inhaler Device: An inhaler device is any device useful in
the administration of the inhalable medicament of the invention.
Examples of inhaler devices include nebulizers, metered dose
inhalers, dry powder inhalers, intermittent positive pressure
breathing apparatuses, humidifiers, bubble environments, oxygen
chambers, oxygen masks and artificial respirators.
[0092] Reactive Groups: Reactive groups are chemical groups capable
of forming a covalent bond. Such reactive groups are coupled or
bonded to a therapeutic or diagnositic agent. Reactive groups will
generally be stable in an aqueous environment and will usually be
carboxy, phosphoryl, or convenient acyl group, either as an ester
or a mixed anhydride, an imidate or maleimide, thereby capable of
forming a covalent bond with functionalities such as an amino
group, a hydroxy or a thiol at the target site on pulmonary
components. For the most part, the esters will involve phenolic
compounds, or be thiol esters, alkyl esters, phosphate esters, or
the like. Prefereably, the reactive group will be a maleimide
group.
[0093] Functionalities: Functionalities are groups on pulmonary
components to which reactive groups on modified therapeutic agents
react to form covalent bonds. Functionalities include hydroxyl
groups for bonding to ester reactive entities; thiol groups for
bonding to maleimides, imidates and thioester groups; amino groups
for bonding to carboxy, phosphoryl or acyl groups and carboxyl
groups for bonding to amino groups.
[0094] IC.sub.50: Concentration of an enzyme inhibitor at which 50%
of the enzymatic activity is inhibited.
[0095] Protective Groups: Protective groups are chemical moieties
utilized to protect reactive entities from reacting with other
functionalities. Various protective groups are disclosed in U.S.
Pat. No. 5,493,007 which is hereby incorporated by reference. Such
protective groups include acetyl, fluorenylmethyloxycarbonyl
(FMOC), t-butyloxy carbonyl (BOC), benzyloxycarbonyl (CBZ), and the
like. For small organic molecules all protecting groups like
tetrahydropyranyl (THP), all silyl derivatives, acetals,
thioacetals and the like.
[0096] Linking Groups: Linking groups are chemical moieties that
link or connect reactive groups to therapeutic agents. Linking
groups may comprise one or more alkyl moeities, alkoxy moeity,
alkenyl moeity, alkynyl moeity or amino moeity substituted by alkyl
moeities, cycloalkyl moeity, polycyclic moeity, aryl moeity,
polyaryl moeities, substituted aryl moeities, heterocyclic
moeities, and substituted heterocyclic moeities. Linking groups may
also comprise poly ethoxy amino acids, such as AEA ((2-amino)
ethoxy acetic acid) or a preferred linking group AEEA ([2-(2-amino)
ethoxy)] ethoxy acetic acid.
[0097] Sensitive functional groups--A sensitive functional group is
a group of atoms that represents a potential reaction site on a
therapeutic agent. If present, a sensitive functional group may be
chosen as the attachment point for the linking group-reactive group
modification. Sensitive functional groups include but are not
limited to carboxyl, amino, thiol, and hydroxyl groups.
[0098] Modified Therapeutic and Diagnostic Agents--Modified
therapeutic and diagnostic agents are agents that have been
modified by attaching a reactive group. The reactive group may be
attached to the therapeutic agent either via a linking group, or
optionally without using a linking group. Modified therapeutic and
diagnostic agents may be administered in vivo such that conjugation
with blood or pulmonary components occurs in vivo, or they may be
first conjugated to blood or pulmonary components in vitro and the
resulting conjugated therapeutic agent (as defined below)
administered in vivo.
[0099] Conjugated Therapeutic and Diagnostic Agents--Conjugated
therapeutic and diagnostic agents are modified therapeutic and
diagnostic agents that have been conjugated to a blood or pulmonary
component via a covalent bond formed between the reactive group of
the modified therapeutic agent and the functionalities of the
pulmonary component, with or without a linking group. As used
throughout this application, the term "conjugated therapeutic
agent" can be made more specific to refer to particular conjugated
therapeutic agents, for example "conjugated antihistamine."
[0100] Taking into account these definitions, the present invention
is directed to modified therapeutic and diagnostic agents capable
of reacting with available functionalities on pulmonary or blood
components via covalent linkages. The invention is also directed to
methods of making the modified agents and their use. The modified
therapeutic agents of the present invention are capable of reacting
in vivo to form conjugates with pulmonary and/or blood components,
such as pulmonary or blood proteins, thereby extending the
half-life and improving bioavailability of the therapeutic agent
without deterioiusly altering the agent's therapeutic effect. In
preferred embodiments of this invention, the functionality on the
protein will be a thiol group and the reactive group on the
modified therapeutic agent will be a maleimido-containing group
such as gamma-maleimide-butyralamide (GMBA), maleimidopropionic
acid (MPA) or maleimide-benzoyl-succinimide (MBS).
[0101] The invention in one aspect contemplates delivery of the
modified agents to the blood of a host for conjugation to blood
components, including blood proteins. While pulmonary
administration is further described as such a route of
administration, it will be understood that the invention is not
limited to such routes of adminstration, and also contemplated
administration of the modified agents to a patient's blood stream
using other methods, including parenterally, such as intravenously
(IV), intraarterially (IA), intramuscularly (IM), subcutaneously
(SC) and the like.
[0102] For pulmonary delivery, a wide variety of devices and
carrier molecules have been utilized to enhance pulmonary drug
delivery and can be used with the modified agents of the present
invention. These devices and methods include metered dosing,
carriers such as liposomes (Meisner et al, 1989) actide/glycolide
copolymer (PLGA) nanospheres (Niwa et al., 1995), albumin
microspheres (Feinstein et al., 1990), and other physical art forms
to created aerosols or nanoparticulates.
[0103] A new type of inhalation aerosol, characterized by particles
of small mass density and large size, has permitted the highly
efficient delivery of inhaled therapeutics (e.g. insulin,
testosterone) into the systemic circulation. Particles with mass
densities less than 0.4 gram per cubic centimeter and mean
diameters exceeding 5 micrometers have been reported to avoid the
lungs' natural clearance mechanisms providing higher
bioavailability than that of conventional inhaled therapeutic
particles. (Edwards et al., 1997). For most of these therapies,
aerosols are designed to comprise small spherical droplets or
particles of mass density near 1 g/cm.sup.3 and mean geometric
diameter between approximately 1 and 3 micron, suitable for
particle penetration into the airways or lung periphery.
[0104] Studies performed primarily with liquid aerosols have shown
that these characteristics of inhaled aerosols lead to optimal
therapeutic effect, both for local and systemic therapeutic
delivery. Inefficient drug delivery can still arise, owing to
excessive particle aggregation in an inhaler, deposition in the
mouth and throat, and overly rapid particle removal from the lungs
by mucocilliary or phagocytic clearance mechanisms.
[0105] To address these problems, particle surface chemistry and
surface roughness are traditionally manipulated. Recent data
indicate that major improvements in aerosol particle performance
may also be achieved by lowering particle mass density and
increasing particle size, since large, porous particles display
less tendency to agglomerate than (conventional) small and
nonporous particles. Also, large, porous particles inhaled into the
lungs can potentially release therapeutic substances for long
periods of time by escaping phagocytic clearance from the lung
periphery, thus enabling therapeutic action for periods ranging
from hours to many days. (Edwards et al., 1998)
[0106] It has been previously reported that specific transport
receptors for albumin (GP60, albondin) exist in the endothelium
that function as unique albumin carriers (U.S. Pat. No. 5,254,342).
These transcytosis proteins facilitate the movement of albumin and
albumin carriers across the lining of the airway and result in
extensive plasma levels of these proteins or protein carriers. As
further described, modified agents according to the present
invention can be prepared that react with albumin, and upon
pulmonary delivery, the resulting conjugates can pass to the
bloodstream via such carriers.
[0107] Pulmonary drug delivery is also advantageous for local
treatment of the lung in that it promotes an increase in drug
retention-time in the lung and more importantly, a reduction in
extrapulmonary side-effects, invariably resulting in enhanced
therapeutic efficacies. (Shek, 1994). A key advantage of pulmonary
delivery includes reduced systemic toxicity and increased drug
concentration at the site of action (e.g. infection or inflammation
site. (Stout and Derendorf, 1987).
[0108] The use of in vivo or ex vivo bioconjugation associated with
pulmonary drug delivery includes the following non-limited
benefits. Retention of the drug at the site of placement is
enhanced due to covalently attachment of the drug to the airway
site. Additionally, prolonged duration of action of the drug is
made possible, both in the lung by in situ attachment to soluble
proteins for localized intrapulmonary activity, as well as systemic
absorption and conjugation to blood proteins.
[0109] Drug stability is improved, both locally and systemically,
as conjugation affords protection against enzymatic degradation
that occurs in the pulmonary mucosal fluid or in the plasma. Also,
in the deep lungs, alveolar macrophages can rapidly deposited
particles; the reactivity of the modified agents of the invention
with epithelial cells will allow for localized retention of the
agent.
[0110] Localized delivery to the pulmonary tissues also reduces
toxicity and reduces systemic exposure as there is no first-pass
liver effect. Systemic delivery also exhibits reduced extravascular
side effects through conjugation to, for example, albumin, due to,
for example, the limitation of hepatic, central nervous system
(CNS) or renal toxicity due to the limited clearance of albumin
into these organs.
[0111] Pulmonary delivery of the modified agents of the invention
also provides advantages of improved patient compliance due to
prolonged duration of action of the modified agents. In turn, cost
benefits can be achieved through, e.g., reduced costs of goods per
course of therapy due to prolonged duration of action, and
outpatient use of medications that would otherwise have limited use
or complicated dosing titration schedules. Pulmonary delivery can
also reduce difficulties associated with oral dosing, including low
solubility, interactions with food, and low bioavailability.
[0112] A further advantage of pulmonary delivery of modified agents
of the present invention is the ability to deliver
systemically-large macromolecule drugs, such as insulin, growth
hormones, beta-interferon, calcitonin, and others that, due to
their large size and instability, are typically delivered by
injection. The present invention provides an alternative and more
convenient route of adminstering these drugs. In addition, many
drugs and therapeutic peptides are more stable in solid, dry form,
rather than solubilized. Dry pulmonary formulations of such drugs
modified according to the present invention provide for a more
stable form of the drug, as well as the convenience of pulmonary
administration.
[0113] 1. Therapeutic Agents
[0114] A wide variety of therapeutic agents are contemplated for
use in the present invention, including peptide therapeutics and
small organic molecules, provided they can be modified as
described.
[0115] In addition to therapeutic agents discussed above, the
following therapeutic agents are within the scope of this
invention.
[0116] Sympathomimetic compounds mimic the action of endogenous
catecholamines (adrenalinelike neurotransmitters) at peripheral
sympathetic neurons in addition to CNS effects. Adrenaline systems
control important body functions like blood pressure regulation and
wakefulness. A vast panoply of compounds has been developed that
allows one to selectively tweak these systems. These include
agonists, used as decongestants and antiasthmatics and antagonists,
used as antihypertensives). 123
[0117] Selective agents include alpha agonists such as
phenylephrine (Neo-Synephrine) and more lipophilic agents such as
naphazoline. Clonidine, an antihypertensive, is sometimes used in
ethanol withdrawal, and has been tried in cigarette smoking
cessation. It probably has a net antagonist effect through
autoreceptors, i.e. presynaptic receptors detect an excess of
adrenergic agonist and decrease norepinephrine release. Ibopamine,
a cardiotonic, has diuretic and dopamine agonist activity.
[0118] The beta agonists are popular antiasthma medications.
Isoproterenol and dobutamine are fairly selective for the beta1
receptor; the latter is used as a cardiotonic. 45
[0119] Alpha blockers such as phentolamine and prazosin are also
used as antihypertensives. Yohimbine, a selective alpha2 blocker,
is a popular aphrodisiac for males, purported to prolong or
intensify erection. Alpha2 receptors are inhibitory, so that
inhibiting them produces a stimulating effect. Alpha2 and alpha3
receptors are alsopresent in fat storage sites; antagonizing them
may provide a way to promote fat catabolism without resorting to
central stimulants.
[0120] Beta antagonists include propranolol, which has been used
for some time to calm the nerves in event-specific anxiety (stage
fright) as well as its more traditional role in hypertension.
Acebutolol is a cardioselective beta blocker used in angina.
Antidepressants
[0121] Antidepressants work by altering the concentration of
catecholamines and/or serotonin in CNS neurons emanating from the
limbic system into the frontal lobe. Raising levels of
catecholamines--excitator- y, adrenalin-type
neurotransmitters--causes stimulation. Elevating serotonin, an
inhibitory neurotransmitter, produces a calming action, and results
in subsequent upregulation of catecholamine systems as a mechanism
of habituation. 6789
[0122] The selective serotonin reuptake inhibitors (SSRIs) inhibit
reuptake of serotonin without significantly affecting adrenergic
systems. The adverse effect profiles are much less than the
tricyclics, since muscarinic, histaminic and adrenergic binding is
much reduced.
[0123] The effect of these serotonin agents on mood has led to more
complex theories of how antidepressants work. According to one
hypothesis, the noradrenergic systems (dopamine, noradrenalin)
which underlie the serotonin systems respond to an increase in the
inhibitory serotonin function by upregulating, or increasing the
number of receptors on the individual post-synaptic neural surface.
This increase in adrenaline-type neural function might then account
for the antidepressant activity which is delayed from the onset of
serotonin reuptake inhibition by several weeks. Another possibility
is that the serotonin receptors take a while to register the excess
serotonin and resopond with similar mechanisms. In addition, recent
evidence suggests interaction with DNA through transcriptases,
increasing production of neurotransmitter by producing more
synthase enzymes, for example.
Antihistamines, Antiasthmatics & Histamine Agonists
[0124] Antihistamines are compounds that block histamine from
activating histamine receptors. Since histamine functions to
mediate allergic response, blocking histamine at H1 receptors stops
the body's characteristic responses, i.e. inflammation and
vasoconstriction. H2 receptors in the stomach regulate the release
of gastric acid; hence the new class of H2-blockers such as Zantac
and Tagamet stop the secretion of acid by selectively blocking
these receptors without affecting the H1 receptors responsible for
allergic response.
[0125] Histamine is concentrated in mast cells, cells whose
function is essentially to release histamine and immunoglobins when
tissue damage occurs. Receptors on the cell surface trigger lysing
(breaking open) of the cell, releasing these allergic mediators.
Mast cells are especially numerous in parts of the body that are
injured often, such as the fingers and toes, or which enjoy
frequent contact with the environment, such as the mucosa of the
lips, nose, etc.
[0126] Histamine is also a neurotransmitter in the CNS and a
typical problem with some antihistamines is drowsiness. The effort
has been to produce compounds that do not enter the brain very
well. 10111213
[0127] Fenethazine represents a tricyclic antihistamine very
similar to Thorazine, a strong antipsychotic dopamine blocker.
Cyproheptadine (Periactin), which also acts at serotonin receptors,
resembles the now-popular Claritin. Periactin has been prescribed
psychiatrically for anxiety. Benadryl is probably the most familiar
of the class; it has strong sedating qualities. Hydroxyzine also
has been prescribed as a sedative. Dimenhydrinate has been marketed
as an anti-nausea medication as Dramamine. Cetirizine (Zyrtec) is a
metabolic product of hydroxyzine; since hydroxyzine is available as
a generic, it is cheaper than the newer drug and just as
effective.
[0128] Azelastine (Astelin) has a novel structure but also acts on
both H1 and H2 receptors. Cromolyn works by a distinct mechanism;
it prevents release of histamine following immunoglobin binding on
mast cells (prevents mast degranulation). 14
[0129] H2-selective antihistamines have become popular as
treatments for excess stomach acid. These histamine blockers are
very similar structurally and mechanistically. All four have about
the same bioavailability, half lives, and antagonist activity.
15
[0130] Omeprazole (Prilosec) and lansoprazole (Prevacid) belong to
a class of antisecretory compounds, the substituted benzimidazoles,
that do not exhibit anticholinergic or histamine H2-receptor
antagonist properties, but that suppress gastric acid secretion by
specific inhibition of the (H+,K+)-ATPase enzyme system at the
secretory surface of the gastric parietal cell. These proton pump
inhibitors have emerged as a therapeutic alternative to histamine
antagonists for the treatment of gastric disorders, especially acid
reflux disease ("heartburn"). 16
[0131] For any receptor system it is possible to find drugs that
act as mimetics or agonists. With histamine receptors these drugs
do not find much clinical usefulness, but it is gratifying--to
some!--to enumerate them nevertheless. 2-methylhistamine acts as a
selective agonist at H1 receptors; 4-methylhistamine is relatively
selective at H2. Impromidine antagonizes H2 receptors but functions
as an agonist at H3.
Antiasthmatics
[0132] Asthma is essentially an accentuated allergic response to
the environment, i.e. an autoimmune disorder. The process of
allergic response is complex, which gives one many points at which
to attack the problem. First, immunoglobin and antigens bind to the
surface of mast cells. Mast cells then release histamine,
leukotrienes, peptides, which bind to tissue receptor sites and
modify intracellular chemical processes governing various functions
such as blood pressure regulation, smooth muscle tone, fluid
disposition, etc. Compounds that inhibit any of these steps can be
used to treat asthma and allergy, beginning with the antihistamines
listed above. 1718
[0133] Perhaps the oldest method for reducing asthma symptoms is
bronchodilation by methylxanthine compounds like caffeine and
theophylline. These are currently outmoded by other compounds that
perform the same function more selectively like the
beta2-adrenergic agonists (beta agonists). Methylxanthines act by
inhibiting the enzyme which effects cAMP degradation
(phosphodiesterase) and by antagonizing adenosine, which causes
bronchoconstriction. The beta agonists, like metaproterenol,
isoetharine, isoproterenol, terbutaline, and albuterol, mimic
adrenaline at a subset of adrenaline receptor which controls the
tone of smooth muscle like that of the bronchi. Another older class
of drugs, antimuscarinics, exemplified by atropine, has enjoyed
some historical use.
[0134] Zafirlukast (Accolate) represents a line of attack on the
leukotriene compounds released along with histamine from mast
cells. Leukotrienes are mediators like histamine which bind to
receptors on tissue cells to signal an allergic reaction. Blocking
them blocks the signal to the (bronchial) cells and thus the
undesirable response.
[0135] The final mechanism one can attack is the slow inflammatory
response to binding by leukotrienes and/or histamine. The body
regulates inflammation with glucocorticoid steroids, and synthetic
compounds such as fluticasone (Flonase) and beclomethasone
(Beclonase) are effective mimetics.
Antihyperlipidemics
[0136] Antihyperlipidemics are relaively new drugs which lower
blood cholesterol levels and help to prevent atherosclerosis by
inhibiting the formation of plaque on arterial and vascular
linings. The formation of this plaque is dependent on the
proportion of various types of blood-fats, particularly on the
ratio of high-density lipoproteins (HDLs) to low-density
lipoproteins (LDLs). This proportion is in turn influenced by
genetics and by the amount of certain substances in the diet,
particularly cholesterol and saturated fat. Cholesterol is
essential in the formation of VLDLs, large lipoproteins produced by
the liver; on catabolysis by lipoprotein lipase, VLDLs produce the
smaller LDLs, which are the so-called "bad cholesterol," HDLs being
termed "good cholesterol" in the common parlance. Because the
production of these lipoproteins is complex, several points can be
targeted for action by various drugs. 19
[0137] The most commonly used antihyperlipidemics are simvastatin
analogs, especially simvastatin (Zocor) itself. These drugs, known
as HMG-COA reductase inhibitors, decrease production of cholesterol
by inhibiting the first step in sterol synthesis which involves
production of mevalonate ion from CoA-S-mevalonate. Decreased
cholesterol formation results in a reduction in VLDLs and hence
LDLs. Pravastatin is the active metabolite of mevastatin;
lovastatin and simvastatin are inactive prodrugs that work through
their hydroxyl derivatives, obtained by similar ring-opening.
[0138] These drugs also stimulate receptor-mediated clearance of
LDLs. LDL receptors undergo upregulation (increase in receptor
density) and VLDL catabolysis is increased.
[0139] Recent studies suggest a reduction in osteoporosis as a
result of treatment with statin drugs. 20
[0140] Other drugs like clofibrate and gemfibrozil increase the
activity of lipoprotein lipase, reducing the level of VLDLs. This
enzyme is an extracellular species present in the blood and gut.
Effects on other lipase enzymes may account for the nausea common
to this drug. Cholesterol production in the liver is reduced,
probably as a secondary effect due to lower VLDL levels, while
fecal cholesterol excretion is enhanced. Clofibrate is fairly toxic
and may be carcinogenic.
Antihypertensives
[0141] The body controls blood pressure by a complex feedback
mechanism between baroreceptors and effector nerves, primarily
adrenergic in nature. This system is modulated by a peptide systems
(angiotensin/renin).
[0142] Pharmacologic control of blood pressure acts through four
basic mechanisms. Sympathoplegics reduce peripheral vascular
resistance, inhibit cardiac function and increase venous pooling by
a number of mechanisms involving adrenergic nerves.
[0143] Direct-acting vasodilators decrease blood pressure by
increasing blood volume. Veins and arteries are forms of smooth
muscle, and relaxing the muscle results in larger volume and lower
pressure. Angiotensin antagonists work on peptide systems with
effects on smooth muscle. Finally, diuretics decrease sodium
content, decreasing blood volume and thus blood pressure. Because
the body responds to exogenous agents by homeostatic regulation,
concomitant use of several agents working on different mechanisms
is frequently used, rather than simply increasing the dose of a
single med. 212223
[0144] Sympathoplegics or sympatholytics antagonize the function of
adrenalin compounds. Beta blockers block beta-1 receptors in hart
muscle, decreasing cardiac output. The include propranolol
(Inderal), metoprolol (Lopressor), labetolol (Normodyne), nadolol
(Corgard), atenolol (Tenormin), and acebutolol (Sectrol). Some
adrenoceptor-activating compounds have a hypotensive effect by
acting centrally at alpha receptors. These include clonidine
(Catapres), guanfacine (Tenex), guanabenz (Wytensin), and
methyldopa. These probably work by activating presynaptic
autoreceptors, decreasing norepinephrine (agonist) release.
[0145] Compounds that block alpha-1 receptors peripherally, in
blood vessels, include prazosin (Minipress) and terazosin
(Hytrin).
[0146] Reserpine (Serpasil) blocks amine uptake, while guanethidine
(Ismelin) and guanadrel (Hylorel) block sympathetic nerve
terminals.
[0147] Propranolol, which was formerly the most prescribed drug of
the class, leads to accumulation of bradykinin, which contributes
to the antihypertensive effect. 24
[0148] Some anticholinergic drugs (ganglion blockers) are effective
as hypotensives, but they are less popular because of side effects.
These include trimethapan (Arfonad) and mecamylamine (Inversine).
25
[0149] Diazoxide acts by opening potassium channels and relaxing
smooth venous and arterial muscle. Despite structural similarities,
it has no diuretic properties. It has a relatively long half-life
of about 24 hours, and is often used parenterally (by injection) in
emergencies. The drug also inhibits insulin release and is used in
diabetes. Minoxidil is mainly used orally, also opening potassium
channels.
[0150] Hydralazine and minoxidil dilate arterioles but not veins.
Nitroprusside (as the sodium salt) dilates both, by a mechanism
involving activation of guanylyl cyclase, resulting in formation of
cGMP and relaxation of smooth muscle. 26
[0151] The calcium channel blockers are a popular means to control
hypertension. Smooth muscle contraction depends on calcium influx
to control muscle tone. The reaction path is complex, involving the
peptides calmodulin and myosin light chain kinase (MLCK). When the
latter enzyme is activated it phosphorylates myosin which acts with
actin to contract muscle. Blocking the channel statistically
prevents calcium ion influx and decreases tension in blood vessel
smooth muscle. Skeletal muscles rely on intracellular calcium ion,
and are not affected by these drugs. Cardiac muscle is highly
dependent on calcium channel action.
[0152] Calcium channels also exist at presynaptic nerve terminals
in adrenergic neurons. These are voltage-dependent ion channels
embedded in nerve membranes at the ends of adrenergic neurons. When
a voltage pulse arrives at the end of a neuron (propagated by
sequential firing of sodium channels), the calcium channels detect
the change in voltage and allow influx of Ca.sup.++ ions. This
triggers binding of vesicles and release of vesicular adrenalin,
noradrenalin or dopamine, along with cotransmitters such as
peptides, ATP, etc. One of the acid tests as to whether a substance
is a neurotransmitter is whether its release is
calcium-dependent.
[0153] Verapamil is the oldest and prototypical calcium channel
blocker. It is highly bound to blood plasma proteins and suffers
about an 80% hepatic first-pass elimination on oral administration.
This means that most of the drug absorbed through the intestine is
removed by the kidney before reaching the target tissues (heart and
major blood vessels). Nifedipine is significantly less active at
cardiac sites than diltiazem or verapamil, and is also highly
plasma-bound. Diltiazem is much less plasma bound. Despite plasma
binding, all three drugs have fairly short half-lives (3-6
hours).
[0154] Ion channel blockers generally act by lodging in an open
channel and blocking it. Similarities in ion channels mean that
some sodium channel blockade occurs with calcium channel blockers.
This is more of a problem with verapamil than the other drugs. In
addition to treatment of hypertension, these drugs are used to
treat angina, migraine, and atherosclerosis. 27
[0155] Diuretics reduce blood volume by causing excretion of water
through the kidney. This reduces blood pressure very effectively.
The drugs used for this purpose are typically thiazides; a main
problem is potassium depletion. Potassium-sparing diuretics have
also been developed. 28
[0156] Recent studies have shown the ACE inhibitors to be extremely
safe drugs. ACE is an enzyme which converts angiotensin I to
angiotensin II, the active form. Angiotensin receptors modulate the
tension of smooth muscle, including venous and arterial tissue.
Inhibiting the enzyme decreases the amount of active peptide extant
in body tissues.
Antipsychotics, Lithium, Mood Stabilizers & Dopamine
Agonists
[0157] Antipsychotics are used to calm mania or racing thoughts, to
control aggression, or to block spurious thoughts in schizophrenia,
including auditory hallucination (hearing voices). They exert their
tranquilizing effects by blocking the excitatory neurotransmitter
dopamine at post-synaptic terminals. Dopamine neurons are abundant
in the limbic system and its projections into the cerebrum,
especially the originating in the substantia nigra. Blocking of
adrenergic, histaminic and serotonergic receptors also contribute
to the CNS effects of these drugs.
[0158] Although they are potent psychotropics, these drugs are not
commonly abused, since they inhibit the brain's pleasure pathways
which emanate from the limbic system into the frontal lobe. 29
[0159] Tricyclic compounds like Thorazine, known as phenothiazines,
are the oldest compounds, and are the least selective, blocking
several subtypes of dopamine receptors. Thorazine was discovered by
accident while seeking better antihistaminic agents. It has shown
efficacy in blocking the effects of LSD, confirming the dopamine
agonist activity of that drug. 3031
[0160] Newer compounds of the haloperidol class (bytyrophenones),
first synthesized in the late 1950s, are more selective for D2
subreceptors. 32
[0161] Clebopride and sulpiride analogs represent another
structural class of antipsychotics with similar actions. The
binding profile of all these groups (indeed, of each compound) will
be slightly different. 3334
[0162] The clozapine analogs represent another structure type with
a different neurological profile. They are not as selective for D2
as the haldol class, but may be more effective at controlling some
types of psychosis. These drugs show significant 5-HT2 receptor
blockade and may be more selective for limbic dopamine systems (as
opposed to those involved in motor control), reducing the
extrapyramidal syndrome (EPS) and dyskinesias common to
antipsychotic meds. The newer agents, such as Zyprexa and Seroquel,
seem not to impart the agranulocytosis common with clozapine.
[0163] Clozapine is extensively metabolized and some of its
metabolites show anti-AIDS activity.
[0164] Other novel structural classes which function as
neuroleptics are represented by butaclamol (a pentacyclic) and
tetrabenazine. The latter is a dopamine depleter, a drug which can
chemically induce depression. 3536
[0165] The use of anticonvulsant medications for psychotropic
purposes has recently grown, primarily to prophylact against manic
and/or panic syndromes. Phenytoin and carbamazepine probably work
by affecting ion-gating systems (particularly sodium channels) in
excitable membranes. Phenytoin structurally resembles the
barbiturates while carbamazepine has a tricyclic structure like the
tricyclic antidepressants.
[0166] Gabapentine and valproic acid probably work on GABA systems.
The latter inhibits the enzyme responsible for degrading GABA at
high concentrations, but probably works by other mechanisms at
lower, therapeutic levels. Lamotrigine is another antiepileptic
used as a mood stabilizer. It reduces release of glutamate, an
excitatory amino acid. Topamax, a fructose derivative, enhances
GABA systems while blocking glutamate.
[0167] Propanolol is a beta-adrenergic blocker prescribed for
performance phobia or stage fright. Clonidine is an alpha agonist
sometimes used to calm peripheral tremor as in alcohol withdrawal.
Verapamil, a calcium-channel blocker, is also used for this
purpose. 37
[0168] Pergolide and naxagolide are dopamine agonists employed
against Parkinsonism, which results from decreasing dopamine
function in the CNS with aging. Apomorphine is a selective D2
agonist, while ibopamine serves agonist function at both dopamine
and adrenergic receptors.
Cholinergic Drugs
[0169] Acetylcholine neurons convey sensory information to the
brain and control muscular tension, including peristalsis and motor
control. Cholinergic neurons are dominant in inhibitory activity
inherent to so-called parasympathetic neurons whic comlpement
dopamine/norepinephrine based neurons in parallel sympathtic
structures. Two cholinergic receptor subtypes have been identified
by selective agonists: nicotinic and muscarinic. At least two
subtypes of muscarinic receptors (M1 and M2) have been
identified.
[0170] In addition to direct agonists, selective antagonists,
enzyme inhibitors, and antidotes to enzyme inhibitors have been
developed. Cholinoceptors also serve as heteroreceptors,
presynaptically governing the release of norepinephrine and other
neurotransmitters. 38
[0171] Nicotine is a selective agonist at nicotinic receptors: it
defines this subset of cholinergic receptors. Muscarine defines the
other subset, with further distinctions of M1 and M2 (at least)
existing. Muscarine is produced in trace amounts in the fly agaric
mushroom. Other species of fungus produce greater amounts. Fly
agaric also contains muscarinic antagonists (atropine) and GABA
agonists (muscimol). Atropine used to be applied as an antidote to
poisoning by muscarine in this fungus, before the role of muscimol
was elucidated.
[0172] The N-hydroxymethyl amide of nicotinic acid is also active
as an agonist at nicotinic cholinoceptors. Carbachol is used
opthalmically as a miotic, i.e. to dilate the pupils. It is also
used in large animals, mainly in atonic conditions of the gut,
since its formal positive charge prevents it from entering the
brain and limits its absorption in the gut. In addition to receptor
action, it probably promotes acetylcholine release. Lachesine is a
selective muscarinic agonist.
[0173] Guanidine exists as the guanidium ion at physiologic pH; it
is used as a pro-cholinergic, antiviral, antifungal, antipyretic
and muscle stimulant. Bethanechol activates M1 and M2 subreceptors,
releases IP3 (inositol triphosphate), and activates guanylyl
cyclase. Again, as a quaternary, positively charged species, it is
used mainly to mimic acetylcholine in the gut. It is sometimes
given to relieve the antimuscarinic constipation caused by
tricyclic antidepressants or other meds. Pilocarpine is a
cholinomimetic which also increases gastric acid secretion.
GABA Drugs
[0174] GABA (gamma-aminobutyric acid) is the most important
inhibitory neurotransmitter in the CNS. By gating negative chloride
(Cl-) ions into the interior of nerve cells, GABA inhibits the
presynaptic release of neurotransmitter due to a positive voltage
polarization pulse. Such inhibition is extremely common: GABA
receptors can be found at 60-80% of CNS neurons.
[0175] Subtypes of GABA receptors can be activated by the mushroom
toxin muscimol (at the A subtype) as well as the antispasmodic
amino acid baclofen (B subtype). These drugs directly mimic the
action of GABA at the receptor.
[0176] Allosteric facilitation of GABA receptors occurs at several
distinct sites; the compounds which bind there are used as
sedatives and anxiolytics. These compounds bend the receptor open
to indirectly facilitate GABA binding. 3940
[0177] Progabide is a pro-drug which decomposes to GABA in the CNS.
It crosses the blood-brain barrier, which GABA itself, being a
zwitterion (doubly-ionized amino acid), does not. Vigabatrin
(gamma-vinyl-GABA) inhibits GABA-aminotransferase (GABA-T), the
enzyme responsible for degrading GABA in the synapse. It thus
prolongs the sojourn of GABA molecules and promotes binding in this
way.
[0178] Depakote (valproic acid) seems to act on nerve membranes to
reduce susceptibility to seizure. At high doses it acts like
vigabatrin to inhibit GABA-T. Gabapentine is another recently
marketed antiepileptic (Neurontin) that is also finding psychiatric
application as a mood stabilizer. The neurological rationale for
this application is that panic attacks (or mania in bipolar
disorder) resemble epilepsy in that they are characterized by a
pre-panic "kindling" phenomenon, characterized by repetitive neural
firings, leading to a critical stage. Gabapentine may encourage
production of or discourage degradation of GABA. Lamotrigine
probably works by reducing release of glutamate, an excitatory
neurotransmitter usually governed by the inhibitory GABA.
[0179] Novel GABA drugs represent one of the most active areas of
psychotropic research. Riluzole, for instance, is a GABA uptake
inhibitor with anticonvulsant and hypnotic properties; it also
blocks sodium channels and inhibits glutamate release.
Opiate Narcotics
[0180] Opiates, derived from the poppy plant, contain alkaloids
which activate the brain's endogenous endorphin receptors to
produce analgesia, euphoria, and respiratory suppression. Poppy
opiates possess a polycyclic phenanthrene nucleus with various
substituents that determine the fit into the receptor. Although
morphinelike compounds have been found in mammalian brain tissue,
it is generally agreed that the enkephalins and endorphins
represent the endogenous compounds which poppy constituents
mimic.
[0181] Opiate receptors of several varieties are responsible for
the major pharmacologic effects. These subtypes are given Greek
names like mu (analgesia, euphoria), sigma (dysphoria, cardiac
stimulation), kappa (sedation, spinal cord analgesia, miosis),
delta, etc. Antitussive properties, emesis (vomiting), and
anticholinergic (constipation) effects also occur, indicating a
wide variety of receptor types and actions. The sigma receptor is
now surmised to be related to glutamate function.
[0182] Opiate receptors exert effects on synaptic transmission by
presynaptically modulating the release of neurotransmitters,
including acetylcholine, norepinephrine, dopamine, serotonin, and
substance P. The latter compound is a peptide neurotransmitter
involved in nociceptive (pain-related) neurons. Opiate receptors
act on G-peptides, transmembranal macromolecules linked to
post-synaptic intracellular enzymes (such as adenylyl cyclase) or
ion channels (such as K.sup.+, Ca.sup.++). In high doses the
opiates cause generalized CNS depression sufficient for surgical
anesthesia. 4142434445
[0183] Codeine is a mild analgesic which retains agonist activity
at other receptor subtypes including those controlling respiration,
peristalsis and euphoria. Morphine is among the most potent of the
phenanthrene class. The less amphoteric heroin crosses the
blood-brain barrier more readily but decomposes into morphine once
there. Oxycodone, the main active constituent in Percodan and
Percocet, is somewhat less potent.
[0184] Meperidine (Demerol) is a synthetic drug that has
approximately the same analgesic activity as morphine. Methadone,
invented by the Nazis and originally named dolophine, is famous for
its use in assuaging the heroin withdrawal syndrome. Its half-life
is substantially greater than that of heroin, and while it is bound
to receptors it blocks newly administered heroin. Its analgesic
activity is also approximately equal to morphine's, but it imparts
less euphoria.
[0185] Fentanyl constitutes one of the most potent synthetics,
propoxyphene (Darvon) one of the least. Methoxy compounds such as
codeine and oxycodone are less susceptible to first-pass reactions
(typically conjugation to a glucuronide) and therefore have a
higher oral-to-parenteral ratio. Less-amphoteric compounds
(compounds with more definite acid or base properties) pass the
blood-brain barrier more easily. 46
[0186] Alteration of the phenanthrene skeleton produces drugs with
mixed agonist/antagonist properties at opiopeptin subreceptors.
These drugs are being used variously as pain killers, aids in
withdrawal from heroin and even alcohol addiction, and (illegally)
to increase athletic stamina. Stadol has been used nasally to
relieve migraines. Although mixed agonists retain analgesic
properties, they often impart dysphoric effects. 47
[0187] Narcotic antagonists are especially useful in cases of
overdose, where they can reverse the CNS depression caused by
opiate agonists. Naloxone is the most often used, most effective,
and prototypal narcotic antagonist. Naloxone, nalmefene, and nadide
are among several other compounds used to antagonize morphine
receptors.
[0188] Naltrexone has recetnly been used to reduce the craving for
alcohol among recovering alcoholics and heroin addicts (as
ReVia).
Nootropics & Smart Drugs
[0189] Nootropics, also known as smart drugs or cognition
activators, are drugs that enhance mental function. Several
mechanisms that affect nerve function may be attacked. Compounds
that are used by the body to manufacture neurotransmitters
constitute one group (precursors). Reuptake and degradation
inhibitors form another. Mimetics of excitatory neurotransmitters
and antagonists of inhibitory ones can both stimulate neural
function. Antianoxics enhance the ability of neurons to bum
glucose. Phospholipid compounds affect the fatty excitable
membranes of nerve cells, which are responsible for transporting a
depolarization pulse down dendrites and axons. Steroid compounds
also affect membrane chemistry. Vasodilators which act in the CNS
increase blood supply to brain cells. Still other drugs increase
the flexibility of red blood cells so they can gain access to more
neurons more often. All these effects be theoretically be used to
enhance neurological function in the CNS. 4849
[0190] Glycine systems perform inhibitory functions in the CNS.
Enhancement of these pathways imparts antianxiety effects and so
stabilizes mood. Glycine itself is a zwitterion and so does not
pass the blood-brain barrier very well. Dimethylglycine is
stabilized by the methyl groups; its greater lipophilicity results
in better transport to the CNS, where it is converted to glycine.
Milacemide is a pro compound which decomposes (via MAO-B) to
glycinamide and then glycine in the CNS.
[0191] Glutamate and aspartate are another group of excitatory
neurotransmitter prominent in the CNS. Since they are acidic amino
acids they have difficulty crossing the blood-brain barrier, but
standard tricks can be used to deliver them to the CNS. Making an
amide out of a carboxy acid is one of these (as in glutamine and
aceglutamide); a somewhat more radical method is to make a covalent
salt with calcium, as in calcium-N-carbamoylaspartate.
[0192] Carnitine is a catabolic (tearing-down) amino acid which
serves as a neuroprotectant at NMDA receptors (a subset of
glutamate/aspartate receptors). Acetylation of the hydroxy group
gives ALC, which again has the effect of promoting transport into
the CNS. 50
[0193] Several steroids have been used to bolster mental function
and libido. Both testosterone and estrogens have been administered
historically to increase vitality and sexual drive as people grow
older (replacement therapy). Precursors to estrogen and androgen
steroids such as DHEA and pregnenolone have recently been marketed
as nutrients. These steroids do not have significant estrogenic or
androgenic properties until converted by the body to active forms.
As with all precursors, one trusts the body's homeostatic
mechanisms to regulate formation of active molecules by
rate-limiting steps, competitive mechanisms, and tachyphylaxis
(tolerance). 5152
[0194] The use of anticonvulsant medications for psychotropic
purposes has recently grown, primarily to prophylact against manic
and/or panic syndromes. Phenytoin and carbamazepine probably work
by affecting ion-gating systems in excitable membranes. Phenytoin
structurally resembles the barbiturates while carbamazepine has a
tricyclic structure like the tricyclic antidepressants. Propanolol,
a beta-blocker, has been used to calm peripheral reactions to
stress, such as stage fright.
[0195] Propanolol is a beta-adrenergic blocker prescribed for
performance phobia or stage fright. Clonidine is an alpha agonist
sometimes used to calm peripheral tremor as in alcohol withdrawal.
Verapamil, a calcium-channel blocker, is also used for this
purpose. 53
[0196] The piracetam group of antianoxic compounds work by several
mechanisms to invigorate neural function. By supplying glutamic
acid analogs to the Krebs cycle they enhance glucose utilization in
aerobic respiration, the major means by which animal cells extract
chemical energy from sugars via ATP formation. This in turn raises
phospholipid cAMP levels, enhancing the function of dopamine and
acetylcholine neurons. Additionally they function as antioxidants
(compare the structure to that of vitamin C) and retard lipofuscin
formation. Experimentally, piracetam has been shown to increase
athletic performance, to reverse alcohol-induces brain
degeneration, and has been tried as a treatment for dyslexia.
5455
[0197] Methylxanthines are used as bronchodilators in the treatment
of asthma (typically theophylline) and in conjunction with
analgesics to treat headache. Pentoxyfylline and propentofylline
have central and peripheral vasodilatory properties. Increased
blood supply to brain tissue probably accounts for whatever
nootropic properties they have. Pentoxfylline also increases the
elasticity of red blood cells, enabling them to better squeeze
through constricted capillaries. Such drugs are called
anti-ischemics, ischemia referring to a lack of blood supply to a
tissue.
[0198] Pyritinol is another vasodilator which has been used against
dementia senilis (senility). Idebenone resembles ubiquinone, a
compound which catalyzes mitochondrial metabolic processes. It
promotes secretion of nerve growth factor (NGF) and may also
protect cell membranes against lipid peroxidation. Ergocryptine is
an ergot alkaloid which has been used to combat age-related memory
loss and Alzheimer's. It dilates blood vessels by blocking
alpha-adrenoceptors. It has been used in accident victims to
increase blood flow to the brain following trauma to prevent tissue
damage by anoxia. Vinpocetine and vincamine are two alkaloids from
the vinca plant which also have anticoagulant and vasodilation
effects.
Non-Steroidal Anti-Inflammatory Drugs (NSAIDS)
[0199] Nonsteroidal anti-inflammatory drugs (NSAIDS) are the drugs
of choice for mild to moderate pain and to reduce fever
(antipyretics). They interfere with the formation of prostaglandins
by inhibiting the enzyme cyclooxygenase, which closes a bond on
arachidonic acid, an essential oil. The cyclical prostaglandin
compounds are potent, short-lived mediators of the inflammation
response. They are not stored in cells but are synthesized as
needed in response to injury or irritation. Interfering with their
production peripherally turns off the inflammation response in the
body.
[0200] Aspirin and the other NSAIDs may also act at a site in the
CNS. Some of the NSAIDs (e.g. ketoprofen) inhibit other enzymes
such as lipoxygenase, further retarding the allergic/inflammation
response. 56
[0201] Aspirin (acetylsalicylic acid) has been used for centuries
along with its relative, methylsalicylate. The latter, known as oil
of wintergreen and often used topically, is even more toxic than
aspirin in overdose. Aspirin interferes with platelet aggregation
and retards coagulation of blood. This property probably accounts
for its use in the long-term prevention of heart attacks.
[0202] In recent years drugs like ibuprofen and ketoprofen have
become available over-the-counter. The more lipophilic of these
drugs, such as naproxen (Aleve, Naprosyn), ketoprofen (Actron) and
nabumetone (Relafen), have longer half-lives, requiring less
frequent dosing, but are probably no more effective analgesics than
ibuprofen. Liver, kidney and GI problems, of varying seriousness
commensurate with dosage history, are common. Penicillamine has
been used as a long-acting NSAID, but it is fairly toxic, causing
reduction of healing and a host of autoimmmune and histological
disorders. 57
[0203] A new class of NSAIDs inhibit cyclooxygenase-2, an enzyme
responsible for interconversion of prostaglandins. These COX-2
inhibitors are intended to preserve the formation of cytoprotective
prostaglandins while targeting inhibition of the compounds
responsible for pain and inflammation, reducing stomach irritation,
Celebrex (celecoxib) is one such drug. Vioxx is another new drug in
this class. 5859
[0204] Recent studies have suggested that a pair of aminated sugar
compounds can assist in repairing damage to cartilage in
osteoarthritis. Glucosamine, a monomer, and chondroitin, a polymer,
are being marketed as nutrients for this purpose. In cartilage,
sugar polymers form a flexible connecting matrix around the tough
protein strands in cartilage (a composite material). 6061
[0205] The benzodiazepine sedatives include Valium, Librium,
Halcion, Xanax, Ativan, Serax, and Klonopin, to name just a few. In
addition to potential effects on lipophosphate nerve membranes,
these drugs work by allosterically enhancing the effect of the
inhibitory neurotransmitter GABA at post-synaptic receptors. That
is, they "bend" the receptor slightly so that GABA molecules attach
to and activate their receptors more effectively and more often.
Their chief advantage over the barbiturates, such as seconal,
nembutal and phenobarbital, is that they do not act directly to
open chloride ion channels.
Serotonin Drugs
[0206] Serotonin is an inhibitory neurotransmitter which
complements excitatory sympathetic systems like adrenaline and
dopamine in the CNS. Like the "fight or flight" adrenaline
compounds, serotonin is released not only at specific synaptic
sites, but also in a broadcast manner into brain tissue from sets
of "diffuse" neurons emanating from the emotional centers in the
limbic system into the frontal lobe. This diffuse release sets the
biochemical tone of large areas of neural functioning, controlling
mood and motivation. Serotonin's inhibitory action is however more
complex and selective than that of GABA sedatives like Valium or
Xanax, which act more globally. Because of their effects on mood,
serotonin-active drugs are used as antidepressants and anxiolytics
(anti-anxiety) drugs. 6263
[0207] Ondansetron is a selective 5-HT3 antagonist. This receptor
subtype is found on cholinergic neurons; when it is activated it
inhibits release of acetylcholine. Along with its chemical
relatives such as granisetron and zatosetron, it may thus be useful
in reviving memory function in the aged. Granisetron is also used
as an antiemetic (Kytril) in chemotherapy.
[0208] Ketanserin, a selective 5-HT2 antagonist, also acts on
alpha-1 adrenoceptors to lower blood pressure. Mescaline, a
hallucinogen, antagonizes 5-HT2 terminals and has been tried as an
alternative to dopamine blocking antipsychotics (without much
success; it facilitates dopamine function). Oxetorone is a
relatively new antagonist used against migraine, as is pizotyline.
Cyproheptadiene is an older serotonin antagonist and
antihistaminic. Mirtazapine (Remeron) causes serotonin release, but
blocks the 5-HT2 and 5-HT3 subreceptors, effectively augmenting
serotonin action at 5-HT1 receptors. Mianserin and
homochlorcyclizine also antagonize serotonin receptors. 6465
[0209] Sumatriptan activates 5-HT1d terminals, and is used against
migraine under the trade name Imitrex. Zolmitriptan (Zomig) and
rizatriptan (Maxal) are similar, recently approved, antimigraine
serotonin drugs.
[0210] Buspirone, ipsapirone and gepirone enjoy 5-HT1 agonist
properties with only weak D2 blocking effects. Buspirone is used
against anxiety as an alternative to GABA-mimetic sedatives.
8-hydroxy-DPAT acts selectively at 5-HT1 a receptors, while
2-methylserotonin activates 5-HT3 terminals.
Steroids & Reproductive Drugs
[0211] Steroids are fat-soluble (lipophilic) hormones with a
tetracyclic base structure. The steroid structure is synthesized
from smaller structures called terpenes to precursor molecules,
which then undergo extensive and subtle alterations for a rich
variety of uses: to control systems, often in fatty tissues, as
diverse as meiosis, carbohydrate metabolism, fat storage, muscle
growth, immune function, and nerve cell membrane chemistry. Because
of their high lipophilicity, they can pass through cell membranes,
which are fatty bilayers, and influence DNA transcription and
thereby alter protein synthesis. By binding to specific sites on
the DNA, they release a kind of molecular "boot" (hsp90) that, in
the absence of steroid, locks up the DNA and prevents a short
sequence from being expressed into a protein. The action of the
enzymes or active peptides generated by the activation of the DNA
can persist for long periods of time, explaining the long duration
of action of many steroids.
[0212] Steroids may be separated into the broad groups of gonadal
compounds and glucocorticoids, depending on the site of synthesis,
which is in the ovary or testis for the gonadal variety and in the
adrenal cortex for glucocorticoids. They may also be divided
according to function, with the usual designations being androgens,
estrogens, and progestogens (typically for the gonadal hormones)
and anabolics and catabolics (typically for the
glucocorticoids).
[0213] It is important to realize, however, that these terms are
not totally exclusive. That is, even the gonadal hormone
testosterone is synthesized in small quantities by the adrenal
gland, and imparts anabolic properties separate from its effects on
gonad function. Moreover, all hormones act in coordination with
other compounds to produce a net result. 66
[0214] Testosterone is the prototype of the androgen group of
gonadal steroids. Androgens impart features typified by males of
mammalian species. These include morphological features such as a
protruding browridge, robust bone structure, and large canines. It
is also responsible for aggression and libido. It also acts as an
anabolic, aiding muscle formation in response to exercise.
Estradiol, meanwhile, is the prototypical estrogen, imparting
female characteristics such as breast growth and storage of
subcutaneous fat. Estrogens also prevent heart disease (women get
it statistically less than men).
[0215] Oral contraceptives reformulate the body's steroid chemistry
to mimic that of pregnancy to prevent ovulation. This is usually
done in accordance with the natural 28 day menstrual cycle,
although it is possible to trick the body into delaying ovulation
for longer periods. This is accomplished by a mixture of an
estrogen and a progestogen. The most popular preparation is
norethindrone (a progestogen) and ethinylestradiol (an estrogen).
This combination, taken in a large dose just after unprotected sex,
can also prevent pregnancy by the same mechanism. Replacement
therapy for gonadal steroids in the form of testosterone for men
and estrogen/progesterones (depot ProVera) and recently also
testosterone for women has been tried to combat the symptoms of
aging, including diminished sex drive. In men, testosterone helps
libido and may improve cardiovascular fitness and general vigor. In
women, the drop in estrogen after menopause imparts some changes,
but the drop is relatively modest (20% or so) compared to the drop
in progesterone, which causes osteoblasts to make new bone tissue
and inhibits cancer cell formation. Since estrogens, being anabolic
or tissue-building compounds, can promote cancer cell growth, modem
replacement formulations should compensate more for progesterones
than estrogens. Non-steroidal soybean estrogens are now marketed as
treatments for menopausal hot flashes, and testosterone creams and
tablets to increase sex drive. 67
[0216] Cholesterol is heavily involved in membrane in metabolic
chemistries. One of its main uses in the body is to decrease the
permeability of phospholipid cell walls to ionic species such as
Na.sup.+, K.sup.+, and Ca.sup.++. In recent years it has been
implicated in aiding the accumulation of plaque on the interior
surfaces of veins and arteries. Animal meat is typically rich in
cholesterol. Through natural selection, carnivores such as cats and
dogs have developed different chemistries adjusted for processing
higher amounts of cholesterol, cholestanol (the saturated from),
and other steroids, which make diets higher in meat safer for them
than for us. Cholesterol is excreted through gall, a fatty
digestive substance secreted from the liver. It is present in high
quantities in gallstones. Bile is also used to excrete fat-soluble
substances such as bilirubin (from the heme group in decomposed red
blood cells). Cholesterol is also used endogenously to synthesize
vitamin D.
[0217] Cortisone, another prototypical glucocorticoid, controls
healing processes associated with the immune system, as well as
regulating membrane and other functions. Hydrocortisone, also known
as cortisol, works similarly to inhibit histamine-mediated allergic
reaction and regulates the body's response to stress by modulating
the chemistry of neuronal excitable membranes. Prednisone is a
synthetic compound used regularly in place of cortisone.
Dexamethasone has been used to diagnose depression, i.e. in the
dexamethasone suppression test, where the body is "stressed" by
introduction of the steroid and its response is measured. 68
[0218] Hexestrol and diethylstilbestrol are two examples of
polycyclic, non-steroid compounds which activate estrogen
receptors. The local structure in the bound configuration resembles
that of steroids. The other main category of non-steroidal
estrogens is the isoflavones.
[0219] Environmental estrogens have become a health concern since
cattle are commonlty fed estrogens due to their anabolic
(weight-gain) properties. Ingestion of meat therefore equates to
absorbing some estrogens. Some pesticides are non-steroidal
estrogens, as is THC, the main psychoactive constituent of
marijuana. Compounds such as diethylstilbestrol have been shown to
be carcinogens, though this is not due to action on DNA. 69
[0220] Recently, two nonsteroidal estrogen agents have shown great
medical promise in several women's health issues. These selective
estrogen receptor modulators (SERMs) mimic the effects of estrogens
in some tissues but not others.
[0221] Tamoxifen has been used for years following detection of
breast cancer. By blocking estrogen receptors, it discourages tumor
growth. New studies show it may also prevent breast cancer,
probably by the same mechanism. However, the benefits of this
prevention must be weighed against the increased risk of uterine
cancer and other potential risks.
[0222] Raloxifene retains the ability to promote bone maintenance
and prevent osteoporosis; it cuts the risk of breast cancer by as
much as 60%, and decreases levels of LDLs ("bad" cholesterol).
70
[0223] Finasteride (Proscar, Propecia) is presently being marketed
as a systemic (oral) anti-baldness medication with a mechanism
distinct from that of minoxidil, which is a topical vasodilator
which stimulates follicular activity by improving blood flow.
Finasteride works by inhibiting the formation of
5-alpha-dihydrotestosterone, a potent androgen, from the less
potent parent compound, testosterone. It has also been used to
treat benign prostatic hypertrophy (enlargement of the
prostate).
[0224] The side effects of reducing androgens in the body can be
essentially termed feminization: atrophy of the male gonads, breast
augmentation, decrease in aggressive behavior, increased risk of
osteoporosis, etc. 71
[0225] Steroids are not confined to the animal kingdom but are
synthesized by plants as well. The well-known cardiotonic digitalis
is derived from the foxglove plant which synthesizes several
glucoside steroids (i.e. steroids bonded to sugar moieties).
[0226] The oleander shrub generates several steroids with similar
effects on cardiac conduction, including oleandrin and
oleandrigenin. 72
[0227] Sildenafil (Viagra) is a erection facilitator. Erection
depends on an interaction of adrenergic and cholinergic neurons in
which muscles must relax to let blood into erectile tissue. The
presence of nitrous oxide (NO) species is involved, and Viagra may
affect the enzymes responsible for generating NO. Viagra is also a
selective inhibitor of cGMP phophodiesterase, which acts in some GI
vascular smooth-muscles. Caffeine, wose central ring structure
resembles Viagra's, works similarly on the more widespread cAMP
phosphodiesterase, a more widespread second messenger system.
Yohimbine, a selective alpha2 adrenergic blocker, has also been
touted as being able to prolong or intensify erection.
[0228] Gossypol, isolated from the cotton plant, has the ability to
inhibit production of viable sperm in men. It damages the
epithelial lining of seminferous vesicles, inhibiting sperm
formation. It also poisons the oxygen-carrying capacity of
blood.
[0229] Among peptide therapeutic candidates are peptides which
demonstrate anti-neoplastic activity, such as the RGD peptides
including peptide SEQ ID NOS. 1-3.
[0230] Also included are peptides that are active against HIV,
including the GP-41 peptides including peptide SEQ ID NOS. 4-6.
[0231] Anti-viral peptides which demonstrate the ability to disrupt
fusogenic events common in viral infections. These include the RSV
peptides which demonstrate the ability to treat or prevent
infection by respiratory syncytial virus (RSV) as well as acquired
immune deficiency syndrome (AIDS) caused by infection of the human
immunodeficiency virus (HIV). Such peptides include peptide SEQ
NOS. 7-9.
[0232] Also included are GLP-1 peptides including those peptides
depicted in SEQ ID NOS. 10-11.
[0233] Also included are Kringle or K5 peptides including those
peptides depicted in SEQ ID NOS. 12-13; BBB peptides (TAT)
including those peptides depicted in SEQ ID NOS. 14-15 and
analgesic peptides, such as dynorphins, are also useful, including
peptide SEQ ID NO. 16.
[0234] 3. Modified Therapeutic Agents
[0235] The modified therapeutic agents of the present invention
comprise therapeutic agents that have been modified by attaching a
reactive group. The reactive group may be attached to the
therapeutic agent via a linking group, or optionally without using
a linking group. The modified therapeutic agents can react with the
available functionalitieson blood or pulmonary components or blood
components via covalent linkages. The invention also relates to
such modifications, such combinations with pulmonary components or
blood components, and methods for their use. These methods include
extending the effective therapeutic life of the conjugated
therapeutic agents as compared to administration of unconjugated
therapeutic agents.
[0236] To form covalent bonds with functionalities on a protein,
one may use as a reactive group a wide variety of active carboxyl
groups, particularly esters, where the hydroxyl moiety is
physiologically acceptable at the levels required to modify the
therapeutic agent. While a number of different hydroxyl groups may
be employed, the most convenient would be N-hydroxysuccinimide
(NHS), N-hydroxy-sulfosuccinimid- e (sulfo-NHS), maleimide,
maleimide acids including but not limited to maleimidopropionic
acid (MPA), and maleimide esters. In the preferred embodiments of
this invention, the functionality on the blood component will be a
thiol group and the reactive group will a maleimide.
[0237] Primary amines are the principal targets for NHS esters.
Accessible .alpha.-amine groups present on the N-termini of
proteins react with NHS esters. However, .alpha.-amino groups on a
protein may not be desirable or available for the NHS coupling.
While five amino acids have nitrogen in their side chains, only the
.epsilon.-amine of lysine reacts significantly with NHS esters. An
amide bond is formed when the NHS ester conjugation reaction reacts
with primary amines releasing N-hydroxysuccinimide as demonstrated
in the schematic below. 73
[0238] In the preferred embodiments of this invention, the
functional group on this protein will be a thiol group and the
chemically reactive group will be a maleimido-containing group such
as MPA or GMBA (gamma-maleimide-butyralamide). The maleimido group
is most selective for sulfhydryl groups on peptides when the pH of
the reaction mixture is kept between 6.5 and 7.4. At pH 7.0, the
rate of reaction of maleimido groups with sulfhydryls is 1000-fold
faster than with amines. A stable thioether linkage between the
maleimido group and the sulfhydryl is formed which cannot be
cleaved under physiological conditions, as demonstrated in the
following schematic. 74
[0239] A. Specific Labeling.
[0240] Preferably, the modified therapeutic agents of this
invention are designed to specifically react with thiol groups on
pulmonary proteins or mobile blood proteins. Such reaction is
preferably established by covalent bonding of the therapeutic agent
modified with a maleimide link (e.g. prepared from GMBS, MPA or
other maleimides) to a thiol group on a pulmonary protein, such as
intra- or extra-cellular albumin, or a mobile blood protein such as
serum albumin or IgG.
[0241] Under certain circumstances, specific labeling with
maleimides offers several advantages over non-specific labeling of
proteins with groups such as NHS and sulfo-NHS. Thiol groups are
less abundant in vivo than amino groups. Therefore, the
maleimide-modified therapeutic agents of this invention, i.e.,
maleimide therapeutic agents, will covalently bond to fewer
proteins. For example, in albumin (the most abundant blood protein)
there is only a single thiol group. Thus, therapeutic
agent-maleimide-albumin conjugates will tend to comprise
approximately a 1:1 molar ratio of therapeutic agent to albumin. In
addition to albumin, IgG molecules (class II) also have free
thiols. In the case of systemic delivery, since IgG molecules and
serum albumin make up the majority of the soluble protein in blood
they also make up the majority of the free thiol groups in blood
that are available to covalently bond to maleimide-modified
therapeutic agents.
[0242] Further, even among free thiol-containing blood proteins,
including IgGs, specific labeling with maleimides leads to the
preferential formation of therapeutic agent-maleimide-albumin
conjugates, due to the unique characteristics of albumin itself.
The single free thiol group of albumin, highly conserved among
species, is located at amino acid residue 34 (Cys.sup.34). It has
been demonstrated recently that the Cys.sup.34 of albumin has
increased reactivity relative to free thiols on other free
thiol-containing proteins. This is due in part to the very low pK
value of 5.5 for the Cys.sup.34 of albumin. This is much lower than
typical pK values for cysteine residues in general, which are
typically about 8. Due to this low pK, under normal physiological
conditions Cys.sup.34 of albumin is predominantly in the ionized
form, which dramatically increases its reactivity. In addition to
the low pK value of Cys.sup.34, another factor which enhances the
reactivity of Cys.sup.34 is its location, which is in a crevice
close to the surface of one loop of region V of albumin. This
location makes Cys.sup.34 very available to ligands of all kinds,
and is an important factor in Cys.sup.34's biological role as free
radical trap and free thiol scavenger. These properties make Cys34
highly reactive with maleimide-therapeutic agents, and the reaction
rate acceleration can be as much as 1000-fold relative to rates of
reaction of maleimide-therapeutic agents with other free-thiol
containing proteins.
[0243] Another advantage of therapeutic agent-maleimide-albumin
conjugates is the reproducibility associated with the 1:1 loading
of therapeutic agent to albumin specifically at Cys.sup.34. Other
techniques, such as glutaraldehyde, DCC, EDC and other chemical
activations of, e.g, free amines, lack this selectivity. For
example, albumin contains 52 lysine residues, 25-30 of which are
located on the surface of albumin and therefore accessible for
conjugation. Activating these lysine residues, or alternatively
modifying therapeutic agents to couple through these lysine
residues, results in a heterogenous population of conjugates. Even
if 1:1 molar ratios of therapeutic agent to albumin are employed,
the yield will consist of multiple conjugation products, some
containing 0, 1, 2 or more therapeutic agents per albumin, and each
having therapeutic agents randomly coupled at any one or more of
the 25-30 available lysine sites. Given the numerous possible
combinations, characterization of the exact composition and nature
of each conjugate batch becomes difficult, and batch-to-batch
reproducibility is all but impossible, making such conjugates less
desirable as a therapeutic. Additionally, while it would seem that
conjugation through lysine residues of albumin would at least have
the advantage of delivering more therapeutic agent per albumin
molecule, studies have shown that a 1:1 ratio of therapeutic agent
to albumin is preferred. In an article by Stehle, et al., "The
Loading Rate Determines Tumor Targeting properties of
Methotrexate-Albumin Conjugates in Rats," Anti-Cancer Drugs, Vol.
8, pp. 677-685 (1988), incorporated herein in its entirety, the
authors report that a 1:1 ratio of the anti-cancer methotrexate to
albumin conjugated via glutaraldehyde gave the most promising
results. These conjugates were preferentially taken up by tumor
cells, whereas conjugates bearing 5:1 to 20:1 methotrexate
molecules had altered HPLC profiles and were quickly taken up by
the liver in vivo. It is postulated that at these higher ratios,
conformational changes to albumin diminish its effectiveness as a
therapeutic carrier.
[0244] Through controlled administration of maleimide-therapeutic
agents in vivo, one can control the specific labeling of albumin
and IgG in vivo. For systemic delivery via pulmonary
administration, in typical administrations, 80-90% of the
administered maleimide-therapeutic agents that reach the
bloodstream will label albumin and less than 5% will label IgG.
Trace labeling of free thiols such as glutathione will also occur.
Such specific labeling is preferred for in vivo use as it permits
an accurate calculation of the estimated half-life of the
administered agent.
[0245] In addition to providing controlled specific in vivo
labeling, maleimide-therapeutic agents can provide specific
labeling of albumin or other proteins ex vivo. Such ex vivo
labeling involves the addition of maleimide-therapeutic agents to a
saline solution containing albumin or other protein. Once
conjugation has occurred ex vivo with the maleimide-therapeutic
agents, the saline solution can be administered via pulmonary
delivery for in vivo treatment.
[0246] In contrast to NHS-therapeutic agents, maleimide-therapeutic
agents are generally quite stable in the presence of aqueous
solutions and in the presence of free amines. Since
maleimide-therapeutic agents will only react with free thiols,
protective groups are generally not necessary to prevent the
maleimide-therapeutic agents from reacting with itself. In
addition, the increased stability of the modified therapeutic agent
permits the use of further purification steps such as HPLC to
prepare highly purified products suitable for in vivo use. Lastly,
the increased chemical stability provides a product with a longer
shelf life.
[0247] B. Non-Specific Labeling.
[0248] The therapeutic agents of the invention may also be modified
for non-specific labeling of pulmonary or blood components. Bonds
to amino groups will also be employed, particularly with the
formation of amide bonds for non-specific labeling. To form such
bonds, one may use as a chemically reactive group a wide variety of
active carboxyl groups, particularly esters, where the hydroxyl
moiety is physiologically acceptable at the levels required. While
a number of different hydroxyl groups may be employed in these
linking agents, the most convenient would be N-hydroxysuccinimide
(NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS).
[0249] Other linking agents which may be utilized are described in
U.S. Pat. No. 5,612,034, which is hereby incorporated herein.
[0250] The various sites with which the chemically reactive group
of the modified therapeutic agents may react in vivo include cells,
particularly the alveolar cells and capillary endothelial cells
that make up the alveoli in the lungs as well as red blood cells
(erythrocytes) and platelets in the blood itself. The agents may
also react with pulmonary proteins, including membrane bound
receptors, intra- and extra-cellular albumin, immunoglobulins,
ferritin, and transferrin, and serum proteins of the blood, such as
immunoglobulins, including IgG and IgM, serum albumin, ferritin,
steroid binding proteins, transferrin, thyroxin binding protein,
.alpha.-2-macroglobulin, and the like. Those receptors with which
the modified therapeutic agents react, which are not long-lived,
will generally be eliminated from the human host within about three
days. The proteins indicated above (including the proteins of the
cells) will remain at least three days, and may remain five days or
more (usually not exceeding 60 days, more usually not exceeding 30
days) particularly as to the half life, based on the concentration
in the blood.
[0251] For the most part, for systemic delivery of the therapeutic
agent, reaction will be with mobile components in the blood,
particularly blood proteins and cells, more particularly blood
proteins and erythrocytes. By "mobile" is intended that the
component does not have a fixed situs for any extended period of
time, generally not exceeding 5 minutes, more usually one minute,
although some of the blood component may be relatively stationary
for extended periods of time. Initially, there will be a relatively
heterogeneous population of functionalized proteins and cells.
However, for the most part, the population within a few days will
vary substantially from the initial population, depending upon the
half-life of the functionalized proteins in the blood stream.
Therefore, usually within about three days or more, IgG will become
the predominant functionalized protein in the blood stream.
[0252] Usually, by day 5 post-administration, IgG, serum albumin
and erythrocytes will be at least about 60 mole %, usually at least
about 75 mole %, of the conjugated components in blood, with IgG,
IgM (to a substantially lesser extent) and serum albumin being at
least about 50 mole %, usually at least about 75 mole %, more
usually at least about 80 mole %, of the non-cellular conjugated
components.
[0253] The desired conjugates of non-specific modified therapeutic
agents to blood components may be prepared in vivo by pulmonary
administration of the modified therapeutic agents to the patient,
which may be a human or other mammal. If desired, the subject
conjugates may also be prepared ex vivo by combining a carrier
protein or protein solution with modified therapeutic agents of the
present invention, allowing covalent bonding of the modified
therapeutic agents to functionalitieson the protein and then
administering the conjugated mixture to the host via pulmonary
delivery.
[0254] 3. Diagnostic Agents and Modified Diagnostic Agents
[0255] Diagnostic agents are agents useful in imaging the mammalian
vascular system and include such agents as position emission
tomography (PET) agents, computerized tomography (CT) agents,
magnetic resonance imaging (MRI) agents, nuclear magnetic imaging
agents (NMI), fluroscopy agents and ultrasound contrast agents.
[0256] The modified diagnostic agent of the present invention will,
for the most part, have the following formula: X-Y-Z.
[0257] In the formula, X is a diagnostic agent selected from PET
agent, CT agents, MRI agents, NMI agents, fluroscopy agents and
ultrasound contrast agents. Diagnostic agents of interest include
radioisotopes of such elements as iodine (I), including .sup.123I,
.sup.125I, .sup.131I, etc., barium (Ba), gadolinium (Gd),
technetium (Tc), including .sup.99Tc, phosphorus (P), including
.sup.31P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr),
including .sup.51Cr, carbon (C), including .sup.14C, or the like,
fluorescently labeled compounds, etc.
[0258] In the formula, Y is a linking group of from 0-30, more
usually of from 2-12, preferably of from 4-12 atoms, particularly
carbon, oxygen, phosphorous and nitrogen, more particularly carbon
and oxygen, where the oxygen is preferably present as oxy ether,
where Y may be alkylene, oxyalkylene, or polyoxyalkylene, where the
oxyalkylene group has from 2-3 carbon atoms, and the like. A
linking group of 0 atoms is preferred when it is desired to place X
as close to Z as possible.
[0259] In the formula, Z is a reactive entity, such as carboxy,
carboxy ester, where the ester group is of 1-8, more usually 1-6
carbon atoms, particularly a physiologically acceptable leaving
group which activates the carboxy carbonyl for reaction with amino
groups in an aqueous system, e.g. N-hydroxysuccinimide (NHS),
N-hydroxysulfosuccinimide, (sulfo-NHS), maleimide, maleimide
esters, maleimide acids, maleimide-benzoyl-succinimi- de (MBS),
gamma-maleimido-butyryloxy succinimide ester (GMBS) and
maleimidopropionic acid (MPA), N-hydroxysuccinimide isocyanate,
isothiocyanate, thiolester, thionocarboxylic acid ester, imino
ester, mixed anhydride, e.g. carbodiimide anhydride, carbonate
ester, etc. and the like. The reactive entity Z will covalently
bond to functionalities in vivo or ex vivo.
[0260] 4. Synthesis of Peptide Therapeutic Agents
[0261] A. Peptide Synthesis
[0262] Therapeutic agents according to the present invention that
are peptides may be synthesized by standard methods of solid phase
peptide chemistry known to those of ordinary skill in the art. For
example, peptides may be synthesized by solid phase chemistry
techniques following the procedures described by Steward and Young
(Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis,
2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984) using an
Applied Biosystem synthesizer. Similarly, multiple peptide
fragments may be synthesized then linked together to form larger
peptides. These synthetic peptides can also be made with amino acid
substitutions at specific locations.
[0263] For solid phase peptide synthesis, a summary of the many
techniques may be found in J. M. Stewart and J. D. Young, Solid
Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963
and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46,
Academic Press (New York), 1973. For classical solution synthesis
see G. Schroder and K. Lupke, The Peptides, Vol. 1, Acacemic Press
(New York). In general, these methods comprise the sequential
addition of one or more amino acids or suitably protected amino
acids to a growing peptide chain. Normally, either the amino or
carboxyl group of the first amino acid is protected by a suitable
protecting group. The protected or derivatized amino acid is then
either attached to an inert solid support or utilized in solution
by adding the next amino acid in the sequence having the
complimentary (amino or carboxyl) group suitably protected and
under conditions suitable for forming the amide linkage. The
protecting group is then removed from this newly added amino acid
residue and the next amino acid (suitably protected) is added, and
the process is repeated.
[0264] After all the desired amino acids have been linked in the
proper sequence, any remaining protecting groups (and any solid
support) are removed sequentially or concurrently to afford the
final polypeptide. By simple modification of this general
procedure, it is possible to add more than one amino acid at a time
to a growing chain, for example, by coupling (under conditions
which do not racemize chiral centers) a protected tripeptide with a
properly protected dipeptide to form, after deprotection, a
pentapeptide.
[0265] A particularly preferred method of preparing compounds of
the present invention involves solid phase peptide synthesis
wherein the amino acid .alpha.-N-terminal is protected by an acid
or base sensitive group. Such protecting groups should hav the
properties of being stable to the conditions of peptide linkage
formation while being readily removable without destruction of the
growing peptide chain or racemization of any of the chiral centers
contained therein. Suitable protecting groups are
9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl ,
t-amyloxycarbonyl, isobornyloxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like.
The 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is
particularly preferred for the synthesis of the peptides of the
present invention. Other preferred side chain protecting groups
are, for side chain amino groups like lysine and arginine,
2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro,
p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and
adamantyloxycarbonyl; for tyrosine, benzyl,
o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl
(t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine,
t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl,
benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for
tryptophan, formyl; for asparticacid and glutamic acid, benzyl and
t-butyl and for cysteine, triphenylmethyl (trityl).
[0266] In the solid phase peptide synthesis method, the
.alpha.-C-terminal amino acid is attached to a suitable solid
support or resin. Suitable solid supports useful for the above
synthesis are those materials which are inert to the reagents and
reaction conditions of the stepwise condensation-deprotection
reactions, as well as being insoluble in the media used. The
preferred solid support for synthesis of .alpha.-C-terminal carboxy
peptides is 4-hydroxymethylphenoxymethyl-copol- y(styrene-1%
divinylbenzene). The preferred solid support for .alpha.-C-terminal
amide peptides is the 4-(2',4'-dimethoxyphenyl-Fmoc-am-
inomethyl)phenoxyacetamidoethyl resin available from Applied
Biosystems (Foster City, Calif.). The .alpha.-C-terminal amino acid
is coupled to the resin by means of N,N'-dicyclohexylcarbodiimide
(DCC), N,N'-diisopropylcarbodiimide (DIC) or
O-benzotriazol-1-yl-N,N,N',N'-tetra-
methyluronium-hexafluorophosphate (HBTU), with or without
4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT),
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate
(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl),
mediated coupling for from about 1 to about 24 hours at a
temperature of between 10.degree. and 50.degree. C. in a solvent
such as dichloromethane or DMF.
[0267] When the solid support is
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl- )phenoxy-acetamidoethyl
resin, the Fmoc group is cleaved with a secondary amine, preferably
piperidine, prior to coupling with the .alpha.-C-terminal amino
acid as described above. The preferred method for coupling to the
deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl-
)phenoxy-acetamidoethyl resin is
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-
uroniumhexafluoro-phosphate (HBTU, 1 equiv.) and
1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of
successive protected amino acids can be carried out in an automatic
polypeptide synthesizer as is well known in the art. In a preferred
embodiment, the .alpha.-N-terminal amino acids of the growing
peptide chain are protected with Fmoc. The removal of the Fmoc
protecting group from the .alpha.-N-terminal side of the growing
peptide is accomplished by treatment with a secondary amine,
preferably piperidine. Each protected amino acid is then introduced
in about 3-fold molar excess, and the coupling is preferably
carried out in DMF. The coupling agent is normally
O-benzotriazol-1-yl-N,N,N',N'-tetrame-
thyluroniumhexafluorophosphate (HBTU, 1 equiv.) and
1-hydroxybenzotriazole (HOBT, 1 equiv.).
[0268] At the end of the solid phase synthesis, the polypeptide is
removed from the resin and deprotected, either in successively or
in a single operation. Removal of the polypeptide and deprotection
can be accomplished in a single operation by treating the
resin-bound polypeptide with a cleavage reagent comprising
thioanisole, water, ethanedithiol and trifluoroacetic acid. In
cases wherein the .alpha.-C-terminal of the polypeptide is an
alkylamide, the resin is cleaved by aminolysis with an alkylamine.
Alternatively, the peptide may be removed by transesterification,
e.g. with methanol, followed by aminolysis or by direct
transamidation. The protected peptide may be purified at this point
or taken to the next step directly. The removal of the side chain
protecting groups is accomplished using the cleavage cocktail
described above. The fully deprotected peptide is purified by a
sequence of chromatographic steps employing any or all of the
following types: ion exchange on a weakly basic resin (acetate
form); hydrophobic adsorption chromatography on underivitized
polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel
adsorption chromatography; ion exchange chromatography on
carboxymethylcellulose; partition chromatography, e.g. on Sephadex
G-25, LH-20 or countercurrent distribution; high performance liquid
chromatography (HPLC), especially reverse-phase HPLC on octyl- or
octadecylsilyl-silica bonded phase column packing.
[0269] Molecular weights of these ITPs are determined using Fast
Atom Bombardment (FAB) Mass Spectroscopy.
[0270] (1) N-Terminal Protective Groups
[0271] As discussed above, the term "N-protecting group" refers to
those groups intended to protect the .alpha.-N-terminal of an amino
acid or peptide or to otherwise protect the amino group of an amino
acid or peptide against undesirable reactions during synthetic
procedures. Commonly used N-protecting groups are disclosed in
Greene, "Protective Groups In Organic Synthesis," (John Wiley &
Sons, New York (1981)), which is hereby incorporated by reference.
Additionally, protecting groups can be used as pro-drugs which are
readily cleaved in vivo, for example, by enzymatic hydrolysis, to
release the biologically active parent. .alpha.-N-protecting groups
comprise loweralkanoyl groups such as formyl, acetyl ("Ac"),
propionyl, pivaloyl, t-butylacetyl and the like; other acyl groups
include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,
trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl,
benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the
like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl
and the like; carbamate forming groups such as benzyloxycarbonyl,
p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,
3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,
4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl- ,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-di- methoxybenzyloxycarbonyl,
benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc),
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and
the like; arylalkyl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like
and silyl groups such as trimethylsilyl and the like.
[0272] (2) Carboxy Protective Groups
[0273] As discussed above, the term "carboxy protecting group"
refers to a carboxylic acid protecting ester or amide group
employed to block or protect the carboxylic acid functionality
while the reactions involving other functional sites of the
compound are performed. Carboxy protecting groups are disclosed in
Greene, "Protective Groups in Organic Synthesis" pp. 152-186
(1981), which is hereby incorporated by reference. Additionally, a
carboxy protecting group can be used as a pro-drug whereby the
carboxy protecting group can be readily cleaved in vivo, for
example by enzymatic hydrolysis, to release the biologically active
parent. Such carboxy protecting groups are well known to those
skilled in the art, having been extensively used in the protection
of carboxyl groups in the penicillin and cephalosporin fields as
described in U.S. Pat. Nos. 3,840,556 and 3,719,667, the
disclosures of which are hereby incorporated herein by reference.
Representative carboxy protecting groups are C.sub.1-C.sub.8
loweralkyl (e.g., methyl, ethyl or t-butyl and the like); arylalkyl
such as phenethyl or benzyl and substituted derivatives thereof
such as alkoxybenzyl or nitrobenzyl groups and the like;
arylalkenyl such as phenylethenyl and the like; aryl and
substituted derivatives thereof such as 5-indanyl and the like;
dialkylaminoalkyl such as dimethylaminoethyl and the like);
alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl,
valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl,
1-(propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl,
1-methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl,
propionyloxymethyl and the like; cycloalkanoyloxyalkyl groups such
as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,
cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the
like; aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl and
the like; arylalkylcarbonyloxyalkyl such as
benzylcarbonyloxymethyl, 2-benzylcarbonyloxyethyl and the like;
alkoxycarbonylalkyl or cycloalkyloxycarbonylalkyl such as
methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl,
1-methoxycarbonyl-1-ethyl and the like; alkoxycarbonyloxyalkyl or
cycloalkyloxycarbonyloxyalkyl such as methoxycarbonyloxymethyl,
t-butyloxycarbonyloxymethyl, 1-ethoxycarbonyloxy-1-ethyl,
1-cyclohexyloxycarbonyloxy-1-ethyl and the like;
aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,
2-(5-indanyloxycarbonyloxy)ethyl and the like;
alkoxyalkylcarbonyloxyalky- l such as
2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;
arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl
and the like; arylalkenyloxycarbonyloxyalkyl such as
2-(3-phenylpropen-2-ylox- ycarbonyloxy)ethyl and the like;
alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and
the like; alkylaminocarbonylaminoalkyl such as
methylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl
such as acetylaminomethyl and the like;
heterocycliccarbonyloxyalkyl such as
4-methylpiperazinylcarbonyloxymethyl and the like;
dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,
diethylaminocarbonylmethyl and the like;
(5-(loweralkyl)-2-oxo-1,3-dioxol- en-4-yl)alkyl such as
(5-t-butyl-2-oxo-1,3-dioxolen4-yl)methyl and the like; and
(5-phenyl-2-oxo-1,3-dioxolen4-yl)alkyl such as
(5-phenyl-2-oxo-1,3-dioxolen4-yl)methyl and the like.
[0274] Representative amide carboxy protecting groups are
aminocarbonyl and loweralkylaminocarbonyl groups.
[0275] Preferred carboxy-protected compounds of the invention are
compounds wherein the protected carboxy group is a loweralkyl,
cycloalkyl or arylalkyl ester, for example, methyl ester, ethyl
ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester,
isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl
ester, phenylethyl ester and the like or an alkanoyloxyalkyl,
cycloalkanoyloxyalkyl, aroyloxyalkyl or an
arylalkylcarbonyloxyalkyl ester. Preferred amide carboxy protecting
groups are loweralkylaminocarbonyl groups. For example, aspartic
acid may be protected at the .alpha.-C-terminal by an acid labile
group (e.g. t-butyl) and protected at the .beta.-C-terminal by a
hydrogenation labile group (e.g. benzyl) then deprotected
selectively during synthesis.
[0276] B. Peptide Modification
[0277] The manner of producing the modified peptides of the present
invention will vary widely, depending upon the nature of the
various elements comprising the peptide. The synthetic procedures
will be selected so as to be simple, provide for high yields, and
allow for a highly purified stable product. Normally, the
chemically reactive group will be created at the last stage of the
synthesis, for example, with a carboxyl group, esterification to
form an active ester. Specific methods for the production of
modified peptides of the present invention are described below.
[0278] Specifically, the selected peptide is modified with the
linking group only at either the N-terminus, C-terminus or interior
of the peptide. The therapeutic activity of this modified
peptide-linking group is then assayed. If the therapeutic activity
is not reduced dramatically (i.e., reduced less than 10-fold), then
the stability of the modified peptide-linking group is measured by
its in vivo lifetime. If the stability is not improved to a desired
level, then the peptide is modified at an alternative site, and the
procedure is repeated until a desired level of therapeutic and
stability is achieved.
[0279] More specifically, each peptide selected to undergo
modification with a linking group and a reactive group will be
modified according to the following criteria: if a terminal
carboxylic group is available on the peptide and is not critical
for the retention of therapeutic activity, and no other sensitive
functional group is present on the peptide, then the carboxylic
acid will be chosen as attachment point for the linking
group-reactive group modification. If the terminal carboxylic group
is involved in therapeutic activity, or if no carboxylic acids are
available, then any other sensitive functional group not critical
for the retention of therapeutic activity will be selected as the
attachment point for the linking group-reactive entity
modification. If several sensetive functional groups are available
on a a peptide, a combination of protecting groups will be used in
such a way that after addition of the linking group/reactive entity
and deprotection of all the protected sensetive functional groups,
retention of therapeutic activity is still obtained. If no
sensetive functional groups are available on the peptide, or if a
simpler modification route is desired, synthetic efforts will allow
for a modification of the original peptide in such a way that
retention of therapeutic is maintained. In this case the
modification will occur at the opposite end of the peptide
[0280] An NHS derivative may be synthesized from a carboxylic acid
in absence of other sensetive functional groups in the peptide.
Specifically, such a peptide is reacted with N-hydroxysuccinimide
in anhydrous CH.sub.2Cl.sub.2 and EDC, and the product is purified
by chromatography or recrystallized from the appropriate solvent
system to give the NHS derivative.
[0281] Alternatively, an NHS derivative may be synthesized from a
peptide that contains an amino and/or thiol group and a carboxylic
acid. When a free amino or thiol group is present in the molecule,
it is preferable to protect these sensetive functional groups prior
to perform the addition of the NHS derivative. For instance, if the
molecule contains a free amino group, a transformation of the amine
into a Fmoc or preferably into a tBoc protected amine is necessary
prior to perform the chemistry described above. The amine
functionality will not be deprotected after preparation of the NHS
derivative. Therefore this method applies only to a compound whose
amine group is not required to be freed to induce the desired
therapeutic effect. If the amino group needs to be freed to retain
the original properties of the molecule, then another type of
chemistry described below has to be performed.
[0282] In addition, an NHS derivative may be synthesized from a
peptide containing an amino or a thiol group and no carboxylic
acid. When the selected molecule contains no carboxylic acid, an
array of bifunctional linking groups can be used to convert the
molecule into a reactive NHS derivative. For instance, ethylene
glycol-bis(succinimydylsuccinate) (EGS) and triethylamine dissolved
in DMF and added to the free amino containing molecule (with a
ratio of 10:1 in favor of EGS) will produce the mono NHS
derivative. To produce an NHS derivative from a thiol derivatized
molecule, one can use N-[-maleimidobutyryloxy]succinimide ester
(GMBS) and triethylamine in DMF. The maleimido group will react
with the free thiol and the NHS derivative will be purified from
the reaction mixture by chromatography on silica or by HPLC.
[0283] An NHS derivative may also be synthesized from a peptide
containing multiple sensetive functional groups. Each case will
have to be analyzed and solved in a different manner. However,
thanks to the large array of protecting groups and bifunctional
linking groups that are commercially available, this invention is
applicable to any peptide with preferably one chemical step only to
modify the peptide (as described above) or two steps (as described
above involving prior protection of a sensitive group) or three
steps (protection, activation and deprotection). Under exceptional
circumstances only, would multiple steps (beyond three steps)
synthesis be required to transform a peptide into an active NHS or
maleimide derivative.
[0284] A maleimide derivative may also be synthesized from a
peptide containing a free amino group and a free carboxylic acid.
To produce a maleimide derivative from a amino derivatized
molecule, one can use N-[.gamma.-maleimidobutyryloxy]succinimide
ester (GMBS) and triethylamine in DMF. The succinimide ester group
will react with the free amino and the maleimide derivative will be
purified from the reaction mixture by crystallization or by
chromatography on silica or by HPLC.
[0285] Finally, a maleimide derivative may be synthesized from a
peptide containing multiple other sensetive functional groups and
no free carboxylic acids. When the selected molecule contains no
carboxylic acid, an array of bifunctional crosslinking reagents can
be used to convert the molecule into a reactive NHS derivative. For
instance maleimidopropionic acid (MPA) can be coupled to the free
amine to produce a maleimide derivative through reaction of the
free amine with the carboxylic group of MPA using HBTU/HOBt/DIEA
activation in DMF.
[0286] Many other commercially available heterobifunctional
crosslinking reagents can alternatively be used when needed. A
large number of bifunctional compounds are available for linking to
entities. Illustrative reagents include: azidobenzoyl hydrazide,
N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio)propionamide),
bis-sulfosuccinimidyl suberate, dimethyl adipimidate,
disuccinimidyl tartrate, N-y-maleimidobutyryloxysuccinimide ester,
N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl
[4-azidophenyl]-1,3'-di- thiopropionate, N-succinimidyl
[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carbo- xylate.
[0287] 5. Synthesis of Modified Organic Therapeutic Agents
[0288] Similar to procedures for modified peptide therapeutics, the
synthetic procedures used to prepare modified organic therapeutics
will also be selected so as to be simple, provide for high yields,
and allow for a highly purified product. Normally, the chemically
reactive group will be created as the last stage, for example, with
a carboxyl group, esterification to form an active ester will be
the last step of the synthesis. Methods for the production and/or
modification of organic therapeutic agents of the present invention
are described in the Examples below.
[0289] Each organic therapeutic agent selected to undergo the
derivatization with a linking group and a reactive group will be
modified according to the following criteria: Generally, the
therapeutic agents are commercially available. If not, they can be
synthesized by procedures well known in the art. As a first step,
the therapeutically active region of the therapeutic agent is
identified. Next, the therapeutic agent is modified at a site
sufficiently far away from the active portion to prevent a
potential interference between the modified drug and the target
site of the drug, such that the modified agent substantially
retains its therapeutic activity, (i.e. the therapeutic activity is
reduced by no more than 10 fold). Finally, keeping constant the
site of chemical modification, optimize the biological activity of
the modified agent by modifying the length and nature of the
linking group.
[0290] If a carboxylic group, not critical for the retention of
pharmacological activity is available on the original molecule and
no other reactive functionality is present on the molecule, then
the carboxylic acid will be chosen as attachment point for the
linking group-reactive entity modification. If no carboxylic acids
are available, then any other functionalities not critical for the
retention of pharmacological activity will be selected as
attachment point for the linking group-reactive entity
modification. If several functionalities are available on a
therapeutic agent, a combination of protecting groups will be used
in such a way that after addition of the linking group/reactive
entity and deprotection of all the protected functionalities,
retention of pharmacological activity is still obtained. If no
functionalities are available on the therapeutic agent, synthetic
efforts will allow for a modification of the original parent drug
in such a way that retention of biological activity and retention
of receptor or target specificity is obtained.
[0291] Where the derivatized therapeutic agent of the present
invention represents a derivatized enzyme inhibitor will generally
have substantially lower IC.sub.50's generally in the range of
about 0.5-0.01 of the IC.sub.50 of the parent molecule. Desirably,
the IC.sub.50 should be not less than 0.05, preferably not less
than about 0.1. In view of the varying IC.sub.50's, the amount of
the derivatized therapeutic agent administered will also vary.
[0292] The determination of the nature and length of the linking
group will be performed through an empirical optimization phase and
will be measured by the retention or the loss of biological
activity. For instance, with a given inhibitor enzyme interactions,
an iteration of the modification of the nature and the length of
the linking group and a measure of the biological enzymatic
activity may be necessary to determine the most favored linking
group length and nature. Preferably a short hydrophilic 4-12 atom
linking group easily synthesized will be favored to start the
iteration process.
[0293] In the case of radiolabeled therapeutic agents, a minimum
distance from the target has to be respected based on the nature of
the isotope and its penetration. The length and nature of the
linking groups are not as important as they are for an enzyme
inhibitor combination. For instance an isotope that emits a beta
rays like .sup.32P should be positioned within 5 mm from the target
to have maximum efficiency (99%) with limited or no effect coming
from a small change on the nature and length of the linking
group.
[0294] 6. Synthesis of Modified Diagnostic Agents
[0295] The manner of producing the modified diagnostic agents of
the present invention will vary widely, depending upon the nature
of the various elements comprising the molecule. The synthetic
procedures will be selected so as to be simple, provide for high
yields, and allow for a highly purified product. Normally, the
chemically reactive group will be created as the last stage, for
example, with a carboxyl group, esterification to form an active
ester will be the last step of the synthesis. Methods for the
production of the diagnostic agents of the present invention are
described in the Examples.
[0296] 7. Pulmonary Formulations and Delivery Methods
[0297] A further aspect of the invention is directed to aerosol
compositions for treatment of the symptoms of pulmonary conditions.
With therapeutic agents capable of forming a covalent bond with a
pulmonary fluid protein. Alternatively, the therapeutic agent may
be first conjugated with the pulmonary solution protein prior to
administration.
[0298] The aerosol compositions may be composed of an aqueous
solution suitable for inhalation consisting of at least 2.5% by
weight (more preferably between about 3% and 10% by weight, and
most preferably at least about 5% by weight) of a derivatized
therapeutic agent. The droplets of the aerosol should be 10.mu. or
less in diameter to maximize deposition in the lung alveoli rather
than the throat or upper respiratory tract.
[0299] The invention also features inhaler devices for
administration of the inhalable compositions (or medicaments) of
the subject invention. In one aspect of the invention, the inhaler
device comprises a housing defining a chamber which contains a dry
powder. The dry powder is composed of therapeutic agent compound
present in an amount that, upon administration will bind to
pulmonary proteins. Alternatively, the agent may be covalently
bonded to pulmonary proteins ex vivo prior to administration. At
least 50% (preferably at least 70%, and more preferably at least
90%) of the powder consists of primary particles which have a
diameter of 10 .mu.m or less, and which may be agglomerated into
larger particles or agglomerates which readily break down into
primary particles upon inhalation from the device. The chamber has
an opening through which the medicament can be drawn by inhalation
by a patient.
[0300] In another aspect of the invention, the inhaler device
comprises a vessel containing an inhalable medicament in the form
of an aqueous solution suspended in a compressed or liquified
propellant gas. At least 2.5% by weight (preferably at least 3%,
more preferably at least 4%, even more preferably at least 5% and
most preferably between 6 and 10%) of the aqueous solution is a
pH-raising buffer compound.
[0301] The inhaler device may also have a housing defining a port
onto which the vessel is mounted, a lumen in communication with the
port, and a mechanism for controllably releasing the propellant
from the vessel into the lumen, thereby releasing the suspended
medicament from the vessel into the lumen. The lumen is configured
to route the medicament suspended in the propellant into the
respiratory system of the patient.
[0302] 8. Therapeutic Uses of Modified Therapeutic Agents
[0303] The modified therapeutic agents of the invention find
numerous uses, as enumerated below.
[0304] A. Therapeutic Uses of Modified Antineoplastic Agent
[0305] The antineoplastic agents of the invention, including but
not limited to those specified in the examples, possess
anti-angiogenic activity. As modified antineoplastic agents having
anti-angiogenic activity, the compounds of the present invention
are useful in the treatment of a variety of diseases, for example
primary and metastatic solid tumors and carcinomas of the breast;
colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach.
Pancreas; liver; gallbladder; bile ducts; small intestine; urinary
tract including kidney, bladder and urothelium; female genital
tract including cervix, uterus, endometrium, ovaries,
choriocarcinoma and festational trophoblastic disease; male genital
tract including prostate, seminal vesicles, testes and germ cells
tumors; endocrine glands including thyroid, adrenal and pituitary;
skin including hmangiomas, melanomas, sarcomas arising from bone of
soft tissues and Karposi's sarcoma, Wilm's tumor, rhabdomyosarcoma;
tumor of the head and neck, brain, nerves, eyes, and meninges
including astrocytomas, gliomas, glioblastomas, retinoblastomas,
neuromas, neuroblastomas; tumors of the bone marrow and
hematopoeitic tumors, solid tumors arising from hematopoietic
malignancies such as leukemias and including chloromas,
plasmocytomas, plagues and tumors of mycosis fungoides and
cutaneous T-cell lymphoma/leukemia; acute lymphotic, actute
granulocytic and chronic granulocytic leukemia; lymphomas including
both Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of
autoimmune diseases including rheumatoid, immune and degenerative
arthritis; ocular diseases including diabethic retinopathy;
retinopathy of prematurity; corneal graft rejection, retrolental
fibroplasia, neovascular glaucoma, rubeosis, retinal
neovascularization due to macular degeneration and hypoxia;
abnormal neovascularization conditions of the eye; skin diseases
including psoriasis; blood vessel diseases including hemagiomas and
capillary proliferatrion withinatherosclerotic plaques; myocardial
angiogenesis; plaque neovascularization; hemophiliac joints;
angiofibroma; wound granulation; dieases charadterized by excessive
or abnormal stimulation of endothelial cells including intestinal
adhesion, Crohn's disease, atheroscelrosis, scleroderma and
hypertrophic scars and diseases which have angiogenesis as a
pathological consequence including ulcers (Helicobacter pilori);
rheumatoid arthritis, osteogenic sarcoma, osteoarthritis,
osteopenias such as osteoporosis, periodontitis, gingivitis,
corneal epidermal or gastric ulceration, and tumor metastasis,
invasion and growth, retinopathies, wound healing (ocular
inflammation, soft and osseous tissue disease,
gingivitis/periodontal disease), vascular disease (restenosis)
annuerysm inflammation, autoimmune diseases, and rare cancers such
as choriocarcinoma.
[0306] The compounds of the present invention may also be useful
for the prevention of metastases from the tumors described above
either when used alone or in combination with radiotherapy and/or
other chemotherapeutic treatments conventionally administered to
patients for treating angiogenic diseases. For example, when used
in the treatment of solid tumors, compounds of the present
invention may be administered with chemotherapeutic agents such as
alpha-inferon, COMP (cyclophosphamnide, vincristine, methotraxate
and prednisone), etoposide, mBACOD (methotraxate, bleomycin,
doxorubicin, cyclophosphamide, vincristine and dexamethasone),
PROMACE/MOPP (prednisone, methotrexate, doxirubicin,
cyclophaophamide, taxol, etoposide/mechloetamine, vincristine,
prednisone and procarbazine), vincristine, vinblastine,
angioinhibins, TNP-470, pentosan polysulfate, platelet factor-4,
angiostatin, LM-609, SU-101, CM-101, techgalan, thalidomide, SP-PG
and the like. Other chemotherapeutic agents include alkylating
agents such as nitrogen mustards including mechloethamine,
melphanchloambucil, cyclophaosphamide, and ifosfamide;
nirrosdoureas including carmustine, lomustine, semustine and
streptozocin; alkyl sulfonates icluding busulfan; triazines
including dacarbazine; ethyenimines including thiotepa na
dhexamethylmelanine; folic acid analogs including methotraxate;
pyrimidine analogs including 5-FU, cytosine arabinoside; purine
analogs including 6-mercaptopurine and 6-thioguanine; antitumor
antibiotics including actinomycin D; the anthraqcyclines including
doxorubicin, bleomycin, mitomycin C and methramycin; hormones and
hormones antagonists including tamoxifen and corticosteroids and
mioscellaneous agnets including cisplatin and brequinar; fragments
of plasminogen (kringle-5) as well as fragments from other
integrin-binding substrates. For insnance, a tumor may be treated
conventionally with surgery, radiation or chemiotherapy and
administration of modified antineoplastic agents to extend the
dormancy of micrometastasis and to inhibit the growth of any
residual primary tumor.
[0307] B. Therapeutic Uses of Modified Matrix Metalloprotease
Inhibitors
[0308] The MMPIs of the invention, including but not limited to
those specified in the examples, possess anti-angiogenic activity.
As modified matrix metalloprotease inhibitors having
anti-angiogenic activity, the compounds of the present invention
are useful in the treatment of a variety of diseases, for example
primary and metastatic solid tumors and carcinomas of the breast;
colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach.
Pancreas; liver; gallbladder; bile ducts; small intestine; urinary
tract including kidney, bladder and urothelium; female genital
tract including cervix, uterus, ovaries, choriocarcinoma and
festational trophoblastic disease; male genital tract including
prostate, seminal vesicles, testes and germ cells tumors; endocrine
glands including thyroid, adrenal and pituitary; skin including
hmangiomas, melanomas, sarcomas arising from bone of soft tissues
and Karposi's sarcoma; tumor of the nrain, nerves, eyes, and
meninges including astrocytomas, gliomas, glioblastomas,
retinoblastomas, neuromas, neuroblastomas; tumors of the bone
marrow and hematopoeitic tumors, solid tumors arising from
hematopoietic malignancies such as leukemias and including
chloromas, plasmocytomas, plagues and tumors of mycosis fungoides
and cutaneous T-cell lymphoma/leukemia; lymphomas including both
Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmune
diseases including rheumatoid, immune and degenerative arthritis;
ocular diseases including diabethic retinopathy; retinopathy of
prematurity; corneal graft rejection, retrolental fibroplasia,
neovascular glaucoma, rubeosis, retinal neovascularization due to
macular degeneration and hypoxia; abnormal neovascularization
conditions of the eye; skin diseases including psoriasis; blood
vessel diseases including hemagiomas and capillary proliferatrion
withinatherosclerotic plaques; myocardial angiogenesis; plaque
neovascularization; hemophiliac joints; angiofibroma; wound
granulation; dieases charadterized by excessive or abnormal
stimulation of endothelial cells including intestinal adhesion,
Crohn's disease, atheroscelrosis, scleroderma and hypertrophic
scars and diseases which have angiogenesis as a pathological
consequence including ulcers (Helicobacter pilori); rheumatoid
arthritis, osteoarthritis, osteopenias such as osteoporosis,
periodontitis, gingivitis, corneal epidermal or gastric ulceration,
and tumor metastasis, invasion and growth, retinopathies, wound
healing (ocular inflammation, soft and osseous tissue disease,
gingivitis/periodontal disease), vascular disease (restenosis)
annuerysm inflammation and and autoimmune diseases. Another use is
as birth control agent which inhibits ovulation and establishment
of the placenta.
[0309] The compounds of the present invention may also be useful
for the prevention of metastases from the tumors described above
either when used alone or in combination with radiotherapy and/or
other chemotherapeutic treatments conventionally administered to
patients for treating angiogenic diseases. For example, when used
in the treatment of solid tumors, compounds of the present
invention may be administered with chemotherapeutic agents such as
alpha-inferon, COMP (cyclophosphamnide, vincristine, methotraxate
and prednisone), etoposide, mBACOD (methotraxate, bleomycin,
doxorubicin, cyclophosphamide, vincristine and dexamethasone),
PROMACE/MOPP (prednisone, methotrexate, doxirubicin,
cyclophaophamide, taxol, etoposide/mechloetamine, vincristine,
prednisone and procarbazine), vincristine, vinblastine,
angioinhibins, TNP-470, pentosan polysulfate, platelet factor-4,
angiostatin, LM-609, SU-101, CM-101, techgalan, thalidomide, SP-PG
and the like. Other chemotherapeutic agents include alkylating
agents such as nitrogen mustards including mechloethamine,
melphanchloambucil, cyclophaosphamide, and ifosfamide;
nirrosdoureas including carmustine, lomustine, semustine and
streptozocin; alkyl sulfonates icluding busulfan; triazines
including dacarbazine; ethyenimines including thiotepa na
dhexamethylmelanine; folic acid analogs including methotraxate;
pyrimidine analogs including 5-FU, cytosine arabinoside; purine
analogs including 6-mercaptopurine and 6-thioguanine; antitumor
antibiotics including actinomycin D; the anthraqcyclines including
doxorubicin, bleomycin, mitomycin C and methramycin; hormones and
hormones antagonists including tamoxifen and corticosteroids and
mioscellaneous agnets including cisplatin and brequinar; fragments
of plasminogen (kringle-5) as well as fragments from other
integrin-binding substrates. For instance, a tumor may be treated
conventionally with surgery, radiation or chemiotherapy and the
modified MMPI molecules of the invention to extend the dormancy of
micrometastasis and to inhibit the growth of any residual primary
tumor.
[0310] It has also been found that hydroxamic acid MMPIs can
inhibit the production of the cytokine tumor necrosis factor (TNF)
(Mohler et al., Nature, 1994, 370, 218-220; Gearing AJH et al.,
Nature 1994, 370, 555-557; McGeehan G M et al., Nature 1994, 370,
558-561). Compounds which inhibit the production or action of TNF
are thought to be potentially useful for the treatment or
prophylaxis of many inflammatory, infectious, immunological or
malignant diseases. These include, but are not restricted to,
septic shock, haemodynamic shock and sepsis syndrome, post
ischaemic reperfusion injury, malaria, Crohn's disease,
mycobacterial infection, meningitis, psoriasis, congestive heart
failure, fibrotic disease, cachexia, graft rejection, cancer,
autoimmune disease, rheumatic arthritis, multiple scleroris,
radation damage, toxicity following administration of
immunosuppressive monoclonal antibodies such as OKT3 or CAMPATH-1
and hyperoxic alveolar injury. Since excessive TNF production has
been noted in several diseases or conditions also characterized by
MMP-mediated tissue degradation, compounds which inhibit both MMPs
and TNF production may have particular advantages in the treatment
or prophylaxis of diseases or conditions in which both mechanisms
are involved.
[0311] The compounds of the present invention inhibit various
enzymes from the matrix metalloproteinase family such as
collagenase, which initiates collagen breakdown, stromelysin
(protoglycanase), and gelatinase, and hence are useful for the
treatment of matrix metallo endoproteinase diseases. There is
evidence implicating collagenase as one of the key enzymes in the
breakdown of articular cartilage and bone in rheumatoid arthritis
(Arthritis and Rheumatism, 20, 1231-1239, 1977). Potent inhibitors
of collagenase and other metalloproteases involved in tissue
degradation are useful in the treatment of rheumatoid arthritis and
related diseases in which collagenolytic activity is important.
Inhibitors of metalloproteases of this type can therefore be used
in treating or preventing conditions which involve tissue
breakdown; they are therefore useful in the treatment of
arthropathy, dermatological conditions, bone resorption,
inflammatory diseases and tumour invasion and in the promotion of
wound healing. Specifically, compounds of the present invention may
be useful in the treatment of osteopenias such as osteoporosis,
rheumatoid arthritis, osteoarthritis, periodontitis, gingivitis,
corneal ulceration and tumour invasion.
[0312] C. Therapeutic Uses of Oxytocin
[0313] A conjugated oxytocin may be used to aid lactation and help
relax the pelvis prior to birth. It could also be used to prevent
post partum uterine hemorrage.
[0314] D. Therapeutic Uses of Cholecystokinin (CCK)
[0315] A conjugated CCK could be used in diagnostic studies of the
gall bladder or in chronic cholecystisis.
[0316] E. Therapeutic Uses of Antihypertensive Agents
[0317] Antihypertensive agents are used to treat hypertension.
[0318] F. Therapeutic Uses of Methylprednisolone
[0319] Methylprednisolone is used to treat a wide range of
disorders such as asthma and arthritis. In gastroenterology, it is
effective in the treatment of several inflammatory conditions such
as ulcerative and microscopic colitis, Crohn's disease and
autoimmune hepatitis. A newer usage is for reduction of
post-traumatic spinal cord edema.
[0320] G. Therapeutic Uses of GP-41 Peptides
[0321] GP-41, an HIV transmembrane protein, can be used to create
therapeutic and diagnostic agents against HIV. For example,
antibodies can be constructed to recognize epitopes of gp41. The
structure of this antibody will provide important information
regarding antibody/antigen interaction, guide chemists in the
selection of superior antigenic peptides for HIV detection, and
will provide important information for future recombinant
experiments with genetically engineered antibodies.
[0322] H. Therapeutic Uses of Blood Brain Barrier (BBB)
Peptides
[0323] As BBB peptides can traverse the blood brain barrier through
protein transduction, these peptides can be covalently linked to
compounds, peptides, antisense peptide nucleic acids or 40-nm iron
beads, or as in-frame fusions with full-length proteins, to allows
these compounds to enter any cell type in a receptor- and
transporter-independent fashion. This effectively delivers these
compounds past the blood brain barrier.
[0324] I. Therapeutic Uses of Modified Cell Adhesion (RGD)
Peptides
[0325] The RGD peptides of the invention and their derivatives and
analogs find multiple uses including use as a treatment for
neoplastic diseases and inflammatory diseases such as rheumatoid
arthritis, lupus.
[0326] 1. Anti-Neoplastic Treatments
[0327] The modified cell adhesion peptides of the invention or
their derivatives or analogs generally will target directly to
cancer cells via peptide-specific receptors. It has been shown that
receptors for these peptides are expressed at elevated levels on
the surface of tumor cells. Thus, the modified peptides or their
derivatives or analogs can be used to preferentially target drugs
to metastatic tumor cells. Therefore, the modified cell adhesion
peptides or their derivatives or analogs are useful as agents for
the treatment of different types of cancers such as breast
carcinoma, melanoma, and fibrosarcoma.
[0328] The use of an effective amount of modified cell adhesion
peptides or their derivatives or analogs as a treatment for cancer
has the advantage of being more potent than non modified cell
adhesion peptides. Since the modified cell adhesion peptides or
their derivatives or analogs are more stable in vivo, smaller
amounts of the molecule can be administered for effective
treatment.
[0329] The derivatives and conjugates of the modified cell adhesion
peptides and their analogs may be used in several different ways
and to achieve several different ends. As mentioned above, these
materials may be used in place of typical cell adhesion peptide
drugs as an anti-adhesive agent. As compared with cell adhesion
peptide drugs currently available, the materials of this invention
can reduce clot formation with less side effects and are available
for reducing clot formation for a substantially longer time than
conventionally administered cell adhesion peptide drugs. In
addition, the derivatized cell adhesion peptides of this invention
may be utilized (in accordance with U.S. Pat. Nos. 5,443.827;
5,439,88 and 5,433,940 and PCT application number WO/97/01093 which
are hereby incorporated by reference) in conjunction with various
other anti-adhesive or anti-cancer therapies. Such anti-cancer
therapies include the use of radiation or treatment with
antineoplastic agents such as, for example, vinca alkaloids,
alkylating agents, doxorubicin, etoposide, methotrexate, tamoxifen,
vinblastine, asparaginase, biclutamide, bleomycin, carboplatin,
carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, daunorubisin, docetaxel,
floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea,
idarubicin, ifosfamide, interferon alpha, irnotecan, leuprolide,
mechlorethamine, megestrol, melphalan, mercaptopurine, mitomycin,
mitoxantrone, paclitaxel, plicamycin, porfirmer, procarbazine,
streptozocin, teniposide, thioguanine, thiotepa, topotecan,
trastuzumab, vincristine, vinorelbine, and the like.
[0330] The present invention also provides for a method for
treating cancer in an individual, wherein said method comprises
providing an amount of modified cell adhesion peptide sufficient to
treat cancer; where the composition contains a modified cell
adhesion peptide.
[0331] 2. Treatment Of Inflammatory Disease
[0332] The modified cell adhesion peptides of the invention and
their derivatives and analogs also find use as anti-inflammatories.
In one aspect of the invention, there is provided a method of
treating a mammalian subject with an abnormality resulting in
increased inflammation of the joints or tissues using the modified
cell adhesion peptides of the invention or their derivatives or
analogs. The method comprises administering a modified cell
adhesion peptide or its derivative or analog to the subject in an
amount sufficient to produce an anti-inflammatory effect on the
subject. The modified cell adhesion peptide may be administered
intracerebroventriculary, orally, subcutaneously, intramuscularly,
or intravenously.
[0333] The peptides of the present invention, their derivatives,
analogs, and conjugates can be used to treat acute or chronic
inflammatory disorders involving ischemia, infection, tissue
swelling, and/or bone and cartilage degradation. Inflammatory
disease refers to a condition in which activation of leukocytes
leads to an impairment of normal physiologic function. Examples of
such conditions include acute and chronic inflammation such as
osteoarthritis, sepsis, ARDS, immune and autoimmune disorders,
rheumatoid arthritis, IBD (inflammatory bowel disease), lupus, MS,
graft rejection, cirrhosis, sarcoidosis, granulomatous lesions,
periodontitis/gingivitis, graft-vs.-host disease, contact
dermatitis, and the like. Included among autoimmune disorders which
may be treated using the present method are chronic active
hepatitis, Graves' disease, insulin-dependent diabetes mellitus
(type I), and Hasshimoto's thyroiditis. Included among inflammatory
disorders which may be treated using the present method are
inflammatory brain disease, inflammatory demyelinating disease,
inflammatory vasculitis, inflammatory myopathies, osteomyelitis,
Crohn's disease and interstitial cystitis. Additional examples of
inflammatory diseases include myocardial diseases, infectious
diseases, pulmonary diseases and graft rejection.
[0334] J. Therapeutic Uses of Modified Insulinotropic Peptides Such
as GLP-1
[0335] The modified insulinotropic peptides (ITPs) such as GLP-1 of
the invention find multiple uses including use as a treatment for
diabetes, a sedative, a treatment of nervous system disorders, use
to induce an anxiolytic effect on the CNS, use to activate the CNS,
use for post surgery treatment and as a treatment for insulin
resistance.
[0336] 1. Diabetes Treatments
[0337] The modified ITPs of the invention generally will normalize
hyperglycemia through glucose-dependent, insulin-dependent and
insulin-independent mechanisms. As such, the modified ITPs are
useful as primary agents for the treatment of type II diabetes
mellitus and as adjunctive agents for the treatment of type I
diabetes mellitus.
[0338] The use of an effective amount of modified ITPs as a
treatment for diabetes mellitus has the advantage of being more
potent than non modified ITPs. Since the modified ITPs are move
stable in vivo, smaller amounts of the molecule can be administered
for effective tratment. The present invention is especially suited
for the treatment of patients with diabetes, both type I and type
II, in that the action of the peptide is dependent on the glucose
concentration of the blood, and thus the risk of hypoglycemic side
effects are greatly reduced over the risks in using current methods
of treatment.
[0339] The present invention also provides for a method for
treating diabetes mellitus in an individual, wherein said method
comprises providing an amount of modified ITP sufficient to treat
diabetes; where the composition contains a modified ITP.
[0340] 2. Treatment of Nervous System Disorders
[0341] The modified ITPs of the invention also find use as a
sedative. In one aspect of the invention, there is provided a
method of sedating a mammalian subject with an abnormality
resulting in increased activation of the central or peripheral
nervous system using the modified ITPs of the invention. The method
comprises administering a modified ITP to the subject in an amount
sufficient to produce a sedative or anxiolytic effect on the
subject. The modified ITP may be administered
intracerebroventriculary, orally, subcutaneously, intramuscularly,
or intravenously. Such methods are useful to treat or ameliorate
nervous system conditions such as anxiety, movement disorder,
aggression, psychosis, seizures, panic attacks, hysteria and sleep
disorders.
[0342] In a related aspect, the invention encompasses a method of
increasing the activity of a mammalian subject, comprising
administering a modified ITP to the subject in an amount sufficient
to produce an activating effect on the subject. Preferably, the
subject has a condition resulting in decreased activation of the
central or peripheral nervous system. The modified ITPs find
particular use in the treatment or amelioration of depression,
schizoaffective disorders, sleep apnea, attention deficit syndromes
with poor concentration, memory loss, forgetfulness, and
narcolepsy, to name just a few conditions in which arousal of the
central nervous system may be advantageous.
[0343] The modified ITPs of the invention may be used to induce
arousal for the treatment or amelioration of depression,
schizoaffective disorders, sleep apnea, attention deficit syndromes
with poor concentration, memory loss, forgetfulness, and
narcolepsy. The therapeutic efficacy of the modified ITP treatment
may be monitored by patient interview to assess their condition, by
psychological/neurologica- l testing, or by amelioration of the
symptoms associated with these conditions. For example, treatment
of narcolepsy may be assessed by monitoring the occurrence of
narcoleptic attacks. As another example, effects of modified ITPs
on the ability of a subject to concentrate, or on memory capacity,
may be tested using any of a number of diagnostic tests well known
to those of skill in art.
[0344] 3. Post Surgery Treatment
[0345] The modified ITPs of the invention may be utilized for post
surgery treatments. A patient is in need of the modified ITPs of
the present invention for about 1-16 hours before surgery is
performed on the patient, during surgery on the patient, and after
the patient's surgery for a period of not more than about 5
days.
[0346] The modified ITPs of the present invention are administered
from about sixteen hours to about one hour before surgery begins.
The length of time before surgery when the compounds used in the
present invention should be administered in order to reduce
catabolic effects and insulin resistance is dependent on a number
of factors. These factors are generally known to the physician of
ordinary skill, and include, most importantly, whether the patient
is fasted or supplied with a glucose infusion or beverage, or some
other form of sustenance during the preparatory period before
surgery. Other important factors include the patient's sex, weight
and age, the severity of any inability to regulate blood glucose,
the underlying causes of any inability to regulate blood glucose,
the expected severity of the trauma caused by the surgery, the
route of administration and bioavailability, the persistence in the
body, the formulation, and the potency of the compound
administered. A preferred time interval within which to begin
administration of the modified ITPs used in the present invention
is from about one hour to about ten hours before surgery begins.
The most preferred interval to begin administration is between two
hours and eight hours before surgery begins.
[0347] Insulin resistance following a particular type of surgery,
elective abdominal surgery, is most profound on the first
post-operative day, lasts at least five days, and may take up to
three weeks to normalize Thus, the post-operative patient may be in
need of administration of the modified ITPs used in the present
invention for a period of time following the trauma of surgery that
will depend on factors that the physician of ordinary skill will
comprehend and determine. Among these factors are whether the
patient is fasted or supplied with a glucose infusion or beverage,
or some other form of sustenance following surgery, and also,
without limitation, the patient's sex, weight and age, the severity
of any inability to regulate blood glucose, the underlying causes
of any inability to regulate blood glucose, the actual severity of
the trauma caused by the surgery, the route of administration and
bioavailability, the persistence in the body, the formulation, and
the potency of the compound administered. The preferred duration of
administration of the compounds used in the present invention is
not more than five days following surgery.
[0348] 4. Insulin Resistance Treatment
[0349] The modified ITPs of the invention may be utilized to treat
insulin resistance independently from their use in post surgery
treatment. Insulin resistance may be due to a decrease in binding
of insulin to cell-surface receptors, or to alterations in
intracellular metabolism. The first type, characterized as a
decrease in insulin sensitivity, can typically be overcome by
increased insulin concentration. The second type, characterized as
a decrease in insulin responsiveness, cannot be overcome by large
quantities of insulin. Insulin resistance following trauma can be
overcome by doses of insulin that are proportional to the degree of
insulin resistance, and thus is apparently caused by a decrease in
insulin sensitivity.
[0350] The dose of modified ITPs effective to normalize a patient's
blood glucose level will depend on a number of factors, among which
are included, without limitation, the patient's sex, weight and
age, the severity of inability to regulate blood glucose, the
underlying causes of inability to regulate blood glucose, whether
glucose, or another carbohydrate source, is simultaneously
administered, the route of administration and bioavailability, the
persistence in the body, the formulation, and the potency.
[0351] K. Therapeutic Uses of Modified Kringle 5 Peptides
[0352] As described earlier, angiogenesis includes a variety of
processes involving neovascularization of a tissue including
"sprouting", vasculogenesis, or vessel enlargement. With the
exception of traumatic wound healing, corpus leuteum formation and
embryogenesis, it is believed that the majority of angiogenesis
processes are associated with disease processes and therefore the
use of the present therapeutic methods are selective for the
disease and do not have deleterious side effects.
[0353] There are a variety of diseases in which angiogenesis is
believed to be important, which may be treatable with the modified
peptides of the invention. These diseases include, but not limited
to, inflammatory disorders such as immune and non-immune
inflammation, chronic articular rheumatism and psoriasis, disorders
associated with inappropriate or inopportune invasion of vessels
such as diabetic retinopathy, neovascular glaucoma, restenosis,
capillary proliferation in atherosclerotic plaques and
osteoporosis, and cancer associated disorders, such as solid
tumors, solid tumor metastases, angiofibromas, retrolental
fibroplasia, hemangiomas, Kaposi sarcoma and the like cancers which
require neovascularization to support tumor growth.
[0354] The modified kringle 5 peptides of the invention find use in
methods which inhibit angiogenesis in a diseased tissue ameliorates
symptoms of the disease and, depending upon the disease, can
contribute to cure of the disease. The modified peptides of the
invention are more stable in vivo and, as such, smaller amounts of
the modified peptide can be administered for effective treatment In
one embodiment, the invention contemplates inhibition of
angiogenesis, per se, in a tissue. The extent of angiogenesis in a
tissue, and therefore the extent of inhibition achieved by the
present methods, can be evaluated by a variety of method, for
detecting .A-inverted..sub.5#.sub.3-immunopositive immature and
nascent vessel structures by immunohistochemistry.
[0355] As described herein, any of a variety of tissues, or organs
comprised of organized tissues, can support angiogenesis in disease
conditions including skin, muscle, gut, connective tissue, joints,
bones and the like tissue in which blood vessels can invade upon
angiogenic stimuli.
[0356] In one related embodiment, a tissue to be treated with the
modified kringle 5 peptides of the invention is an inflamed tissue
and the angiogenesis to be inhibited is inflamed tissue
angiogenesis where there is neovascularization of inflamed tissue.
In this class the method contemplates inhibition of angiogenesis in
arthritic tissues, such as in a patient with chronic articular
rheumatism, in immune or non-immune inflamed tissues, in psoriatic
tissue and the like.
[0357] The patient treated in the present invention in its many
embodiments is desirably a human patient, although it is to be
understood that the principles of the invention indicate that the
invention is effective with respect to all mammals, which are
intended to be included in the term "patient." In this context, a
mammal is understood to include any mammalian species in which
treatment of diseases associated with angiogenesis is desirable,
particularly agricultural and domestic mammalian species.
[0358] In another related embodiment, a tissue to be treated with
the modified kringle 5 peptides of the invention is a retinal
tissue of a patient with diabetic retinopathy, macular degeneration
or neovascular glaucoma and the angiogenesis to be inhibited is
retinal tissue angiogenesis where there is neovascularization of
retinal tissue.
[0359] In an additional related embodiment, a tissue to be treated
with the modified kringle 5 peptides of the invention is a tumor
tissue of a patient with a solid tumor, a metastases, a skin
cancer, a breast cancer, a hemangioma or angiofibroma and the like
cancer, and the angiogenesis to be inhibited is tumor tissue
angiogenesis where there is neovascularization of a tumor tissue.
Typical solid tumor tissues treatable by the present methods
include lung, pancreas, breast, colon, laryngeal, ovarian, and the
like tissues.
[0360] Inhibition of tumor tissue angiogenesis is a particularly
preferred embodiment because of the important role
neovascularization plays in tumor growth. In the absence of
neovascularization of tumor tissue, the tumor tissue does not
obtain the required nutrients, slows in growth, ceases additional
growth, regresses and ultimately becomes necrotic resulting in
killing of the tumor.
[0361] The present invention thus provides for a method of
inhibiting tumor neovascularization by inhibiting tumor
angiogenesis according to the present methods using the modified
kringle 5 peptides of the invention. Similarly, the invention
provides a method of inhibiting tumor growth by practicing the
angiogenesis-inhibiting methods. The methods are also particularly
effective against the formation of metastases because (1) their
formation requires vascularization of a primary tumor so that the
metastatic cancer cells can exit the primary tumor and (2) their
establishment in a secondary site requires neovascularization to
support growth of the metastases.
[0362] In a related embodiment, the invention contemplates the
practice of the method in conjunction with other therapies such as
conventional chemotherapy directed against solid tumors and for
control of establishment of metastases. The administration of the
modified kringle 5 peptides of the invention is typically conducted
during or after chemotherapy, although it is preferably to inhibit
angiogenesis after a regimen of chemotherapy at times where the
tumor tissue will be responding to the toxic assault by inducing
angiogenesis to recover by the provision of a blood supply and
nutrients to the tumor tissue. In addition, it is preferred to
administer the modified kringle 5 peptides after surgery where
solid tumors have been removed as a prophylaxis against metastases.
Insofar as the present methods apply to inhibition of tumor
neovascularization, the methods can also apply to inhibition of
tumor tissue growth, to inhibition of tumor metastases formation,
and to regression of established tumors using the modified kringle
5 peptides of the invention.
[0363] Restenosis is a process of smooth muscle cell (SMC)
migration and proliferation at the site of percutaneous
transluminal coronary angioplasty which hampers the success of
angioplasty. The migration and proliferation of SMC's during
restenosis can be considered a process of angiogenesis which is
inhibited by the modified kringle 5 peptides of the present
invention. Therefore, the invention also contemplates inhibition of
restenosis by inhibiting angiogenesis in a patient following
angioplasty procedures. For inhibition of restenosis, the modified
kringle 5 peptide is typically administered after the angioplasty
procedure for from about 2 to about 28 days, and more typically for
about the first 14 days following the procedure.
[0364] The present method for inhibiting angiogenesis in a tissue
comprises contacting a tissue in which angiogenesis is occurring,
or is at risk for occurring, with a composition comprising a
therapeutically effective amount of a modified kringle 5 peptide.
The dosage ranges for the administration of the modified kringle 5
peptide depend upon the form of the peptide, and its potency, as
described further herein, and are amounts large enough to produce
the desired effect in which angiogenesis and the disease symptoms
mediated by angiogenesis are ameliorated. The dosage should not be
so large as to cause adverse side effects, such as hyperviscosity
syndromes, pulmonary edema, congestive heart failure, and the like.
Generally, the dosage will vary with the age, condition, sex and
extent of the disease in the patient and can be determined by one
of skill in the art. The dosage can also be adjusted by the
individual physician in the event of any complication.
[0365] As angiogenesis inhibitors, such modified kringle 5 peptides
are useful in the treatment of both primary and metastatic solid
tumors and carcinomas of the breast; colon; rectum; lung;
oropharynx; hypopharynx; esophagus, stomach; pancreas; liver;
gallbladder; bile ducts; small intestine; urinary tract including
kidney, bladder and urothelium; female genital tract including
cervix, uterus, ovaries, choriocarcinoma and gestational
trophoblastic disease; male genital tract including prostate,
seminal vesicles, testes and germ cell tumors; endocrine glands
including thyroid, adrenal, and pituitary; skin including
hemangiomas, melanomas, sarcomas arising from bone or soft tissues
and Kaposi's sarcoma; tumors of the brain, nerves, eyes, and
meninges including astrocytomas, gliomas, glioblastomas,
retinoblastomas, neuromas, neuroblastomas, Schwannomas and
meningiomas; solid tumors arising from hematopoietic malignancies
such as leukemias and including chloromas, plasmacytomas, plaques
and tumors of mycosis fungoides and cutaneous T-cell
lymphoma/leukemia; lymphomas including both Hodgkin's and
non-Hodgkin's lymphomas; prophylaxis of autoimmune diseases
including rheumatoid, immune and degenerative arthritis; ocular
diseases including diabetic retinopathy, retinopathy of
prematurity, corneal graft rejection, retrolental fibroplasia,
neovascular glaucoma, rubeosis, retinal neovascularization due to
macular degeneration and hypoxia; abnormal neovascularization
conditions of the eye; skin diseases including psoriasis; blood
vessel diseases including hemagiomas and capillary proliferation
within atherosclerotic plaques; Osler-Webber Syndrome; myocardial
angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; wound granulation; diseases
characterized by excessive or abnormal stimulation of endothelial
cells including intestinal adhesions, Crohn's disease,
atherosclerosis, scleroderma and hypertrophic scars (i.e. keloids)
and diseases which have angiogenesis as a pathologic consequence
including cat scratch disease (Rochele minalia quintosa) and ulcers
(Helicobacter pylori). Another use is as a birth control agent
which inhibits ovulation and establishment of the placenta.
[0366] The modified kringle 5 peptides of the present invention may
also be useful for the prevention of metastases from the tumors
described above either when used alone or in combination with
radiotherapy and/or other chemotherapeutic treatments
conventionally administered to patients for treating angiogenic
diseases. For example, when used in the treatment of solid tumors,
the modified kringle 5 peptides of the present invention may be
administered with chemotherapeutic agents such as alpha inteferon,
COMP (cyclophosphamide, vincristine, methotrexate and prednisone),
etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,
cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP
(prednisone, methotrexate (w/leucovin rescue), doxorubicin,
cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine,
prednisone and procarbazine), vincristine, vinblastine,
angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,
angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG
and the like. Other chemotherapeutic agents include alkylating
agents such as nitrogen mustards including mechloethamine, melphan,
chlorambucil, cyclophosphamide and ifosfamide; nitrosoureas
including carmustine, lomustine, semustine and streptozocin; alkyl
sulfonates including busulfan; triazines including dacarbazine;
ethyenimines including thiotepa and hexamethylmelamine; folic acid
analogs including methotrexate; pyrimidine analogues including
5-fluorouracil, cytosine arabinoside; purine analogs including
6-mercaptopurine and 6-thioguanine; antitumor antibiotics including
actinomycin D; the anthracyclines including doxorubicin, bleomycin,
mitomycin C and methramycin; hormones and hormone antagonists
including tamoxifen and cortiosteroids and miscellaneous agents
including cisplatin and brequinar. For example, a tumor may be
treated conventionally with surgery, radiation or chemotherapy and
kringle 5 administration with subsequent kringle 5 administration
to extend the dormancy of micrometastases and to stabilize and
inhibit the growth of any residual primary tumor.
[0367] L. Therapeutic Uses of Modified Opioid Molecules and
Analgesic Agents
[0368] The derivatives and conjugates of the opioid molecules and
analgesic agents may be used in several different ways and to
achieve several different ends. These materials may be used in
place of typical antinociceptive agents for alleviating pain. As
compared with drugs currently available, the materials of this
invention can alleviate pain without central mediated side effects
or potential of addiction or loss of efficacy, and are available
for alleviating pain for a substantially longer time than
conventionally administered drugs. Opioid derivatives and
conjugates of this invention also may be utilized (in accordance
with U.S. Pat. No. 5,482,930) as anti-inflammatory and/or
anti-irritation agents or in general to inhibit vascular leakage
from tissues. In addition, as is known in the art, these materials
may be used to treat hosts which are or have become tolerant to
morphine (or to treat patients undergoing methadone treatment
programs), as well as treatment of narcotics withdrawal in
general.
[0369] M. Therapeutic Uses of Modified Immuno-Suppressants
[0370] A variety of immuno-suppressant agents such as cyclosporin
and derivatives, corticosteroids, sulfasalazine, thalidomide,
methotrexate, OKT3, peptide-T, or agents that inhibit T-cell
activation or adhesion would be useful to prior to transplantation
to mask immune responsiveness and organ rejection. Such agents
could be applied at the time of tissue harvest (e.g. heart, lung,
liver harvest) or immediately prior to restitution of blood flow in
the recipient. Such immuno-suppressant agents would prevent the
recognition of foreign antigen from the donor tissue that would
facilitate short term acceptance and facilitate longer term ability
for the host to accommodate the transplanted organ.
[0371] N. Therapeutic Uses of Modified Antibiotics:
[0372] The modified antibiotics of the invention find use in
treating infections.
[0373] O. Therapeutic Uses of Modified Antidepressants
[0374] The modified antidepressants of the invention are useful for
treating depression.
[0375] P. Therapeutic Uses of Modified Anti-Viral and
Anti-Fusogenic Peptides HIV and Anti-HIV Peptides:
[0376] The human immunodeficiency virus (HIV), which is responsible
for acquired immune deficiency syndrome (AIDS), is a member of the
lentivirus family of retroviruses. There are two prevalent types of
HIV, HIV-1 and HIV-2, with various strain of each having been
identified. HIV targets CD-4+ cells, and viral entry depends on
binding of the HIV protein gp41 to CD-4+ cell surface
receptors.
[0377] Modified anti-viral or anti-fusogenic peptides of the
invention may be used as a therapeutic agent in the treatment of
patients who are suffering from HIV infection, and can be
administered to patients according to the methods described below
and other methods known in the art. Effective therapeutic dosages
of the modified peptides may be determined through procedures well
known by those in the art and will take into consideration any
concerns over potential toxicity of the peptide.
[0378] The modified peptides can also be administered
prophylactically to previously uninfected individuals. This can be
advantageous in cases where an individual has been subjected to a
high risk of exposure to a virus, as can occur when individual has
been in contact with an infected individual where there is a high
risk of viral transmission. This can be expecially advantageous
where there is no known cure for the virus, such as the HIV virus.
As a example, prophylactic administration of a modified anti-HIV
peptide would be advantageous in a situation where a health care
worker has been exposed to blood from an HIV-infected individual,
or in other situations where an individual engaged in high-risk
activities that potentially expose that individual to the HIV
virus.
[0379] 1. SIV and anti-SIV peptides: Simian immunodeficiency
viruses (SIV) are lentiviruses that cause acquired immunodeficiency
syndrome (AIDS)-like illnesses in susceptible monkeys. Modified
anti-viral peptides according to the invention can be used for the
treatment of infected animals or as a prophylactic in a similar
fashion as for HIV.
[0380] 2. RSV: Respiratory syncytial virus (RSV) is a respiratory
pathogen, especially dangerous in infants and small children where
it can cause bronchiolitis (inflammation of the small air passages)
and pneumonia. RSVs are negative sense, single stranded RNA viruses
and are members of the Paramyxoviridae family of viruses. The route
of infection of RSV is typically through the mucous membranes by
the respiratory tract, i.e., nose, throat, windpipe and bronchi and
bronchioles. Antiviral peptides according to the invention can be
used for prevention and treatment of RSV related diseases.
[0381] 3. HPV: Human parainfluenza virus (HPIV or HPV), like RSV,
is another leading cause of respiratory tract disease, and like
RSVs, are negative sense, single stranded RNA viruses that are
members of the Paramyxoviridae family of viruses. There are four
recognized serotypes of HPIV--HPIV-1, HPIV-2, HPIV-3 and HPIV-4.
HPIV-1 is the leading cause of croup in children, and both HPIV-1
and HPIV-2 cause upper and lower respiratory tract illnesses.
HPIV-3 is more often associated with bronchiolitis and pneumonia.
Antiviral peptides according to the invention can be used for
treatment of HPV related diseases.
[0382] 4. MeV: Measles virus (MV or MeV) is an enveloped negative,
single-stranded RNA virus belonging to the Paramyxoviridae family
of viruses. Like RSV and HPV, MeV causes respiratory disease, and
also produces an immuno-suppression responsible for additional,
opportunistic infections. In some cases, MeV can establish
infection of the brain leading to severe neurlogical complications.
Antiviral peptides according to the invention can be used for
treatment of RSV related diseases.
[0383] Q. Therapeutic Uses of Modified Antihistamine Agents
[0384] Modified anthistamine agents find use in treating excess
histamine formed in body tissues including allergic reactions.
[0385] R. Therapeutic Uses of Modified Anti-Angina Agents
[0386] Modified anthi-angina agents find use in treating angina
including treatment of choking and suffocating sensations.
[0387] Angina results from insufficient blood supply to the heart,
and is often caused by blockages in the arteries that feed the
heart muscle with blood (coronary artery stenoses due to
atherosclerosis). "Unstable" angina conditions, can develop into
acute coronary syndromes (ACS), including myocardial infarction.
Antianginal therapies include treatment with nitroglycerin and the
use of aspirin and heparin.
[0388] Platelet activation and aggregation play an important and
essential role in the formation of intracoronary thrombus in acute
coronary syndromes (ACS). Glycoprotein IIb/IIIa receptor inhibitors
are currently used in connection with heparin and aspirin in ACS.
Glycoprotein IIb/IIIa receptor inhibitors block the final step for
platelet aggregation and fibrinogen binding, thus preventing
thrombus formation. Tirofiban is a potent, synthetic, non-peptide
and specific glycoprotein IIb/IIIa receptor inhibitor and has shown
to be well tolerated and to reduce the risk of ischaemic
complications in patients with unstable angina, non-Q-wave
myocardial infarction and high-risk patients undergoing
revascularisation when used in combination with aspirin and
heparin. Other GP IIb/IIIa receptor inhibitors include abciximab
and eptifibatide.
[0389] S. Use of Modified Thyroxine Molecules
[0390] Thyroxine, an amino acid of the thyroid gland (Merck Index,
1989, 9348:1483) and thyroxine analogues are well-known in the art.
It is well established in the literature that thyroid hormones,
specifically thyroxines T3 and T4, have two distinct types of
biological actions: one on cell metabolism, the second on cell
differentiation and development (Jorgensen, 1978, "Thyroid Hormones
and Analogues II. Structure-Activity Relationships," In: Hormonal
Proteins and Peptides, Vol. VI, pp.107-204, C. H. Li, ed., Academic
Press, New York). For example, thyroxine suppresses uptake of
iodine by the thyroid (Money et al., 1959, "The Effect of Various
Thyroxine Analogues on Suppression of Iocline-131 Uptake by the Rat
Thyroid," Endocrinology 64:123-125) and induces cell
differentiation as studied by tadpole metamorphosis (Money et al.,
1958, "The Effect of Change in Chemical Structure of Some Thyroxine
Analogues on the Metamorphosis of Rana Pipiens Tadpoles,"
Endocrinology 63:20-28). Additionally, thyroxine and certain
thyroxine analogues depress growth of non-malignant mouse pituitary
thyrotropic tumors (Kumaoka et al., 1960, "The Effect of Thyroxine
Analogues on a Transplantable Mouse Pituitary Tumor," Endocrinology
66:32-38; Grinberg et al., 1962, "Studies with Mouse Pituitary
Thyrotropic Tumors. V. Effect of Various Thyroxine Analogs on
Growth and Secretion," Cancer Research 22:835-841).
[0391] The structural requirements of thyroxine and thyroxine
analogues for metabolic stimulation and induction of cell
differentiation are not identical (see Jorgensen, 1978, "Thyroid
Hormones and Analogues II. Structure-Activity Relationships," In:
Hormonal Proteins and Peptides, Vol. VI, p. 150, C. H. Li, ed.,
Academic Press, New York). For example, Money et al. have found
that there is no correlation between suppression of thyroid iodine
uptake and induction of tadpole metamorphosis (Money et al., 1958,
"The Effect of Change in Chemical Structure of Some Thyroxine
Analogues on the Metamorphosis of Rana Pipiens Tadpoles,"
Endocrinology 63:20-28). Based on these observations, it was
conceived that as yet unidentified cellular responses may be
altered or induced by certain thyroxine analogues which do not
exhibit either mode of action (metabolic or differentiating)
exhibited by thyroxine T3 and T4.
[0392] Deficiency of thyroid activity, whether occurring
spontaneously or resulting from surgical removal of thyroid gland,
thyroiditis, or decreased function secondary to pituitary
degeneration results in clinical hypothyroidism. Whatever the
cause, the symptom is treated by replacement therapy using the
modified thyroxine molecules of the invention.
[0393] The present invention also relates to a method for the
treatment of anemia which is associated with rheumatoid arthritis
and of the anemia present in patients having a viral or bacterial
infection wherein symptoms of rheumatoid arthritis are additionally
present using the modified thyroxine molecules of the
invention.
[0394] According to the invention, the associated anemia which is
characterized as being moderately hypochromic and normocytic is
treated by administering to a patient in need of treatment a
composition for increasing the thyroxine in the blood stream and
thereby increasing the ceiling on the number of red cells maturing
from the stem cells in the blood stream. The composition can
include the presence of an anti-inflammatory agent so as to treat
the inflammation present and reduce any pain.
[0395] T. Use of Modified Bronchodialators and Anti-Asthmatic
Agents
[0396] Anti-asthmatic agents find use in the treatment of asthma
and other lung diseases. Such anti-asthmatic agents include
bronchodialators like albuterol (Proventil or Ventolin) and
maleimidopropionamyl-1-theobrominea- cetamide.
[0397] U. Uses of Modified Diagnostic Agents
[0398] The diagnostic agent employed and the vascular protein or
proteins targeted will depend upon whether one wishes to
diagnostically image the anatomic compartment over an extended
period of time, whether one wishes to preferentially image only a
specific cell type or compartment, or both. Applications for
covalently bonding a diagnostic agent of interest to a long-lived
vascular protein for diagnostic imaging of the vascular space over
an extended period of time are numerous and include enhancing the
ability to detect abnormalities in blood flow throughout the entire
mammalian vascular system, including the detection of internal
injury causing abnormal bleeding or, alternatively, the presence of
thromboses. For example, one may wish to image the vascular space
over an extended period of time to detect the effects of a
particular treatment while they occur, i.e., detecting the
disappearance of an embolism, the stoppage of internal bleeding, or
the like.
[0399] Diagnostically imaging the vascular space over an extended
period of time also allows for the detection of various diseases
associated with the vascular system, i.e., such as arterial
blockage in the heart. Thus, diagnostically imaging the vascular
system over an extended period of time may be employed to
non-invasively detect a consistently reduced blood flow to the
heart. Such a method also provides a means for quantitatively
measuring cardiac efficiency and ventricular output volume over an
extended period of time, i.e., during extended periods of exercise,
or the like.
[0400] Other applications for such a method arise from the ability
to non-invasively visualize anatomical structures of the mammalian
vascular system and the effects on those anatomical structures over
time of the administration of various drugs, such as vasodilators,
vasoconstrictors, or the like. Such may allow for the early
detection of developmental vascular abnormalities, injuries, or the
like.
[0401] Additional applications arising from the ability to
diagnostically image the vascular space over an extended period of
time include functional assessment of the cardiovascular system as
routinely utilized in nuclear medicine for single measurements.
[0402] Applications for preferentially bonding a diagnostic agent
of interest to a specific protein or proteins present in the
vascular system so as to diagnostically image only a specific cell
type or compartment are also numerous. For example, having the
ability to preferentially direct a diagnostic agent of interest to
a specific cell type in the vascular system can allow for the
non-invasive and early detection of lesions or various tumors
associated with the mammalian vascular system by directing the
bifunctional anchor molecule to a tumor specific cell surface
protein.
[0403] Additionally, diagnostic agents can be directed to cell
surface proteins of specific cell types predominantly associated
with specific anatomic compartments, allowing one to preferentially
diagnostically image such compartments as lymph nodes, Peyer's
patches, kidney glomeruli, liver, pancreas, tonsil, or any other
organ to which mobile cells in the vasculature will migrate.
[0404] Other applications for preferentially diagnostic imaging a
specific cell type or compartment of the vascular system include
diagnosis and treatment of stenosis or plaque, vascular shunt
reendothelialization or shunt failure due to tissue growths, or
organ rejection due to tissue migration.
[0405] The diagnostic agents of the invetion may be delivered to a
local site via a local delivery device. Delivery devices include
catheters, syringes, trocars and endoscopes. Delivery of the agent
to a local site allows imaging of the specific area of delivery.
The agents that find particular use in localized delivery are the
non-specific diagnostic agents such as NHS-derivatives.
[0406] The invention can be more clearly illustrated by the
following non-limiting examples.
EXAMPLE 1
Preparation of Modified RGD Peptide AGYKPEGKRGDAK
[0407] RGD peptide AGYKPEGKRGDAK (SEQ ID NO:1) was synthesized and
modified to include a linking group and a maleimide group according
to the synthesis scheme set forth below.
[0408] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Ala-OH. They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). In last elongation step, the synthesis was then
re-automated for the addition of the 3-maleimidopropionic acid
(Step 2). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 3). The product was purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B)) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 75
EXAMPLE 2
Preparation of Modified RGD Peptid KRGDACEGDSGGPFC
[0409] RGD peptide KRGDACEGDSGGPFC (SEQ ID NO:2) was synthesized
and modified to include a linking group and a maleimide group
according to the synthesis scheme set forth below.
[0410] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,
Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C), Fmoc-Ala-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH
They were dissolved in N,N-dimethylformamide (DMF) and, according
to the sequence, activated using O-benzotriazol-1-yl-N,N,N',N'--
tetramethyl-uronium hexafluorophosphate (HBTU) and
Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group
was achieved using a solution of 20% (V/V) piperidine in
N,N-dimethylformamide (DMF) for 20 minutes (step 1). C are cyclized
cysteine. The cyclisation was achieved by cyclization by treatment
with TI(TFA).sub.3 (3 equiv. on 175 ummol scale) when the coupling
was paused at last lysine residue (step 2). After cyclization, In
last elongation step, the synthesis was then re-automated for the
addition of the 3-maleimidopropionic acid (Step 3). Between every
coupling, the resin was washed 3 times with N,N-dimethylformamide
(DMF) and 3 times with isopropanol. The peptide was cleaved from
the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et.sub.2O (Step 4). The
product was purified by preparative reversed phased HPLC using a
Varian (Rainin) preparative binary HPLC system: gradient elution of
30-55% B (0.045% TFA in H.sub.2O (A) and 0.045% TFA in CH.sub.3CN
(B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10.mu.
phenyl-hexyl, 21 mm.times.25 cm column and UV detector (Varian
Dynamax UVD II) at .lambda. 214 and 254 nm to afford the desired
protein in >95% purity, as determined by RP-HPLC. 76
EXAMPLE 3
Preparation of Modified RGD Peptide KRGDACEGDSFFPFC
[0411] RGD peptide KRGDACEGDSFFPFC (SEQ ID NO:3) was synthesized
and modified to include a linking group and a maleimide group
according to the synthesis scheme set forth below.
[0412] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,
Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C), Fmoc-Ala-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-AEEA-OH, Fmoc-AEEA-OH, They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). C are cyclized cysteine. The cyclisation was
achieved by, cyclization by treatment with TI(TFA).sub.3 (3 equiv.
on 175 ummol scale) when the coupling was paused at last lysine
residue (step 2). After cyclization, In last elongation step, the
synthesis was then re-automated for the addition of the linking
group s and the 3-maleimidopropionic acid (Step 3). Between every
coupling, the resin was washed 3 times with N,N-dimethylformamide
(DMF) and 3 times with isopropanol. The peptide was cleaved from
the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et.sub.2O (Step 4). The
product was purified by preparative reversed phased HPLC using a
Varian (Rainin) preparative binary HPLC system: gradient elution of
30-55% B (0.045% TFA in H.sub.2O (A) and 0.045% TFA in CH.sub.3CN
(B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10.mu.
phenyl-hexyl, 21 mm.times.25 cm column and UV detector (Varian
Dynamax UVD II) at .lambda. 214 and 254 nm to afford the desired
peptidein >95% purity, as determined by RP-HPLC. 77
EXAMPLE 4
Preparation of Modified GP-41A Peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW- F
[0413] GP-41A peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID
NO:4) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0414] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). In the last elongation step, the synthesis was
automated for the addition of the 3-maleimidopropionic acid (Step
2). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 3). The product was purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B)) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 78
EXAMPLE 5
Preparation of Modified GP-41B Peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW- F
[0415] GP-41 B peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID
NO:5) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0416] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH.
[0417] They were dissolved in N,N-dimethylformamide (DMF) and,
according to the sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethy- l-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The selective deprotection of the Lys (Aloc)
group is performed manually and accomplished by treating the resin
with a solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL).
[0418] In the last elongation step, the synthesis was automated for
the addition of the 3-maleimidopropionic acid (Step 3). Between
every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product was purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B)) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 79
EXAMPLE 6
Preparation of Modified GP-41C Peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW- F
[0419] GP-41C peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID
NO:6) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0420] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1).
[0421] The selective deprotection of the Lys (Aloc) group is
performed manually and accomplished by treating the resin with a
solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL).
[0422] In the last elongation step, the synthesis was automated for
the addition of the Fmoc-AEEA-OH and finally 3-maleimidopropionic
acid (Step 2). Between every coupling, the resin was washed 3 times
with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 3). The product was purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B)) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 80
EXAMPLE 7
Preparation of Modified RSV Peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
[0423] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID
NO:7) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0424] Solid phase peptide synthesis on a 100 .mu.mole scale is
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Rink Amide MBHA. The following
protected amino acids are sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and,
according to the sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group is achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1).
[0425] The amino group of the final amino acid is acetylated using
Acetic Acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). The
selective deprotection of the Lys (Aloc) group is performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
CHCl.sub.3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then
washed with CHCl.sub.3 (6.times.5 mL), 20% HOAc in DCM (6.times.5
mL), DCM (6.times.5 mL), and DMF (6.times.5 mL). The synthesis is
then re-automated for the addition of the 3-maleimidopropionic acid
(Step 3).
[0426] Between every coupling, the resin is washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide is cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product is purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 81
EXAMPLE 8
Preparation of Modified RSV Peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
[0427] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID
NO:8) was synthesized and modified to include a linking group and a
maleimide group to produce the modified peptide depicted below.
[0428] Solid phase peptide synthesis on a 100 .mu.mole scale is
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Rink Amide MBHA. The following
protected amino acids are sequentially added to resin: Fmoc-Val-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH,
Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Val-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Aloc)-OH. They are
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using O-benzotriazol-1-yl-N,N,N',N'--
tetramethyl-uronium hexafluorophosphate (HBTU) and
Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group
is achieved using a solution of 20% (V/V) piperidine in
N,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino
group of the final amino acid is acetylated using Acetic Acid,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-ur- onium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The
selective deprotection of the Lys (Aloc) group is performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
CHCl.sub.3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then
washed with CHCl.sub.3 (6.times.5 mL), 20% HOAc in DCM (6.times.5
mL), DCM (6.times.5 mL), and DMF (6.times.5 mL). The synthesis is
then re-automated for the addition of the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin is washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide is cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product is purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda.214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 82
EXAMPLE 9
Preparation of Modified RSV Peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
[0429] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID
NO:9) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0430] Solid phase peptide synthesis on a 100 .mu.mole scale is
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Rink Amide MBHA. The following
protected amino acids are sequentially added to resin: Fmoc-Val-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Aloc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH,
Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Val-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH. They are dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group is achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid is
acetylated using Acetic Acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The
selective deprotection of the Lys (Aloc) group is performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
CHCl.sub.3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then
washed with CHCl.sub.3 (6.times.5 mL), 20% HOAc in DCM (6.times.5
mL), DCM (6.times.5 mL), and DMF (6.times.5 mL). The synthesis is
then re-automated for the addition of the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin is washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide is cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product is purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC. 83
EXAMPLE 10
Preparation of Modified GLP-1 Peptide
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK
[0431] GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID NO:10)
was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth
below.
[0432] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin: Fmoc-Lys(Aloc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH,
Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH,
Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,
Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Ala-OH, Fmoc-His(Boc)-OH, The following protected amino acids
were sequentially added to resin: They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-- tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The selective deprotection of the Lys (Aloc)
group is performed manually and accomplished by treating the resin
with a solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL).The synthesis was then re-automated for the addition
of the 3-maleimidopropionic acid (Step 3). Between every coupling,
the resin was washed 3 times with N,N-dimethylformamide (DMF) and 3
times with isopropanol. The peptide was cleaved from the resin
using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4). The product was
purified by preparative reversed phased HPLC using a Varian
(Rainin) preparative binary HPLC system: gradient elution of 30-55%
B (0.045% TFA in H.sub.2O (A) and 0.045% TFA in CH.sub.3CN (B))
over 180 min at 9.5 mL/min using a Phenomenex Luna 10.mu.
phenyl-hexyl, 21 mm.times.25 cm column and UV detector (Varian
Dynamax UVD II) at .lambda. 214 and 254 nm to afford the desired
molecule in >95% purity, as determined by RP-HPLC. 84
EXAMPLE 11
Preparation of Modified GLP-1 Peptide
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK
[0433] GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID NO:11)
was synthesized and modified to include a linking group and a
maleimide group, as described below.
[0434] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin: Fmoc-Arg(Pbf)-OH,
Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH,
Fmoc-His(Boc)-OH, The following protected amino acids were
sequentially added to resin: They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uroni- um
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The selective deprotection of the Lys (Aloc)
group is performed manually and accomplished by treating the resin
with a solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). The linking group Fmoc-AEEA-OH was added and then
the Fmoc was removed in the usual fashon. This procedure was redone
to add a second AEEA linking group.
[0435] The synthesis was then re-automated for the addition of the
3-maleimidopropionic acid (Step 3). Between every coupling, the
resin was washed 3 times with N,N-dimethylformamide (DMF) and 3
times with isopropanol. The peptide was cleaved from the resin
using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4).
[0436] The product was purified by preparative reversed phased HPLC
using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H.sub.2O (A) and 0.045% TFA in
CH.sub.3CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna
10.mu. phenyl-hexyl, 21 mm.times.25 cm column and UV detector
(Varian Dynamax UVD II) at .lambda. 214 and 254 nm to afford the
desired molecule in >95% purity, as determined by RP-HPLC.
EXAMPLE 12
Preparation of Modified K5 Peptide PRKLYDYK
[0437] K5 peptide PRKLYDYK (SEQ ID NO:12) was synthesized and
modified to include a linking group and a maleimide group according
to the synthesis scheme set forth below.
[0438] Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin (0.48
mmol/mg) (250 .mu.mol scale): Fmoc-Lys(Aloc)OH, Fmoc-Tyr(tBu)OH,
Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH. Each coupling was accomplished using
2 equivalents of amino acid, 1 equivalent HBTU, and 2 equivalents
DIEA and performed twice for 30 min. The Fmoc group of the
N-terminal amino acid (Pro) was removed using 20% piperidine/DMF
(3.times.10min).
[0439] The resin was subsequently washed with 6.times.4 mL DMF,
3.times.3 mL EtOH and 6.times.4 mL DMF. Acetylation of the
N-terminus was accomplished manually on the Symphony by adding 4 mL
of 15 equivalents HOAc, 2 equivalents DIEA and 4 equivalents HBTU
in DMF. Acetic capping was performed 2.times.30 min. The resin was
subsequently washed with 3.times.4 mL CH.sub.2Cl.sub.2, 6.times.4
mL 0.5% DIEA/CH.sub.2Cl.sub.2, 3.times.4 mL EtOH and 6.times.4 mL
DMF Selective deprotection of the Lys(Aloc) group was performed
manually on the Symphony by treating the resin with a solution of 3
equivalents of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
CHCl.sub.3:Benzene (1:1) with 2.5% NMM (v/v) and 5% HOAc for 2 h.
The resin was then washed with CHCl.sub.3 (6.times.5 mL), 0.5% DIEA
in CH.sub.2Cl.sub.2 (6.times.5 mL), 0.02 M sodium
diethylthiocarbamate in DMF (6.times.5 mL), EtOH (3.times.4 mL) and
DMF (6.times.5 mL).
[0440] Coupling of 3-maleimidoproprionic acid (MPA) was performed
by resuming automation on the Symphony, which involves delivery of
2 equivalents of MPA, 2 equivalents DIEA and 1 equivalent HBTU to
the reaction vessel. The coupling was carried out twice at 30 min.
Washing was conducted using 6.times.4 mL DMF, 3.times.3 mL EtOH and
6.times.4 mL DMF. Cleavage from the resin was performed by
automation using 10 mL of the following cleavage mixture: 85%
TFA/5% triisopropyl silane/5% thioanisol/5% phenol. After the
peptide was cleaved from the resin for 2 hrs, the resin was washed
with TFA and CH.sub.2Cl.sub.2. The combined cleavage and washing
liquors concentrated to 1-2 mL using a rotovap with mild heating
(30.degree. C.) and the peptide was precipitated with Et.sub.2O.
The precipitate was collected by filtration using a SPPS manifold
and washed with 10 mL of ethyl acetate and 30 mL of Et.sub.2O. The
precipitate was subsequently dissolved in 10 mL of water containing
5% acetonitrile (0.04% TFA) in water (0.04% TFA) for
chromatographic purification. 85
EXAMPLE 13
Preparation of Modified K5 Peptide RNPDGDVGGPWAWTTAPRKLYDY
[0441] K5 peptide RNPDGDVGGPWAWTTAPRKLYDY (SEQ ID NO:13) was
synthesized and modified to include a linking group and a maleimide
group according to the synthesis scheme set forth below.
[0442] Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin (0.48
mmol/mg) (100 .mu.mol scale): Fmoc-Tyr(tBu)OH, Fmoc-Asp(tBu)-OH,
Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH,
Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Asp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Arg(Pbf)-OH, MPA.
[0443] Each coupling was accomplished using 5 equivalents of amino
acid, 1 equivalent HBTU, and 2 equivalents DIEA and performed twice
for 30 min. Cleavage from the resin was performed by automation
using 10 mL of the following cleavage mixture: 85% TFA/5%
triisopropyl silane/5% thioanisol/5% phenol. After the peptide was
cleaved from the resin for 2 hrs, the resin was washed with TFA and
CH.sub.2Cl.sub.2.
[0444] The combined cleavage and washing liquors concentrated to
1-2 mL using a rotovap with mild heating (30.degree. C.) and the
peptide was precipitated with Et.sub.2O. The precipitate was
collected by filtration using a SPPS manifold and washed with 10 mL
of ethyl acetate and 30 mL of Et.sub.2O. The precipitate was
subsequently dissolved in 10 mL of water containing 5% acetonitrile
(0.04% TFA) in water (0.04% TFA) for chromatographic purification.
Purification of all the peptides was performed using a Phenomenex
Luna 10.mu. phenyl-hexyl, 21 mm.times.250 mm column equilibrated
with a water/TFA mixture (0.045% TFA in H.sub.2O; Solvent A).
[0445] Elution was achieved at 18 mL/min by running a 10-30%
acetonitrile gradient over 60 min (0.045% TFA in CH.sub.3CN;
Solvent B). Peptides were detected by UV absorbance (Varian Dynamax
UVD II) at 214 and 254 nm. Fractions were collected in 9 mL
aliquots. Fractions containing the desired product were identified
by mass after direct injection onto LC/MS. The selected fractions
were subsequently analyzed by analytical HPLC (10-40% solvent B
over 20 min; Phenomenex Luna 5.mu. phenyl-hexyl, 10 mm.times.250 mm
column, 0.5 mL/min) to identify fractions with .gtoreq.95% purity
for pooling. The pool was freeze-dried using dry ice and acetone
and subsequently lyophilized for at least 2 days to yield a white
powder. 86
EXAMPLE 14
Preparation of Modified BBB Peptide YGRKKRRQRRRL
[0446] BBB peptide YGRKKRRQRRRL (SEQ ID NO:14) was synthesized and
modified to include a linking group and a maleimide group according
to the synthesis scheme set forth below.
[0447] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved
in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). After the tyrosine deprotection, Biotin was
anchored at the N-terminus via regular activation and coupling
conditions. The selective deprotection of the Lys (Aloc) group is
performed manually and accomplished by treating the resin with a
solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL).The synthesis was then re-automated for the addition
of the 3-maleimidopropionic acid (Step 3). Between every coupling,
the resin was washed 3 times with N,N-dimethylformamide (DMF) and 3
times with isopropanol. The peptide was cleaved from the resin
using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4). The product was
purified by preparative reversed phased HPLC using a Varian
(Rainin) preparative binary HPLC system: gradient elution of 30-55%
B (0.045% TFA in H.sub.2O (A) and 0.045% TFA in CH.sub.3CN (B))
over 180 min at 9.5 mL/min using a Phenomenex Luna 10.mu.
phenyl-hexyl, 21 mm.times.25 cm column and UV detector (Varian
Dynamax UVD II) at .lambda. 214 and 254 nm to afford the desired
molecule in >95% purity, as determined by RP-HPLC. 87
EXAMPLE 15
Preparation of Modified BBB Peptide YGRKKRRQRRRL
[0448] BBB peptide YGRKKRRQRRRL (SEQ ID NO:15) was synthesized and
modified to include a linking group and a maleimide group according
to the synthesis scheme set forth below.
[0449] Solid phase peptide synthesis on a 100 .mu.mole scale was
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids were sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved
in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The selective deprotection of the Lys (Aloc)
group is performed manually and accomplished by treating the resin
with a solution of 3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6 CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for
2 h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). After the aloc deprotection, Biotin was anchored at
the .epsilon.-N terminal of the deprotected lysine via regular
activation and coupling conditions. The Fmoc removal of the
N-terminus was then achieved with standard conditions. The
synthesis was then re-automated for the addition of the
3-maleimidopropionic acid at the end terminus (Step 3). Between
every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product was purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B)) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC.
EXAMPLE 16
Preparation of Modified dynorphin Peptide YGGFLRRIRPKLK
[0450] Dynorphin peptide YGGFLRRIRPKLK (SEQ ID NO:16) was
synthesized and modified to include a linking group and a maleimide
group according to the synthesis scheme set forth below.
[0451] Solid phase peptide synthesis on a 100 .mu.mole scale is
performed using manual solid-phase synthesis, a Symphony Peptide
Synthesizer and Fmoc protected Ramage Resin. The following
protected amino acids are sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,
Boc-Tyr(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF)
and, according to the sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group is achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid is
acetylated using Acetic Acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The
selective deprotection of the Lys (Aloc) group is performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
CHCl.sub.3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then
washed with CHCl.sub.3 (6.times.5 mL), 20% HOAc in DCM (6.times.5
mL), DCM (6.times.5 mL), and DMF (6.times.5 mL). The synthesis is
then re-automated for the addition of the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin is washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol. The
peptide is cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4). The product is purified by preparative
reversed phased HPLC using a Varian (Rainin) preparative binary
HPLC system: gradient elution of 30-55% B (0.045% TFA in H.sub.2O
(A) and 0.045% TFA in CH.sub.3CN (B) over 180 min at 9.5 mL/min
using a Phenomenex Luna 10.mu. phenyl-hexyl, 21 mm.times.25 cm
column and UV detector (Varian Dynamax UVD II) at .lambda. 214 and
254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC.
EXAMPLE 17
2-[2-[4-[4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropio-
nylacetamide. (Modified Cetirizine)
[0452] A mixture of 1-[(4-chlorophenylmethyl]-piperazine 1, methyl
(2-chloroethoxy)-acetate 2 and sodium carbonate in anydrous xylene
is heated under reflux with good stirring as indicated in the
schematic below. The reaction mixture is then cooled and filtered
and the solid is washed with benzene, the washed solid being
discarded. The filtrate is evaporated to dryness and the
evaporation residue is purified by chromatography on a column of
silica (eluent: chloroform:methanol 97:3 v/v). This generated
methyl 2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-pipe-
razinyl]ethoxy]-acetate 3. The compound is dissolved in of absolute
ethanol. 1 N ethanolic solution of potassium hydroxide is then
added thereto and the reaction mixture is heated under reflux for 4
hours. It is cooled and the precipitate removed by filitration,
after washing with diethyl ether. The filtrate is evaporated to
dryness and the evaporation residue is triturated with diethyl
ether and left to crystallize. The compound potassium
2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyl]e-
thoxy]-acetate is then obtained. The potassium salt is dissolved in
water and adjusted with 10% hydrochloric acid to a pH of 4. The
solution is extracted with chloroform and the organic phase is
dried over anhydrous magnesium sulfate, whereafter it is evaporated
to dryness. The evaporation residue is triturated with diethyl
ether and left to crystallize to produce
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperaziny-
l]ethoxy]-acetic acid 4.
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazin-
yl]ethoxy]-acetic acid 4 is then placed in DMF and activated with
and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropylamine. The reaction is
stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, triturated with cold ether and
left to crystallize. This last step generated 5
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-m-
aleimidopropionylacetamide, a modified antihistamine molecule.
88
EXAMPLE 18
11-(N-maleimidopropionyl4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[-
5.6]-cyclohepta-[1,2-b]-pyridine (Modified Loratidine).
[0453]
11-(N-8-chloro-4-piperidylidene)-6,11-dihydro-5H-benzo-[5,6]-cycloh-
epta-[1,2-b]-pyridine 1 is placed is placed in DMF and activated
with and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as
indicated in the schematic below. To the reaction mixture is added
3-maleimidopropionic acid. The reaction is stirred for 3 hours. The
organic phase is then washed with water and brine, dried over
MgSO.sub.4, chromatographied triturated with cold ether and left to
crystallize to generate 2,11-(N-maleimidopropionyl-
-4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-
-pyridine, a modified antihistamine molecule. 89
EXAMPLE 19
Modified Tirofiban
[0454] A four-neck round bottom flask equipped with a mechanical
stirrer, condenser, nitrogen inlet, HCl trap, heating unit and a
thermometer probe is purged with nitrogen overnight and then
charged with L-tyrosine 1, CH.sub.3CN,
N,O-bis-trimethylsilyl-trifluoromethyl-acetamide. The suspension is
heated to gentle reflux for 2 h. The resulting clear solution
O,O'-bis-trimethylsilyl-(L)-tyrosine 2, is cooled pyridine and
n-BuSO.sub.2Cl are slowly added over 30 minutes as indicated in the
schematic below. The reaction mixture is then stirred at room
temperature. Almost all the solvent is removed in a batch
concentrator, and the resulting oily residue is treated with
15%/KHSO4 and stirred vigorously for 1 hour. The mixture is
extracted with i-propyl acetate. The combined organic layer is
treated with Ecosorb TM S-402 and stirred at room temperature
overnight. Ecosorb TM is removed by filtration and the filter cake
is washed with i-propyl acetate. The filtrate is evaporated to
dryness and the resulting yellow oil is dissolved in hot EtOAc.
Hexane is added slowly to the stirring solution and the resulting
slurry is stirred at room temperature overnight. The solid is
collected by filtration and the filter cake is washed with
EtOAc/hexane. After drying under vacuum is obtained as a white
solid.
[0455] To a four-neck round bottom flask equipped with a mechanical
stirrer, condenser, nitrogen inlet and a thermometer probe is
charged N-n-butanesulfonyl-(L)-tyrosine 3,4-(4-pyridinyl)-butyl
chloride HCl 4 and DMSO. With vigorous stirring, 3 N aq. KOH is
added over 15 min.
[0456] The temperature is maintained in the 30-40.degree. C. range
for this operation using cooling water. Potassium iodide is added,
and the mixture is heated for 36 h. After cooling to room
temperature, the mixture is diluted with 0.25 N NaOH and extracted
once with t-butyl methyl ether. The aqueous layer is treated with
Ecosorb S-402 and Nuchar SA and the resulting mixture is
mechanically stirred for 1 h. The mixture is filtered through a
coarse-porosity sintered funnel and the filtered cake is washed
with water. The combined filtrate is placed in a vessel equipped
with a pH meter probe and a mechanical stirrer. With vigorous
stirring, NaCl is added, stirred for 30 min, and then 50% aq.
acetic acid wash added until pH 4.80, and stirring continued for
2-3 h. The resulting slurry is filtered through a coarse-porosity
sintered funnel, and the cake is washed with water. The crude
product is dried under house-vacuum under a positive nitrogen
pressure to give beige solid 5 having a wt % purity of 95%.
[0457] Selective hydrogenation of the pyridine ring to piperidine
ring is accomplished by using 5 wt % of 10% Pd/C in AcOH at 60C to
give the target product cleanly without reduction of the phenolic
ring. Filtration of the reaction mixture, evaporation of acetic
acid followed by crystallizing the product 6 from 6%
AcOH/water.
[0458] To a RB flask equipped with a thermometer probe and addition
funnel is charged the crude and 0.25 N NaOH. After complete
dissolution, the solution is cooled to room temperature, and
adjusted to pH 7 by slow additon of 1 N HCl .The solution is
further brought down to pH 5.5 by slow addition of 0.5 N HCl.
Stirring is continued for 1 h, then the slurry is filtered through
a coarse funnel padded with a sheet of shark-skin paper and a
polypropylene pad (10 mu m) and the cake is washed with water. The
solid is dried under house vacuum with nitrogen sweep to give a
beige solid. The compound is then placed in DMF and activated with
and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropylamine. The reaction is
stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, triturated with cold ether and
left to crystallize to generate the modifed tirofiban 7. Tirofiban
is an anti-angina agent. 90
EXAMPLE 20
N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropion-
ilamide (modifed-Enalapril)
[0459] Ethyl 2-oxo-4-phenylbutyrate 1 and L-alanyl-L-proline 2 are
dissolved in a 1:1 ethanol-water solvent as indicated in the
schematic below. A solution of sodium cyanoborohydride in
ethanol-water is added dropwise at room temperature over the course
of two hours. When reaction is complete, the product is absorbed on
strong acid ion-exchange resin and eluted with 2% pyridine in
water. The product-rich cuts are freeze dried to give crude
N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolin- e 4 and the
compound is purified by chromatography to yield the desired isomer.
The compound 4 is then placed in DMF and activated with and
O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (H BTU) and Diisopropylethylamine (DIEA). To
the reaction mixture is added 3-maleimidopropylamine. The reaction
is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, triturated with cold ether and
left to crystallize to produce 5
N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropio-
nilamide, a modified anti-hypertensive agent. 91
EXAMPLE 21
Maleimidopropynamyl-.epsilon.-(3,4,5-trimethoxybenz-amido)-caproicamide
(Modified-Capobenic Acid)
[0460] 3,4,5-trimethoxybenzoyl chloride 1 is added along with
amino-hexanoic acid 2 in a solution of 1N NaOH as indicated in the
schematic below. The resulting solution is preferably treated with
char to decolorize it, the char is filtered, and the filtrate
neutralized with dilute HCl to Congo red indicator end-point. The
resulting precipitate is separated by filtration washed with water,
dried, then recrystallized from ethanol to genarate 3. The compound
is then placed in DMF and activated with and
O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (H BTU) and Diisopropylethylamine (DIEA). To
the reaction mixture is added 3-maleimidopropylamine. The reaction
is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, triturated with cold ether and
left to crystallize in order to produce 4
Maleimidopropynamyl-.epsilon.-(3,4,5-trimethoxybenz-am-
ido)-caproicamide, an anti-arrhthymetic agent 92
EXAMPLE 22
Maleimidopropionamyl-1-theobromineacetamide (Modified
1-theobromineacetic acid)
[0461] 1-theobromineacetic acid 1 is placed in DMF and activated
with O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as
indicated in the schematic below. To the reaction mixture is added
3-maleimidopropylamine. The reaction is stirred for 3 hours. The
organic phase is then washed with water and brine, dried over
MgSO.sub.4, chromatographied, triturated with cold ether and left
to crystallize to produce 2 Maleimidopropionamyl-1-th-
eobromineacetamide, a modifed bronchodilator. 93
EXAMPLE 23
4-anilino-1-(2-phenethyl)piperidin (Modified-Fentanyl)
[0462] 1-phenylethyl-4-piperidone 1 was placed in 1,2,
dichloroethane along with aniline 2, sodiumcyanoborohydride and it
is refluxed for 18 hours. The reaction is then cooled to RT and the
reaction is extracted with brine to generate 3 as indicated in the
schematic below. Finally The compound is then placed in DMF and
activated with and
O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropionic acid. The reaction
is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, triturated with cold ether and
left to crystallize to generate 4, a modified pain killer (opioid
molecule). 94
EXAMPLE 24
Maleimidopropamyl
2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide
(Modified-Loxoprofen).
[0463] Ethyl 2 cyclopentanonecarboxylate 1 and ethyl
2-(4-iodomethylphenyl)propionate 2 are placed in
N,N,dimethylformamide along with potassium hydroxyde as indicated
in the schematic below. The solution is stirred at room temperature
for 5 hours and at 50.degree. C. for 1 hour. The reaction is cooled
and acidified with acetic acid and N,N,dimethylformamide is removed
by vacuum. The residue is extracted with ether and the organic
phase is washed with water and dried on Mg.sub.2SO.sub.4 to afford
3 Finally, the compound 3 is then placed in DMF and activated with
and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethylu- ronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropylamine. The reaction is
stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, chromatographied, triturated with
cold ether and left to crystallize to generate 4 Maleimidopropamyl
2-[4-(2-oxocyclopentan-1-ylme- thyl)phenyl]propionamide to produce
the modified anti-inflammatory agent. 95
EXAMPLE 25
N-maleimidopropionyl-N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropyla- mine (Modified
Fluoxetine)
[0464] .beta.-dimethylaminopropiophenone hydrochloride 1 is
converted to the corresponding free base by the action of aqueous
sodium hydroxide. The liberated free base is taken up in ether, the
ether layer separated and dried, and the ether removed therefrom in
vacuo. The residual oil comprising
.beta.-dimethylaminopropiophenone is dissolved in tetrahydrofuran,
and the resulting solution added in dropwise fashion with stirring
to a solution of diborane in tetrahydrofuran. The reaction mixture
is stirred overnight at room temperature. Next, aqueous
hydrochloric acid is added to decompose any excess diborane
present. The tetrahydrofuran is removed by evaporation. The acidic
solution is extracted twice with benzene, and the benzene extracts
are discarded. The acidic solution is then made basic with an
excess of 5 N aqueous sodium hydroxide. The basic solution is
extracted three times with benzene. The benzene extracts are
separated and combined, and the combined extracts washed with a
saturated aqueous sodium chloride and then dried to produce 2. A
solution containing N,N,-dimethyl 3-phenyl-3-hydroxypropylamine 2
in chloroform is saturated with dry gaseous hydrogen chloride.
Thionyl chloride is then added to the chloroform solution at a rate
sufficient to maintain reflux. The solution is refluxed an
additional 5 hours. Evaporation of the chloroform and other
volatile constituents in vacuo yielded N,N-dimethyl
3-phenyl-3-chloropropylamine hydrochloride 3 which is collected by
filtration, and the filter cake washed twice with acetone.
P-trifluoromethylphenol 4, solid sodium hyroxide and methanol are
placed in a round-bottom flask equipped with magnetic stirrer,
condenser and drying tube. The reaction mixture is stirred until
the sodium hydroxide had dissolved. Next, N,N-dimethyl
3-phenyl-3-chloropropylamine hydrochloride is added. The resulting
reaction mixture is refluxed for about 5 days and then cooled. The
methanol was then removed by evaporation, and the resulting residue
taken up in a mixture of ether and 5 N aqueous sodium hydroxide.
The ether layer is separated and washed twice with 5 N aqueous
sodium hydroxide and three times with water. The ether layer is
dried, and the ether removed by evaporation in vacuo to yield as a
residue N,N-dimethyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 5. A solution
containing cyanogen bromide in benzene and toluene is placed in a
three-neck round-bottom flask equipped with thermometer, addition
funnel, drying tube and inlet tube for nitrogen. The solution is
cooled and nitrogen gas is bubbled thru the solution. Next, a
solution of N,N-dimethyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 5 dissolved in
benzene is added in dropwise fashion. The temperature of the
reaction mixture is allowed to rise slowly to room temperature, at
which temperature stirring is continued overnight while still
maintaining a nitrogen atmosphere.The reaction mixture is washed
twice with water, once with 2 N aqueous sulfuric acid and then with
water until neutral. The organic layer is dried, and the solvents
removed therefrom by evaporation in vacuo to yield N-methyl-N-cyano
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 6. A solution of
potassium hydroxide, water, ethylene glycol and of N-methyl-N-cyano
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine is placed in a
three-neck, round-bottom flask equipped with magnetic stirrer and
condenser. The reaction mixture is heated to refluxing temperature
for 20 hours, and is then cooled. The reaction mixture is extracted
with ether. The ether extracts are combined, and the combined
extracts washed with water. The water wash is discarded. The ether
solution is next contacted with 2 N aqueous hydrochloric acid. The
acidic aqueous layer is separated. A second aqueous acidic extract
with 2 N hydrochloric acid is made followed by three aqueous
extracts and an extract with saturated aqueous sodium chloride. The
aqueous layers are all combined and made basic with 5 N aqueous
sodium hydroxide. N-methyl 3-(p-trifluoromethylphe-
noxy)-3-phenylpropylamine 7, formed in the above reaction, is
insoluble in the basic solution and separated. The amine is
extracted into ether. The ether extracts are combined, and the
combined extracts washed with saturated aqueous sodium chloride and
then dried. Evaporation of the ether in vacuo yielded N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylprop- ylamine 7. Finally, the
compound 7 is then placed in DMF and activated with and
O-(benzotriazol-1-yl)-N,N',N',N'-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropionic acid. The reaction
is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgSO.sub.4, chromatographied, triturated with
cold ether and left to crystallizeto produce 8 to produce the
modified anti-depressant molecule 96
EXAMPLE 26
Maleimidopropionamyl-3,5-3',5' tetraiodothyroninamid
(Modified-Thyroxine)
[0465] N-t-Boc-3,5-3',5' tetraiodothyronine 1 is placed in DMF and
activated with and
O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as
indicated in the schematic below. To the reaction mixture is added
3-maleimidopropylamine. The reaction is stirred for 3 hours. The
organic phase is then washed with water and brine, dried over
MgSO.sub.4, triturated with cold ether and left to crystallize to
produce 2. Finally the compound is placed in a 25% solution of TFA
in CH.sub.2Cl.sub.2 for 15 minutes and the CH.sub.2Cl.sub.2 is
removed invacuo. The oily residue is then lyophilized to yield the
desired compound 3, a modified thyroxine for treament of thyroid
deficiency, i.e., an anti-thyroid deficiency agent. 97
EXAMPLE 27
2S-hydroxy-3R-[1S-(MEEA-EDA-carbonyl)-2,2-dimethyl-propylcarbamoyl]-5-meth-
ylhexanohydroxamic acid (Modified MMPI)
[0466] The compound 1 and 3,4-dihydro-2H-pyran in CH.sub.2Cl.sub.2
and pyridinium p-toluenesulfornate are stirred at room temperature
for 12 h as indicated in the schematic below. Then the solution is
diluted with EtOAc and washed with half-saturated brine to remove
the catalyst. The solvent is evaporated and the residue is treated
with NaOH(1N) and EtOH for 30 min. The solution is acidified with
AcOH, and the product is extracted with EtOAc. The EtOAc solution
is dried, evaporated to give the THP ether 2. The compound 2, DCC
and HOBT in CH.sub.2Cl.sub.2 are stirred at room temperature for 60
min. Then MEEA-EDA HCl (N-(2-aminoethyl)
[2-(2-maleiimidoethoxy)ethoxy]acetamide) and N-methylmorpholine are
added. The reaction is stirred for 2 h. and then quenched by
addition of AcOH. The precipitate is removed by filtration. The
filtrate is washed with diluted HCl, NaHCO.sub.3 and dried. The
crude product is used for the next step.The crude product is
treated with 2N HCl H.sub.2O/EtOH 1:1) for 30 min. EtOH is
evaporated. The product is extracted with CH.sub.2Cl.sub.2. The
combined CH.sub.2Cl.sub.2 layers are washed with NaHCO.sub.3 and
dried. Evaporation of the solvent gives a residue, which is
purified by flash column chromatography to afford 3. This compound
can be purified further by HPLC on a reverse phase column and
lyophilized to produce the modified MMPI. 98
EXAMPLE 28
Preparation of Rhodamine NHS Ester
[0467] Rhodamine Green.TM.-X, succinimidyl ester, hydrochloride
mixed isomers is commercially available from Molecular Probes
(Eugene Oregon) as illustrated below: 99
EXAMPLE 29
In Vivo Addition of NHS-Rhodamine
[0468] New Zealand rabbits (2 Kg), male or female, were
intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50
mg/kg) and Acepromazine (0.75 mg/kg) prior to surgical exposure of
left carotid artery. Both carotid arteries were isolated and blood
flows were measured. A catheter (22G) was inserted in the arterial
segment and rinsed with 0.9% sodium chloride via catheter until
there was no more visible evidence of blood in the segment.
[0469] A 1-cm incubation chamber was created by ligatures in the
segment area. The incubation chamber was flushed three times with 1
mL of 0.9% sodium chloride. A solution of 100 .mu.l of 500 .mu.M
NHS-Rhodamine was prepared and incubated in the incubation chamber
for 3 minutes. The excess of rhodamine was withdrawn with a 1 mL
syringue. The incubation chamber was washed once again with 3 times
100 mL of 0.9% sodium chloride. The incubation chamber was then
removed from the rabbit, cut in three pieces and dipped in 10%
formalin for further evaluation. The NHS-Rhodamine treated arteries
exhibited dramatic levels of fluorescence whereas those arteries
treated solely with Rhodamine exhibited little fluorescence over
background. These results demonstrate that Rhodamine was covalently
bonded to a local delivery site.
EXAMPLE 30
Preparation of [.sup.3H]-NHS-Propionate
[0470] [.sup.3H]-NHS-propionate is available from Amersham Canada
Ltd. (Oakville, Ontario, Canada) and can be prepared from the
tritiated propionic acid through known to the art condensation of
N-hydrosuccinimide in presence of EDC in DMF or methylene
chloride.
EXAMPLE 31
In Vivo Pharmacokinetics Studies of [.sup.3H]-NHS-Propionate
[0471] New Zealand rabbits (2 kg), male or female, were
intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50
mg/kg) et Acepromazine (0.75 mg/kg) prior to surgical exposure of
left carotid artery. Segments of 10 mm of carotids, were
transiently isolated by temporary ligatures and rinsed with 0.9%
sodium chloride via a cannula until there was no more visible
evidence of blood components.
[0472] A catheter (18G) was inserted in the arterial segment and
served to introduce the angioplasty balloon (2.5 mm of diameter,
over the wire/Boston Scientific Inc.). A vascular damage
(angioplasty) was performed on the isolated segment in order to
eliminate the layer of endothelial cells. The angioplasty balloon
was serially inflated at different atmospheres (4, 6, 8 and 10)
during 1 minute, with 45 seconds of delay between inflations. At 4
atmospheres a balloon traction was performed 5 times and 1000 U/kg
of heparin were infused in the blood circulation.
[0473] The angioplasty balloon was then retrieved from the artery
and the catheter was reintroduced. The arterial segment was rinsed
3 times with saline, and 100 .mu.M of [.sup.3H]-NHS-propionate was
incubated within the isolated segment of the artery for either 30
seconds, 3 minutes or 30 minutes. At the end, the excess of
incubation liquid was withdrawn from the artery, and the segment
was rinsed 5 times with saline. The treated artery was immediately
harvested, and incorporation of [.sup.3H]-labeled compounds within
the artery was evaluated by scintillation counting. After 30
seconds of incubation, we recorded an association efficiency of
2.55%. At 3 min and 30 min, we recorded an association efficiency
of 5.5 and 6.5%, respectively. We decided that a 3 min incubation
time was sufficient to treat the artery in an efficient way.
[0474] When evaluating the retention levels, 100 .mu.M of
[.sup.3H]-NHS-propionate or [.sup.3H]-propionate were incubated
with the artery for a period of 3 minutes, after which the segment
has been rinsed 5 times with saline. The catheter was then removed
and the arteriotomy site was closed with microsutures, thus
reestablishing the blood flow within the carotid. Finally, the neck
wound was closed with sutures, and animals are allowed to
recuperate. Three days following the treatment, the animals are
sacrificed with an overdose of sodium pentobarbital, the carotid
segments are removed and examined for compound's presence by
scintillation counting. 10.94% retention of
[.sup.3H]-NHS-propionate was monitored after three days following a
3 minute incubation period based on residual radioactivity in the
artery. The difference in retention efficiency between covalently
and non covalently bound propionate after a 3 minutes incubation
period was determined. An outstanding 12 fold enhancement in
retention was recorded (0.6% of total amount incubated against
0.046% for the non covalently bound) in favor of the
NHS-propionate. This indicates that the tissue association of a
compound is dramatically enhanced by the covalent attachment in
vivo. Subsequent restitution of blood flow demonstrated retention
[.sup.3H]-NHS-propionate of approximately 10% of the material 72
hours after injury. This represents excessive tissue retention
using the embodied technology of agents markedly beyond that seen
with all drug delivery technologies as exemplified in the
literature for standard non covalent agents (Circulation 1994 89
(4) 1518-1524).
EXAMPLE 32
Synthesis of [.sup.32P]NHS Derivative
[0475] To a solution of protected R and R' (both R and R') can be
alkyl, phenyl or alkoxy groups, and X is either O or S, alkoxy,
alkyl and any other functionality stable under these conditions)
phosphodiester (0.1 mmol) and N-hydroxysuccinimide (0.2 mmol) is
added diisopropylethylamine 0.11 mmol), followed by addition of
HBTU (0.22 mmol). The reaction mixture is stirred at room
temperature for 36 hours. DMF is removed by vacuum distillation and
the residue is dissolved in MeOH (10 mL). The MeOH solution is
filtered to remove the insolubles, the filtrate is concentrated in
vacuo, and the residue is dissolved in a minimum amount of MeOH.
Water is then added to induce precipitation and the precipitate is
dried on vacuum to give the desired compound. 100
[0476] The yield of the reaction can usually be improved by using
EDC as the coupling reagent, as exemplified below. To a solution of
R and R' phosphodiester (0.054 mmol) and N-hydroxysuccinimide
(0.115 mmol) in anhydrous DMF (3 mL), is added EDC (31 mg, 0.162
mmol). The solution is stirred at room temperature for 24 hours.
DMF is removed by vacuum distillation and the residue is further
dried on high vacuum. The residue is then dissolved in a minimum
amount of MeOH (0.12 mL) and H.sub.2O (3.2 mL) is added to induce
precipitation. The precipitates are washed with H.sub.2O
(3.times.0.8 mL) and dried on vacuum to give a solid product.
[0477] Any protected phosphonate derivatives may undergo similar
transformation.
EXAMPLE 33
[0478] New Zealand rabbits (2 kg), male or female, were
anesthetized with xylazine (20 mg/kg), ketamine (50 mg/kg) and
acepromazine (0.75 mg/kg) intramuscularly prior to surgical
exposure of left carotid artery. Carotid arteries were surgically
dissected and segments of approximately 10 mm length were isolated.
The vessels were cannulated and rinsed with 0.9% sodium chloride
until there was no more visible evidence of blood components.
[0479] A catheter (18G) was inserted in the arterial segment and
served to introduce the angioplasty balloon (2.5 mm of diameter,
over the wire/Boston Scientific Inc.). Vascular damage
(angioplasty) was performed on the isolated segment in order to
eliminate the layer of endothelial cells. The angioplasty balloon
was serially inflated at different atmospheres (4, 6, 8 and 10) for
1 minute, with 45 seconds of delay between inflations. At 4
atmospheres a balloon traction was performed 5 times and 1000 U/kg
of heparin were infused in the blood circulation.
[0480] The angioplasty balloon was then retrieved from the artery
and the catheter was reintroduced. The arterial segment was rinsed
3 times with saline, and 100 .mu.M of [.sup.32P]-NHS-[linking
group] was incubated within the isolated segment of the artery for
3 minutes. At the end, the excess of incubation liquid was
withdrawn from the artery, and the segment was rinsed 5 times with
saline. The vessel was sutured closed, blood flow restored and
surgical wounds repaired. Animals were returned to the vivarium for
periods up to four weeks. Tissue retention of
[.sup.32P]-NHS-[linking group] was evaluating using whole animal
radiography at selected periods of time after injury.
EXAMPLE 34
Synthesis of [.sup.131I]-NHS Derivative
[0481] To a solution of protected amino protected
[.sup.131I]-iodotyrosine (0.1 mmol) and N-hydroxysuccinimide (0.2
mmol) is added diisopropylethylamine (0.11 mmol), followed by
addition of HBTU (0.22 mmol). The reaction mixture is stirred at
room temperature for 12 hours. DMF is removed by vacuum
distillation and the residue is dissolved in MeOH (10 mL). The MeOH
solution is filtered to remove the insolubles, the filtrate is
concentrated in vacuo, and the residue is dissolved in a minimum
amount of MeOH. Water is then added to induce precipitation and the
precipitate is dried on vacuum to give the desired compound.
[0482] The yield of the reaction can usually be improved by using
EDC as the coupling reagent, as exemplified below. To a solution of
[.sup.131I]-iodotyrosine (0.054 mmol) and N-hydroxysuccinimide
(0.115 mmol) in anhydrous DMF (3 mL), is added EDC (31 mg, 0.162
mmol). The solution is stirred at room temperature for 24 hours.
DMF is removed, by vacuum distillation and the residue is further
dried on high vacuum. The residue is then dissolved in a minimum
amount of MeOH (0.12 mL) and water (3.2 mL) is added to induce
precipitation. The precipitates are washed with H.sub.2O
(3.times.0.8 mL) and dried on vacuum to give a solid product.
101
EXAMPLE 35
In Vivo Pharmacology of .sup.131I Derivative
[0483] New Zealand rabbits (2 Kg), male or female, were
anesthetized with xylazine (20 mg/kg), ketamine (50 mg/kg) and
acepromazine (0.75 mg/kg) intramuscularly prior to surgical
exposure of left carotid artery. Carotid arteries were surgically
dissected and segments of approximately 10 mm length were isolated.
The vessels were cannulated and rinsed with 0.9% sodium chloride
until there was no more visible evidence of blood components.
[0484] A catheter (18G) was inserted in the arterial segment and
served to introduce the angioplasty balloon (2.5 mm of diameter,
over the wire/Boston Scientific Inc.). Vascular damage
(angioplasty) was performed on the isolated segment in order to
eliminate the layer of endothelial cells. The angioplasty balloon
was serially inflated at different atmospheres (4, 6, 8 and 10) for
1 minute, with 45 seconds of delay between inflations. At 4
atmospheres a balloon traction was performed 5 times and 1000 U/kg
of heparin were infused in the blood circulation.
[0485] The angioplasty balloon was then retrieved from the artery
and the catheter was reintroduced. The arterial segment was rinsed
3 times with saline, and 100 .mu.M of [.sup.131I]-NHS-[linking
group] was incubated within the isolated segment of the artery for
3 minutes. At the end, the excess of incubation liquid was
withdrawn from the artery, and the segment was rinsed 5 times with
saline. The vessel was sutured closed, blood flow restored and
surgical wounds repaired. Animals were returned to the vivarium for
periods up to four weeks. Tissue retention of
[.sup.131I]-NHS-[linking group] was evaluated using whole animal
radiography at selected periods of time after injury
EXAMPLE 36
Intrapulmonary Delivery of
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperaz-
inyl]ethoxy]-maleimidopropionylacetamide. (Modified-Cetirizine)
[0486] A Bird Micronebulizer in line with a Bird Mark 7 respirator
may be charged with 5-10 ml of a solution of 12 mg/ml
2-[2-[4-[(4-chloropheny)ph-
enylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide in
mannitol/phosphate buffer. The Micronebulizer may then bes used to
simultaneously ventilate and dose a patient at 22 cm H.sub.2O at a
rate of 1.8 mg/min for 30 min. At this pressure the patient shouldl
ventilate at approximately normal inspiratory volume. The patient
should be allowed to exhale normally after each ventilated breath.
In addition, the patient should be positioned supine for dosing.
After the first dosing period the pateint should be allowed to
breathe normally for another 20 minutes. After the 20 minute
period, a second dosing should be performed in the same way as the
first. Blood plasma samples should be taken at the initiation time
of the first dose and thereafter to monitor the levels of
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidoprop-
ionylacetamide .
EXAMPLE 37
Intrapulmonary Delivery of
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperaz-
inyl]ethoxy]-maleimidopropionylacetamide (Modified-Cetirizine)
Using the Spiros DPI System
[0487] The Spiros DPI is an aerosol generation system that is
largely independent of the inspiratory flow rate and its use is
described in U.S. Pat. No. 6,060,069.
[0488] A modified beclomethasone dipropionate (BDP) formulation may
be prepared by first micronizing through conventional means (e.g.,
a jet mill) to produce a range of particle sizes that are likely to
undergo sedimentation in the human airway. Generally, fine
particles in the range of 0.5 to 5.8 microns in diameter are
thought to undergo sedimentation between the oropharynx and small
bronchioles. Particles within this general size category are
thought to be in the "respirable range." Such micronized materials
have excessive surface free energy, and as a result have a tendency
to adhere strongly to many surfaces, most especially to
themselves.
[0489] Lactose particles in the size range of 20 to 100 microns may
be mixed with the smaller diameter micronized drug particles to
create a homogenous blend. Each lactose particle will generally
bind to a number of smaller drug particles in the blend. The blend
flows more easily during the packaging and dose metering
process.
[0490] The formulation may be then filled into cassettes, each
containing 30 individual doses. The cassettes may then packaged in
sealed foil pouches.
[0491] The following steps using the Spiros BPI system may be used
to deliver a dose of inhaled drug: 1. The Spiros DPI does not need
to be primed; 2. The blue plastic cap is removed from the
mouthpiece; 3. The inhaler is held level; 4. The lid of the DPI is
opened as far back as possible (The lid will click when it has
reached the correct angle); 5. The lid is then closed completely;
6. Before bringing the inhaler up to the mouth, the patient
breathes out, making sure not to breathe into the inhaler.; 7. The
inhaler is brought up to the mouth in a level position; 8. The lips
are sealed fully around the mouthpiece, making sure there is no gap
between the mouthpiece and the lips; 9. The patient breathes in
through the mouth for about 4 seconds, preferably at a flow rate of
about 20 LPM. The motor will turn on and the patient may taste/feel
the drug as it is inhaled; 10. The patient holds their breath for
as long as possible, up to 10 seconds. 11. The Spiros DPI is held
in a level position during loading and dosing.
Sequence CWU 1
1
16 1 13 PRT Artificial Sequence Synthetic Peptide 1 Ala Gly Tyr Lys
Pro Glu Gly Lys Arg Gly Asp Ala Lys 1 5 10 2 15 PRT Artificial
Sequence Synthetic Peptide 2 Lys Arg Gly Asp Ala Cys Glu Gly Asp
Ser Gly Gly Pro Phe Cys 1 5 10 15 3 15 PRT Artificial Sequence
Synthetic Peptide 3 Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly
Pro Phe Cys 1 5 10 15 4 36 PRT Artificial Sequence Synthetic
Peptide 4 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn
Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 5 36 PRT Artificial
Sequence Synthetic Peptide 5 Tyr Thr Ser Leu Ile His Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 6
36 PRT Artificial Sequence Synthetic Peptide 6 Tyr Thr Ser Leu Ile
His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp
Asn Trp Phe 35 7 36 PRT Artificial Sequence Synthetic Peptide 7 Val
Tyr Pro Ser Asp Glu Tyr Asp Ala Ser Ile Ser Gln Val Asn Glu 1 5 10
15 Glu Ile Asn Gln Ala Leu Ala Tyr Ile Arg Lys Ala Asp Glu Leu Leu
20 25 30 Glu Asn Val Lys 35 8 36 PRT Artificial Sequence Synthetic
Peptide 8 Lys Val Tyr Pro Ser Asp Glu Tyr Asp Ala Ser Ile Ser Gln
Val Asn 1 5 10 15 Glu Glu Ile Asn Gln Ala Leu Ala Tyr Ile Arg Lys
Ala Asp Glu Leu 20 25 30 Leu Glu Asn Val 35 9 35 PRT Artificial
Sequence Synthetic Peptide 9 Val Tyr Pro Ser Asp Glu Tyr Asp Ala
Ser Ile Ser Gln Val Asn Glu 1 5 10 15 Glu Ile Asn Gln Ala Leu Ala
Tyr Ile Arg Lys Ala Asp Glu Leu Leu 20 25 30 Glu Asn Val 35 10 31
PRT Artificial Sequence Synthetic Peptide 10 His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys 20 25 30 11 30 PRT
Artificial Sequence Synthetic Peptide 11 His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 12 8 PRT
Artificial Sequence Synthetic Peptide 12 Pro Arg Lys Leu Tyr Asp
Tyr Lys 1 5 13 23 PRT Artificial Sequence Synthetic Peptide 13 Arg
Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Ala Trp Thr Thr Ala 1 5 10
15 Pro Arg Lys Leu Tyr Asp Tyr 20 14 12 PRT Artificial Sequence
Synthetic Peptide 14 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Lys 1 5 10 15 12 PRT Artificial Sequence Synthetic Peptide 15 Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys 1 5 10 16 13 PRT
Artificial Sequence Synthetic Peptide 16 Tyr Gly Gly Phe Leu Arg
Arg Ile Arg Pro Lys Leu Lys 1 5 10
* * * * *