U.S. patent application number 14/018271 was filed with the patent office on 2014-03-06 for bche albumin fusions for the treatment of cocaine abuse.
This patent application is currently assigned to TEVA PHARMACEUTICAL INDUSTRIES, LTD.. The applicant listed for this patent is Hussein Hallak, David Lafleur, Victor Piryatinsky, Viktor Roschke, Moti Rosenstock, Liron Shemesh-Darvish, Liora Sklair-Tavron. Invention is credited to Hussein Hallak, David Lafleur, Victor Piryatinsky, Viktor Roschke, Moti Rosenstock, Liron Shemesh-Darvish, Liora Sklair-Tavron.
Application Number | 20140065126 14/018271 |
Document ID | / |
Family ID | 44145885 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065126 |
Kind Code |
A1 |
Sklair-Tavron; Liora ; et
al. |
March 6, 2014 |
BChE ALBUMIN FUSIONS FOR THE TREATMENT OF COCAINE ABUSE
Abstract
A method of attenuating a biological effect of cocaine exposure
in a primate. Such method includes administering to the primate an
amount of a BChE-albumin fusion protein comprising the amino acid
substitutions A227S, S315G, A356W, and Y360G, wherein the amount of
the fusion protein is effective to cause attenuation of the
biological effect of cocaine exposure in the primate.
Inventors: |
Sklair-Tavron; Liora;
(Zichron Yaakov, IL) ; Rosenstock; Moti; (Menashe,
IL) ; Shemesh-Darvish; Liron; (Ramat-Gan, IL)
; Hallak; Hussein; (Jerusalem, IL) ; Piryatinsky;
Victor; (Netanya, IL) ; Roschke; Viktor;
(Bethesda, MD) ; Lafleur; David; (Washington,
DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sklair-Tavron; Liora
Rosenstock; Moti
Shemesh-Darvish; Liron
Hallak; Hussein
Piryatinsky; Victor
Roschke; Viktor
Lafleur; David |
Zichron Yaakov
Menashe
Ramat-Gan
Jerusalem
Netanya
Bethesda
Washington |
MD
DC |
IL
IL
IL
IL
IL
US
US |
|
|
Assignee: |
TEVA PHARMACEUTICAL INDUSTRIES,
LTD.
Petach-Tikva
IL
|
Family ID: |
44145885 |
Appl. No.: |
14/018271 |
Filed: |
September 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12962410 |
Dec 7, 2010 |
8541373 |
|
|
14018271 |
|
|
|
|
61283791 |
Dec 8, 2009 |
|
|
|
61412205 |
Nov 10, 2010 |
|
|
|
Current U.S.
Class: |
424/94.6 |
Current CPC
Class: |
C12N 9/18 20130101; A61K
38/00 20130101; A61P 25/30 20180101; A61P 25/36 20180101; C07K
14/765 20130101; C07K 14/001 20130101; C12Y 301/01084 20130101;
C07K 2319/31 20130101 |
Class at
Publication: |
424/94.6 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C07K 14/765 20060101 C07K014/765; C12N 9/18 20060101
C12N009/18 |
Claims
1. A method of attenuating a biological effect of a cocaine
exposure in a primate comprising administering to the primate an
amount of a fusion protein comprising: (a) a mutant
butyrylcholinesterase (BChE) polypeptide comprising the sequence
TABLE-US-00027 (SEQ ID NO: 1)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKV,
(b) a human serum albumin (HSA) polypeptide comprising the sequence
TABLE-US-00028 (SEQ ID NO: 2)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE
CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA
KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE
DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,
and (c) a signal peptide comprising the sequence
MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:3), wherein the amount of the
fusion protein is effective to cause attenuation of the biological
effect of the cocaine exposure in the primate.
2. The method of claim 1, wherein the fusion protein comprises the
sequence TABLE-US-00029 (SEQ ID NO: 4)
MRPTWAWWLFLVLLLALWAPARGEDDIIIATKNGKVRGMNLTVFGGTVTA
FLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQSFPGFH
GSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQTGTSSLHV
YDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQW
VQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSF
NAPWAVTSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEA
FVVPYGTPLGVNFGPTVDGDFLTDMPDILLELGQFKKTQILVGVNKDEGT
WFLVGGAPGFSKDNNSIITRKEFQEGLKIFFPGVSEFGKESILFHYTDWV
DDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSK
LPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANFAKY
GNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFP
KVDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE
FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPER
NECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY
FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRL
KCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEM
PADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLR
LAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQL
GEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC
AEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV
PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM
DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.
3-45. (canceled)
46. A method of attenuating a biological effect of a cocaine
exposure in a primate comprising administering to the primate an
amount of a fusion protein comprising: (a) a mutant
butyrylcholinesterase (BChE) polypeptide comprising the sequence
TABLE-US-00030 (SEQ ID NO: 1)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKV,
and (b) a human serum albumin (HSA) polypeptide comprising the
sequence TABLE-US-00031 (SEQ ID NO: 2)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE
CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA
KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE
DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,
wherein the amount of the fusion protein is effective to cause
attenuation of the biological effect of the cocaine exposure in the
primate.
47. The method of claim 46, wherein the fusion protein comprises
the sequence TABLE-US-00032 (Residues 24 to 1137 of SEQ ID NO: 4)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKVDAHKSEVAHRFKDLGEENFKA
LVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG
DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVD
VMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA
ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQ
RFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSI
SSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESEDVCKNYAE
AKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA
KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP
TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSD
RVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKER
QIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEE
GKKLVAASQAALGL.
48. The method of claim 46, wherein the fusion protein is
administered by intramuscular injection.
49. A pharmaceutical composition comprising: (I) a fusion protein
comprising: (a) a mutant butyrylcholinesterase (BChE) polypeptide
comprising the sequence TABLE-US-00033 (SEQ ID NO: 1)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKV,
and (b) a human serum albumin (HSA) polypeptide comprising the
sequence TABLE-US-00034 (SEQ ID NO: 2)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE
CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA
KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE
DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,
and (II) a formulation buffer comprising 10 mM sodium phosphate,
200 mM mannitol, 60 mM trehalose, and 0.01% (w/v) polysorbate 80,
pH 7.2.
Description
[0001] This application is a continuation of U.S. Ser. No.
12/962,410, filed Dec. 7, 2010, now U.S. Pat. No. 8,541,373, issued
Sep. 24, 2013, which claims the benefit of U.S. Provisional
Application Nos. 61/283,791, filed Dec. 8, 2009 and 61/412,205,
filed Nov. 10, 2010, each of which is hereby incorporated by
reference in its entirety.
[0002] Throughout this application various publications, published
patent applications, and patents are referenced. The disclosures of
these documents in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art to which this invention pertains.
BACKGROUND
[0003] Cocaine abuse and dependence have disastrous medical and
social consequences which have made the development of an effective
treatment a high priority (Pan, Y., Gao, D., Yang, W., Cho, H.,
Yahg, G., Tai, H., Zhan, C., "Computational redesign of human
butyrylcholinesterase for anticocaine medication," PNAS,
102(46):16656-61, 2005). However, as stated by Brimijoin et al.:
"there is no reliable means to treat cocaine overdose or reduce the
likelihood of relapse in users who have achieved abstinence. Human
plasma butyrylcholinesterase (BChE) contributes to normal cocaine
metabolism and has been considered for use in treating cocaine
toxicity" (Brimijoin, S., Gao, Y., Anker, J., Gliddon, L., LaFleur,
D., Shah, R., Zhao, Q., Singh, M., Carroll, M., "A Cocaine
Hydrolase Engineered from Human Butyrylcholinesterase Selectively
Blocks Cocaine Toxicity and Reinstatement of Drug Seeking in Rats,"
Neuropsychopharmacology, 33:2715-25, 2008).
[0004] Wild-type BChE, while important for cocaine metabolism in
the body, has low catalytic efficiency with cocaine. The low
cocaine hydrolase activity of wild-type BChE would require the use
of prohibitively large quantities of purified enzyme for treatment
of cocaine abuse or overdose. Mutagenesis performed on human BChE
with the goal of enhancing the cocaine hydrolase activity resulted
in the development of the double mutant A328W/Y332A (residues 356
and 360 relative to full length BChE) that has a k.sub.cat that is
40-fold higher than wild-type BChE, with only a slightly increased
K.sub.M (Sun H., Shen M., Pang Y., Lockridge O., Brimijoin S.,
"Cocaine Metabolism Accelerated by a Re-Engineered Human
Butyrylcholinesterase" Journal of Pharmacology and Experimental
Therapeutics, 302(2):710-716, 2002).
[0005] Further experimentation utilizing molecular dynamics to
simulate the transition state for the first chemical reaction step
of BChE catalyzed hydrolysis of cocaine resulted in the BChE mutant
A227S/S315G/A356W/Y360G that has a catalytic efficiency which is
500-fold greater than wild-type BChE and is greater than other
previously designed BChE mutants (Pan, Y., Gao, D., Yang, W., Cho,
H., Yang, G., Tai, H., Zhan, C., "Computational redesign of human
butyrylcholinesterase for anticocaine medication," PNAS,
102(46):16656-61, 2005).
[0006] To obtain a form of the A227S/S315G/A356W/Y360G BChE mutant
that may be suitable for therapeutic use, the BChE mutant designed
by Pan et al. was fused at its C terminus to human serum albumin
(HSA) because it has been observed that similar fusions exhibit
favorable pharmacokinetic properties with high stability and
extended plasma half lives. It was observed that the BChE-albumin
fusion comprising the above mutations retains high catalytic
efficiency with cocaine and exhibits a plasma half-life of 8 hours
after i.v. injection to rats. (Brimijoin S., Gao, Y., Anker J.,
Gliddon L., LaFleur D., Shah R., Zhao, Q., "A Cocaine Hydrolase
Engineered from Human Butyrylcholinesterase Selectively Blocks
Cocaine Toxicity and Reinstatement of Drug Seeking in Rats"
Neuropsychopharmacology, 33:2715-25, 2008).
[0007] To date, there has been no effective method for treating
cocaine abuse or overdose in primates developed that utilizes a
BChE-albumin fusion comprising the mutations A227S, S315G, A356W,
and Y360G.
BRIEF SUMMARY OF THE INVENTION
[0008] The subject invention provides a method of attenuating a
biological effect of a cocaine exposure in a primate comprising
administering to the primate an amount of a fusion protein
comprising [0009] (a) a mutant butyrylcholinesterase (BChE)
polypeptide comprising the sequence
TABLE-US-00001 [0009] (SEQ ID NO: 1)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKV,
[0010] (b) a human serum albumin (HSA) polypeptide comprising the
sequence
TABLE-US-00002 [0010] (SEQ ID NO: 2)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE
CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA
KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE
DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,
[0011] and [0012] (c) a signal peptide comprising the sequence
MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:3), wherein the amount of the
fusion protein is effective to cause attenuation of the biological
effect of the cocaine exposure in the primate.
DESCRIPTION OF THE FIGURES
[0013] FIG. 1 (SEQ ID NO: 4) shows the amino acid sequence of
AlbuBChE, a BChE-albumin fusion protein comprising the mutations
A227S, S315G, A356W, and Y360G.
[0014] FIG. 2 shows AlbuBChE serum concentration-time profile in
individual cynomolgus monkeys following a single IM administration
of 0.2, 1 or 5 mg/kg AlbuBChE dose (FIG. 2A: linear scale; FIG. 2B:
semi-logarithmic scale).
[0015] FIG. 3 shows mean AlbuBChE serum concentration-time profile
in cynomolgus monkeys following a single IM administration of 0.2,
1 or 5 mg/kg AlbuBChE dose (FIG. 3A: linear scale; FIG. 3B:
semi-logarithmic scale).
[0016] FIG. 4 shows mean cocaine concentration vs. time in control
animals (n=2) and as a function of time post-AlbuBChE dose (n=3)
(FIG. 4A: control and AlbuBChE at 0.2 mg/kg dose; FIG. 4B: control
and AlbuBChE at 1.0 mg/kg dose; FIG. 4C: control and AlbuBChE at
5.0 mg/kg dose).
[0017] FIG. 5 shows mean benzoylecgonine concentration vs. time in
control animals (n=2) and as a function of time post-AlbuBChE dose
(n=3) (FIG. 5A: control and AlbuBChE at 0.2 mg/kg dose; FIG. 5B
control and AlbuBChE at 1.0 mg/kg dose; FIG. 5C: control and
AlbuBChE at 5.0 mg/kg dose).
[0018] FIG. 6 shows mean ecgonine methyl ester concentration vs.
time in control animals (n=2) and as a function of time
post-AlbuBChE dose (n=3) (FIG. 6A: control and AlbuBChE at 0.2
mg/kg dose; FIG. 6B control and AlbuBChE at 1.0 mg/kg dose; FIG.
5C: control and AlbuBChE at 6.0 mg/kg dose).
[0019] FIG. 7 shows a comparison of mean cocaine concentration-time
profile for Day 1 cocaine control group with Day 11 (240 hr)
post-AlbuBChE administration at 0.2, 1 or 5 mg/kg.
[0020] FIG. 8 shows AlbuBChE PK/PD analysis in cynomolgus monkeys
following a single IM administration of 0.2, 1 or 5 mg/kg AlbuBChE
dose. Cocaine was administered IV at a dose of 1 mg/kg in control
animals or at 2, 48, 96, 120 and 240 hr post-AlbuBChE dose.
[0021] FIG. 9 shows cocaine metabolic pathways.
[0022] FIG. 10 shows mean cocaine AUC.sub.(0-t) following 1 mg/kg
IV cocaine dose in control animals (n=2) and as a function of time
post-AlbuBChE dose (n=3).
[0023] FIG. 11 shows mean benzoylecgonine AUC.sub.(0-t) following 1
mg/kg IV cocaine dose in control animals (n=2) and as a function of
time post-AlbuBChE dose (n=3).
[0024] FIG. 12 shows AlbuBChE serum concentration-time profile in
individual squirrel monkeys following a single IM administration of
5 mg/kg AlbuBChE dose (semi-logarithmic scale).
[0025] FIG. 13 shows mean cocaine concentration vs. time in control
animals (n=2) and as a function of time post-AlbuBChE dose
(n=3).
[0026] FIG. 14 shows mean ecgonine methyl ester concentration at 5
min following 1 mg/kg IV cocaine dose in control animals (n=2) and
as a function of time post-AlbuBChE dose (n=3).
[0027] FIG. 15 shows mean benzoylecgonine concentration at 5 min
following 1 mg/kg IV cocaine dose in control animals (n=2) and as a
function of time post-AlbuBChE dose (n=3).
[0028] FIG. 16 shows a summary of squirrel monkey blood levels of
cocaine and the cocaine metabolites ecgonine methyl ester (EME) and
benzoylecgonine (BZ) at 5 minutes (top panel) and 30 minutes
(bottom panel) following cocaine injection.
[0029] FIG. 17 shows AlbuBChE PK/PD analysis in Squirrel Monkeys
following a single IM administration of 5 mg/kg AlbuBChE dose.
Cocaine was administered IV at a dose of 1 mg/kg in control animals
(n=2) or at 2, 72 and 96 hr post-AlbuBChE dose (n=3).
[0030] FIG. 18 shows mean heart rate vs. time in cynomolgus monkeys
prior to and following a single IM administration of 15 mg/kg
AlbuBChE dose or formulation buffer.
[0031] FIG. 19 shows mean heart rate pressure product vs. time in
cynomolgus monkeys prior to and following a single IM
administration of 15 mg/kg AlbuBChE dose or formulation buffer.
[0032] FIG. 20 shows mean arterial blood pressure vs. time in
cynomolgus monkeys prior to and following a single IM
administration of 15 mg/kg AlbuBChE dose or formulation buffer.
[0033] FIG. 21 shows mean diastolic blood pressure vs. time in
cynomolgus monkeys prior to and following a single IM
administration of 15 mg/kg AlbuBChE dose or formulation buffer.
[0034] FIG. 22 shows mean systolic blood pressure vs. time in
cynomolgus monkeys prior to and following a single IM
administration of 15 mg/kg AlbuBChE dose or formulation buffer.
[0035] FIG. 23 shows mean body temperature vs. time in cynomolgus
monkeys prior to and following a single IM administration of 15
mg/kg AlbuBChE dose or formulation buffer.
[0036] FIG. 24A shows mean respiration rate vs. time in cynomolgus
monkeys prior to and following a single IM administration of 15
mg/kg AlbuBChE dose or formulation buffer. Data for male cynomolgus
monkeys is shown in FIG. 24B, top panel, and data for female
cynomolgus monkeys is shown in FIG. 24B, bottom panel.
[0037] FIG. 25A shows mean SPO2 levels vs. time in cynomolgus
monkeys prior to and following a single IM administration of 15
mg/kg AlbuBChE dose or formulation buffer. Data for male cynomolgus
monkeys is shown in FIG. 25B, top panel, and data for female
cynomolgus monkeys is shown in FIG. 25B, bottom panel.
[0038] FIG. 26A shows mean ETCO2 levels vs. time in cynomolgus
monkeys prior to and following a single IM administration of 15
mg/kg AlbuBChE dose or formulation buffer. Data for male cynomolgus
monkeys is shown in FIG. 26B, top panel, and data for female
cynomolgus monkeys is shown in FIG. 26B, bottom panel.
[0039] FIG. 27 shows mean arterial blood pressure vs. time in
cynomolgus monkeys following a cocaine dose of 1 mg/kg administered
IV three hours post 15 mg/kg AlbuBChE dose. Cocaine dose was
administered at t=0.
[0040] FIG. 28 shows mean diastolic blood pressure vs. time in
cynomolgus monkeys following a cocaine dose of 1 mg/kg administered
IV three hours post 15 mg/kg AlbuBChE dose. Cocaine dose was
administered at t=0.
[0041] FIG. 29 shows mean systolic blood pressure vs. time in
cynomolgus monkeys following a cocaine dose of 1 mg/kg administered
IV three hours post 15 mg/kg AlbuBChE dose. Cocaine dose was
administered at t=0.
[0042] FIG. 30 shows mean heart rate vs. time in cynomolgus monkeys
following a cocaine dose of 1 mg/kg administered IV three hours
post 15 mg/kg AlbuBChE dose. Cocaine dose was administered at
t=0.
[0043] FIG. 31 shows mean rate pressure product vs. time in
cynomolgus monkeys following a cocaine dose of 1 mg/kg administered
IV three hours post 15 mg/kg AlbuBChE dose. Cocaine dose was
administered at t=0.
[0044] FIG. 32 shows mean body temperature vs. time in cynomolgus
monkeys following a cocaine dose of 1 mg/kg administered IV three
hours post 15 mg/kg AlbuBChE dose. Cocaine dose was administered at
t=0.
[0045] FIG. 33 shows squirrel monkey response rate (top panels) and
number of injections (bottom panels) during self administration
sessions over consecutive days where either cocaine or saline was
available for self-administration. Responding was tracked for five
days following vehicle or AlbuBChE administration (5 mg/kg).
[0046] FIG. 34 shows levels of reinstatement of cocaine
self-administration following administration of AlbuBChE or
AlbuBChE vehicle. The AlbuBChE or vehicle was given i.m. Two, 48
and 96 hrs later 0.3 mg/kg cocaine i.v. (left panels) or 0.1 mg/kg
cocaine i.v. (right panels) was given 5 min before a saline
substitution session.
[0047] FIG. 35 shows the modulation of cocaine's
discriminative-stimulus effects in methamphetamine trained subjects
by AlbuBChE.
[0048] FIG. 36 shows methamphetamine's discriminative-stimulus
effects in methamphetamine trained subjects after AlbuBChE
administration.
[0049] FIG. 37 shows the relative serum concentration of an albumin
fusion compared to a non-fusion peptide where the albumin fusion is
dosed once-weekly and the non-fusion peptide is dosed daily.
[0050] FIG. 38 shows a SDS-PAGE of AlbuBChE purified from CHO
cells. Lane 1 is a molecular weight marker, and lanes 2 and 4 show
purified AlbuBChE under reducing and non-reducing conditions,
respectively.
[0051] FIG. 39 shows the number of beam crossings vs. time in rats
treated with wild-type BChE or Albu-CocH prior to a cocaine dose,
rats given a cocaine dose alone, and rats given neither cocaine nor
BChE. Locomotor activity was assessed by detecting infrared beam
breaks as described in Brimijoin et al.
[0052] FIG. 40 shows the concentration of AlbuBChE vs. time in mice
following a dose of AlbuBChE and also shows cocaine hydrolysis
(represented as the percentage of cocaine converted to benzoic acid
(% BA) within 60 minutes of cocaine administration) vs. time post
AlbuBChE dose.
[0053] FIG. 41 shows the cocaine content in brain and heart
collected from rats (n=6) given an AlbuBChE (3 mg/kg) dose or
saline, through the tail vein, followed ten minutes later by 30
.mu.Ci 3H-cocaine (3.5 mg/kg), also through the tail vein. Brains
and hearts were collected 10 minutes post 3H-cocaine dose. Left to
right, control data is bars 1 and 3, while AlbuBChE data is bars 2
and 4.
[0054] FIG. 42 shows cocaine (left plot) and benzoate levels (right
plot) in brain, heart and plasma collected from rats given a 3
mg/kg AlbuBChE dose or saline 10 minutes prior to a 30 .mu.Ci
3H-cocaine dose (3.5 mg/kg). Brain, heart and plasma were collected
10 minutes post cocaine dose.
[0055] FIG. 43 shows the experimental design of a study examining
the ability of AlbuBChE to protect from lethal overdose.
[0056] FIG. 44 shows the percentage of rats exhibiting
hyperactivity, seizures, and death in response to a dose of 100
mg/kg cocaine given ten minutes after a dose of 0, 1, 3, or 10
mg/kg AlbuBChE.
[0057] FIG. 45 shows the concentration of cocaine vs. time in
Sprague Dawley rats given an IV dose of AlbuBChE at 0, 2, and 10
mg/kg followed five minutes later by an IP dose of cocaine at 60 or
100 mg/kg.
[0058] FIG. 46 shows a behavioral model of addiction and relapse.
"S" and "D" represent saline and the drug cocaine, respectively.
Animals are trained to emit a lever press for a cocaine infusion.
After stabilization (maintenance), saline is substituted for
cocaine and the behavior is allowed to extinguish. In the
subsequent reinstatement phase, priming injections of cocaine are
given, alternating with saline.
[0059] FIG. 47 shows the selective blockade of cocaine-primed
reinstatement of drug-seeking behavior resulting from AlbuBChE
treatment. Rats that had previously self-administered cocaine and
extinguished when cocaine was replaced with saline were primed with
an IV injection of saline (S), cocaine (C, 10 mg/kg), or
amphetamine (A, 2 mg/kg). AlbuBChE was administered IV (E, 2 mg/kg)
two hours before the behavioral session.
[0060] FIG. 48 shows the number of lever presses per session in
rats following saline injection, cocaine injection (10 mg/kg, IV),
or AlbuBChE (2 mg/kg, IV) followed by cocaine (10 mg/kg, IV) after
an addiction phase and forced abstinence.
[0061] FIG. 49 shows AlbuBChE bioavailability, half-life, and time
for maximum concentration, T.sub.max, in cynomolgus monkeys
following a single intravenous (IV), subcutaneous (SC), or
intramuscular (IM) injection. AlbuBChE absolute bioavailability for
IM and SC routes of administration was calculated relative to IV at
the 3 mg/kg dose level.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The subject invention provides a method of attenuating a
biological effect of a cocaine exposure in a primate comprising
administering to the primate an amount of a fusion protein
comprising [0063] (a) a mutant butyrylcholinesterase (BChE)
polypeptide comprising the sequence
TABLE-US-00003 [0063] (SEQ ID NO: 1)
EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLT
KWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIP
APKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGA
LGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESSG
AASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLT
GCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDGDFLT
DMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEF
QEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFIC
PALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPL
ERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKY
LTLNTESTRIMTKLRAQQCRFWTSFFPKV,
[0064] (b) a human serum albumin (HSA) polypeptide comprising the
sequence
TABLE-US-00004 [0064] (SEQ ID NO: 2)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFA
KTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE
CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA
DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA
KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE
DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPK
EFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,
and [0065] (c) a signal peptide comprising the sequence
MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:3), wherein the amount of the
fusion protein is effective to cause attenuation of the biological
effect of the cocaine exposure in the primate.
[0066] In an embodiment of the method, fusion protein comprises the
sequence
TABLE-US-00005 (SEQ ID NO: 4)
MRPTWAWWLFLVLLLALWAPARGEDDIIIATKNGKVRGMNLTVFGGTVTA
FLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQSFPGFH
GSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQTGTSSLHV
YDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQW
VQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSF
NAPWAVTSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEA
FVVPYGTPLGVNFGPTVDGDFLTDMPDILLELGQFKKTQILVGVNKDEGT
WFLVGGAPGFSKDNNSIITRKEFQEGLKIFFPGVSEFGKESILFHYTDWV
DDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSK
LPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANFAKY
GNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFP
KVDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE
FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPER
NECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY
FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRL
KCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEM
PADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLR
LAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQL
GEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC
AEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV
PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM
DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.
[0067] In another embodiment of the method, the fusion protein is
administered prior to the cocaine exposure.
[0068] In another embodiment, the fusion protein is administered up
to one day prior to the cocaine exposure.
[0069] In another embodiment, the fusion protein is administered up
to 216 hours prior to the cocaine exposure. The fusion protein may
be administered 1, 2, 3, 4, 5, 6, 7, 8, or 9 days prior to the
cocaine exposure.
[0070] In yet another embodiment of the method, the fusion protein
is administered after the cocaine exposure. In a further
embodiment, the fusion protein is administered up to six hours
following the cocaine exposure. In another embodiment, the fusion
protein is administered up to one hour after the cocaine
exposure.
[0071] In yet another embodiment of the method, the cocaine
exposure is a single cocaine exposure.
[0072] In yet another embodiment of the method, the cocaine
exposure is a recurring cocaine exposure. In a further embodiment
of the method, the recurring cocaine exposure is cocaine abuse or
cocaine dependence.
[0073] In yet another embodiment of the method, the recurring
cocaine exposure comprises at least six single cocaine exposures in
a twelve month period.
[0074] In yet another embodiment of the method, the recurring
cocaine exposure comprises at least twenty single cocaine exposures
during the primate's lifetime.
[0075] In yet another embodiment of the method, the biological
effect is caused by cocaine overdose in the primate and the
attenuating is treating or preventing the biological effect.
[0076] In yet another embodiment of the method, the biological
effect is an increase in blood pressure.
[0077] In a further embodiment of the method, the duration of the
increase in blood pressure is reduced by 60-90%.
[0078] In a further embodiment of the method, the duration of the
increase in blood pressure is reduced by about 78%.
[0079] In yet another embodiment of the method, the biological
effect is an increase in heart rate or body temperature. In a
further embodiment of the method, the degree of attenuation is
45%-70%.
[0080] In a further embodiment of the method, the degree of
attenuation is 57%.
[0081] In yet another embodiment of the method, the biological
effect is cocaine seeking behavior in the primate.
[0082] In yet another embodiment of the method, the cocaine seeking
behavior occurs during a period of cocaine abstinence following the
cocaine exposure.
[0083] In yet another embodiment of the method, the cocaine seeking
behavior follows a relapse.
[0084] In yet another embodiment of the method, administration of
the fusion protein two hours before the relapse attenuates cocaine
seeking behavior by the primate immediately following the
relapse.
[0085] In yet another embodiment of the method, administration of
the fusion protein two hours before the cocaine exposure results in
a 50% to 100% reduction in the cocaine seeking behavior by the
primate.
[0086] In yet another embodiment of the method, attenutation of
cocaine seeking behavior is observed up to four days following
administration of the fusion protein.
[0087] In yet another embodiment of the method, the administration
of the fusion protein results in a lowering of total cocaine
exposure in the primate than without the administration.
[0088] In yet another embodiment of the method, attenuating cocaine
seeking behavior results in a period of cocaine abstinence in the
primate. In a further embodiment of the method, the period of
abstinence is 2 weeks to 3 weeks.
[0089] In yet another embodiment of the method, attenuating cocaine
seeking behavior results in a larger proportion of days in which
the primate is not exposed to cocaine than without the
administration.
[0090] In yet another embodiment of the method, attenuating cocaine
seeking behavior results in a larger number of consecutive days in
which the primate is not exposed to cocaine than without the
administration.
[0091] In yet another embodiment of the method, attenuating cocaine
seeking behavior results in a lessening of severity of cocaine
dependence or abuse as evaluated by the cocaine selective severity
assessment (CSSA) or Diagnostic and Statistical Manual of Mental
Disorders IV (DSM-IV).
[0092] In yet another embodiment of the method, the effective
amount of the fusion protein is an amount which reduces the
primate's serum cocaine level to about 0 ng/ml within about 30
minutes of a 1 mg/kg intravenous cocaine dose.
[0093] In yet another embodiment of the method, the administration
of the fusion protein reduces the primate's serum cocaine level to
less than 12% of the serum cocaine level without the administration
within about 5 minutes of a 1 mg/kg intravenous cocaine dose.
[0094] In yet another embodiment of the method, the administration
of the fusion protein reduces the primate's serum cocaine level to
7% of the serum cocaine level without the administration within
about 5 minutes of a 1 mg/kg intravenous cocaine dose.
[0095] In yet another embodiment of the method, the fusion protein
is administered only once, daily, semi-weekly, weekly, bi-weekly,
or monthly.
[0096] In another embodiment, the fusion protein is administered
weekly or twice weekly.
[0097] In another embodiment, the fusion protein is administered as
a single dose following the cocaine exposure.
[0098] In another embodiment, the fusion protein is administered as
a single dose following a cocaine overdose.
[0099] In yet another embodiment of the method, the fusion protein
is administered by intramuscular injection or subcutaneous
injection.
[0100] In yet another embodiment of the method, the fusion protein
is in a formulation buffer comprising 10 mM sodium phosphate, 200
mM mannitol, 60 mM trehalose, and 0.01% (w/v) polysorbate 80, pH
7.2.
[0101] In yet another embodiment of the method, the fusion protein
is present in the formulation at a concentration of at least 30
mg/ml.
[0102] In yet another embodiment of the method, attenuation of the
biological effect is observed up to 72 hours after administration
of the fusion protein.
[0103] In yet another embodiment of the method, the primate is a
human.
[0104] In a further embodiment of the method, the cocaine exposure
is a single cocaine exposure of 10 mg to 60 mg or a recurring
cocaine exposure wherein each single cocaine exposure of the
recurring cocaine exposure is 10 mg to 60 mg.
[0105] In yet another embodiment of the method, the effective
amount of the fusion protein is an amount which reduces the human's
serum cocaine level to about 0 ng/ml within about 30 minutes of a
40 mg intravenous cocaine dose.
[0106] In yet another embodiment of the method, the effective
amount of the fusion protein is 0.06 mg/kg to 5 mg/kg. In another
embodiment of the method, the effective amount of the fusion
protein is 0.06 mg/kg, 0.3 mg/kg, 1.6 mg/kg, or 4.8 mg/kg.
[0107] In yet another embodiment of the method, the effective
amount of the fusion protein is 50 mg to 300 mg. In another
embodiment of the method, the effective amount of the fusion
protein is 50 mg, 100 mg, 150 mg, or 300 mg.
[0108] An embodiment of the BChE-albumin fusion protein comprising
the amino acid substitutions A227S, S315G, A356W, and Y360G is
shown in FIG. 1 (SEQ ID NO: 4). The amino acid sequence shown in
FIG. 1 comprises a heterologous signal peptide, shown by
underlining, a BChE domain comprising amino acids E29 to V529 of
human BChE, and human serum albumin (HSA), shown in italics. The
amino acid substitutions A227S, S315G, A356W, and Y360G are shown
in bold and are underlined in FIG. 1. The numbering of the
substitutions is relative to that of full length BChE. The protein
encoded by the amino acid sequence of FIG. 1 is referred to as
"AlbuBChE" throughout this application.
[0109] It is understood that all combinations of the above
described embodiments of the invention are within the scope of the
invention.
[0110] As used herein, "primate" refers to any of an order of
mammals that are characterized especially by advanced development
of binocular vision, specialization of the appendages for grasping,
and enlargement of the cerebral hemispheres and that include
humans, apes, monkeys, and related forms.
[0111] As used herein, "degree of attenuation" refers to the
decrease in a biological effect of cocaine exposure that is
observed following administration of a BChE-albumin fusion protein
as compared to the biological effect of cocaine exposure observed
in the absence of the BChE-albumin fusion protein. The degree of
attenuation is calculated by the following formula:
degree of attenuation = .DELTA. BChE - albumin absent - .DELTA.
BChE - albumin present .DELTA. BChE - albumin absent
##EQU00001##
[0112] For example, if cocaine exposure raises a baseline
temperature of 38.degree. C. to 38.7.degree. C. in the absence of a
BChE-albumin fusion protein, and cocaine exposure raises a baseline
temperature of 38.degree. C. to 38.3.degree. C. in the presence of
a BChE-fusion protein, the degree of attenuation is 57.1%
((0.7.degree. C.-0.3.degree. C.)/0.7.degree. C.).
[0113] As used herein, "a single cocaine exposure" refers to one
exposure of cocaine isolated from any other exposure of cocaine. "A
recurring cocaine exposure" refers to more than one single cocaine
exposure. The recurring cocaine exposure may be a regular or an
irregular pattern of single cocaine exposures beginning with the
second or subsequent single cocaine exposure in the subject. An
individual experiencing recurring cocaine exposure may meet the
criteria for cocaine dependence or cocaine abuse of the Diagnostic
and Statistical Manual of Mental Disorders IV (DSM-IV).
[0114] As used herein, the term "total cocaine exposure" refers to
the aggregate cocaine exposure during a given time interval. Total
cocaine exposure may be measured during or after a period of a
treatment designed to attenuate cocaine seeking behavior or other
biological effect of cocaine exposure.
[0115] As used herein, the term "a period of cocaine abstinence"
refers to a period of time following cocaine exposure where the
primate does not experience a new cocaine exposure.
[0116] As used herein, the term "relapse" refers to a cocaine
exposure following a period of cocaine abstinence.
[0117] It is understood that where a parameter range is provided,
all tenths of integers within that range are provided by the
invention. For example, "0.2 mg/kg to 15 mg/kg" includes 0.2 mg/kg,
0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg etc. up to and including 15.0
mg/kg.
[0118] An animal dose may be converted to a human equivalent dose
(HED) by using the conversion table found in the publication
"Guidance for Industry: Estimating the Maximum Safe Starting Dose
in Initial Clinical Trials for Therapeutics in Adult Healthy
Volunteers," U.S. Department of Health and Human Services, Food and
Drug Administration Center for Drug Evaluation and Research (CDER),
July 2005. Doses for cynomolgus monkeys in mg/kg may be converted
to an HED in mg/kg by dividing the cynomolgus monkey dose by 3.1.
Doses for squirrel monkeys in mg/kg may be converted to an HED in
mg/kg by dividing the squirrel monkey dose by 5.3.
LIST OF ABBREVIATIONS
[0119] AUC.sub.(0-t) Area under the plasma concentration-time curve
from time zero up to time of last detectable concentration (tz)
[0120] AUC.sub.(0-.infin.) Area under the plasma concentration-time
curve from time zero to infinity [0121] % AUC.sub.ex Percentage of
AUC that is due to extrapolation from tz to infinity [0122] BChE
butyrycholinesterase [0123] BQL Below Quantitation Limit [0124] CL
clearance [0125] C.sub.max maximum concentration [0126] CocH
cocaine hydrolase [0127] ELISA enzyme linked immunosorbent assay
[0128] GFR glomerular filtration rate [0129] hr hour(s) [0130] HRP
horseradish peroxidase [0131] IM intramuscular [0132] IV
intravenous [0133] LLOQ lower limit of quantitation [0134] min
minute(s) [0135] NCA noncompartmental analysis [0136] NIDA National
Institute on Drug Abuse [0137] PBS phosphate buffered saline [0138]
PD pharmacodynamics [0139] PK pharmacokinetics [0140] RT room
temperature [0141] Rsq Coefficient of determination [0142] % RSD
Percent Relative Standard Deviation [0143] SD Standard Deviation
[0144] SC subcutaneous [0145] SOP standard operating protocol
[0146] t.sub.1/2 half-life of the terminal phase [0147] t.sub.max
time of maximum concentration [0148] V.sub.z volume of distribution
from the terminal phase
[0149] The BChE-albumin fusion protein shown in FIG. 1 (AlbuBChE)
was applied to trials as described in the examples below.
Example 1
A Pharmacokinetic and Pharmacodynamic Study Following a Single
Intramuscular Administration of AlbuBChE and Multiple Intravenous
Doses of Cocaine to Cynomolgus Monkeys
Objectives
[0150] The objective of this trial was to determine the
pharmacokinetic (PK) profile of AlbuBChE in male cynomolgus monkeys
following a single intramuscular administration at 0.2, 1 or 5
mg/kg AlbuBChE dose levels and to determine AlbuBChE activity as a
function of time by intravenous administration of 1 mg/kg dose of
cocaine at 2, 48, 96, 120, and 240 hours after AlbuBChE dose
administration.
Rationale
[0151] AlbuBChE is a fusion protein of human serum albumin (HSA)
and a genetically modified form of human butyrylcholinesterase
(BChE) that exhibits high catalytic efficiency for the hydrolysis
of cocaine to benzoic acid. AlbuBChE is under development as a
potential intervention in preventing relapse to drug-seeking
behavior. Fusion of a protein to albumin has been shown to improve
the pharmacokinetic properties of the protein by reducing the
clearance and extending the half-life. A longer half-life is
expected to translate into a longer dosing interval and better
compliance with a drug regimen.
Rationale for Species Selection
[0152] Cynomolgus monkeys (macaca mulatta) were selected for this
study based on anatomical, physiological, and biochemical
similarities to humans, which may facilitate extrapolation of
observed pharmacokinetic and pharmacodynamic properties to humans.
Monkeys are known to express butyrylcholinesterase, which is a
component of this drug. Monkeys are also physiologically responsive
to cocaine.
Rationale for Dose Level and Route
[0153] In a previous study, AlbuBChE pharmacokinetic profile was
evaluated in cynomolgus monkeys. In that study, the test article
was administered SC (7.8, 2.4 and 0.78 mg/kg), IM (2.4 mg/kg) and
IV (2.4 mg/kg). The pharmacokinetic properties of AlbuBChE were
linear throughout the SC doses measured. The choice of IM for this
study was based on the greater bioavailability in IM as compared to
SC (79% and 35-39% respectively) observed in the previous study. In
addition, AlbuBChE IM dose levels of 0.2, 1 and 5 mg/kg were
selected to define AlbuBChE pharmacodynamic range in cynomolgus
monkeys.
[0154] The dose of cocaine at 1 mg/kg IV was selected as a dose
sufficiently high to yield a physiologically relevant response and
achieve measureable concentration of cocaine in blood, while not
excessively stimulating the animals.
[0155] The number of animals in each group is the minimum number of
animals per group necessary for assessment of inter animal
variability. As this is a pilot study, only one sex (males) was
evaluated.
Test Solutions
[0156] The test article, AlbuBChE, was stored in a stock solution
as a frozen liquid formulation containing 29.9 mg/mL at
-70.+-.15.degree. C. Prior to dosing, the test article formulation
was thawed at room temperature. When the test article formulation
was completely thawed, the container was mixed by gentle inversion
and diluted with the appropriate volume of test article diluent to
achieve a concentration of 20 mg/mL. This formulation was serially
diluted with the test article diluent to achieve concentrations of
4 and 0.8 mg/mL.
[0157] Cocaine hydrochloride, the pharmacodynamic test article, was
purchased from Sigma. Dose levels were expressed as the
hydrochloride salt.
Experimental Design
[0158] The study included a total of 11 nauve, adult male
cynomolgus monkeys dividied into four dose groups:
[0159] a control group of two cynomologus monkeys that only
received a single IV dose of cocaine;
[0160] three dose groups that were treated with a single IM dose of
AlbuBChE at 0.2, 1 or 5 mg/kg. Three naive, adult male cynomolgus
monkeys were used in each dose group. Following AlbuBChE treatment,
a 1 mg/kg IV dose of cocaine was administered at 2, 48, 96, 120,
and 240 hours after AlbuBChE dose administration.
[0161] A summary of the dose designation and dose levels can be
found in Table 1. Individual doses were calculated based on body
weights recorded on the day of dose administration. Animals were
not fasted prior to dose administration.
TABLE-US-00006 TABLE 1 Group Designations and Dose Levels AlbuBChE
Dose Dose Level Concentration Dose Volume Number of Group Treatment
(mg/kg) (mg/mL) (mL/kg) Animals 1 Single IV dose of Cocaine 1 1.0 1
2 2 Single IM dose of 0.2 0.8 0.25 3 AlbuBChE Single IV dose of
Cocaine 1 1.0 1 at 2, 48, 96, 120, and 240 hours after AlbuBChE
dose administration.sup.1 3 Single IM dose of 1 4 0.25 3 AlbuBChE
Single IV dose of Cocaine 1 1.0 1 at 2, 48, 96, 120, and 240 hours
after AlbuBChE dose administration.sup.1 4 Single IM dose of 5 20
0.25 3 AlbuBChE Single IV dose of Cocaine 1 1.0 1 at 2, 48, 96,
120, and 240 hours after AlbuBChE dose administration.sup.1
.sup.1All times will be within .+-.5 minutes of the listed
times
Samples Collection for AlbuBChE
[0162] A single intramuscular (IM) administration of AlbuBChE was
administered on study day 1 and blood was collected at the time
points outlined in Table 2. Approximately 0.5 mL of whole blood was
collected from the femoral vein into serum separate tubes
(SST).
TABLE-US-00007 TABLE 2 Pharmacokinetics for AlbuBChE Time Point
Sample Volume Collection Device Pre-dose, 1, 3, 6, 12, 24, 36, 48,
~0.5 mL SST 72, 96, 128, 144, 168, 216, and 264 hours post dose
Samples Collection for Cocaine
[0163] Approximately 0.5 mL of whole blood was collected from the
femoral vein at 5, 10, 15, 20, 30, 40 and 60 minutes following each
dose of cocaine (Study Day 1 of the control group and Study Day 1
(2 hr), 3 (48 hr), 5 (96 hr), 6 (120 hr), and 11 (240 hr)
post-AlbuBChE dose administration). Blood was placed into K2EDTA
blood collection tubes with esterase inhibitor
diisopropylfluorophosphate (DFP). Tubes were inverted several times
and placed on wet ice upon collection. Samples were centrifuged at
2-8.degree. C. within 45 minutes of collection. The resultant
plasma was recovered and a single 200 .mu.L aliquot of plasma was
placed into polypropylene tubes. Plasma samples were frozen over
dry ice and stored at -75.+-.15.degree. C.
Assay Methods
[0164] The following outline describes the ELISA-based assay
employed in the measurement of AlbuBChE concentrations in monkey
serum samples obtained pre-administration and at different times
after administration.
[0165] Immulon 4 HBX plates are coated with 100 .mu.L of anti-BChE
mAb 002-01 (Abcam ab17246) at 1 ug/mL in PBS, overnight at
4.degree. C. Blocking is done with 2% Casein in 1.times.PBS, 200
.mu.L/well, 2 hours at room temperature. After washing, 100 .mu.L
of diluted serum samples are added to the plates along with
standards. Standards are generated through 3.64 fold, serial
dilution of AlbuBChE from 2000 to 3.1 ng/mL. Serum samples and
standards are maintained at 10% serum by dilution with buffer
containing pooled cynomolgus monkey serum. A wash step precedes
detection with 100 .mu.L of anti-HSA mAb-6502-HRP at 0.04 .mu.g/mL
for 1 hour. Wells are washed again, prior to developing with 100
.mu.l of TMB substrate. After 15 minutes, the reaction is
terminated with 100 .mu.L/well of 1N H2SO4 and read on SpectraMax
plate reader at 450/570 nm. Values for unknown serum samples are
calculated by interpolation of standard curve generated by
4-parameter fit of AlbuBChE standards. The limit of quantitation in
cynomolgus serum was 21.1 ng/mL.
LC-MS Quantitation of Cocaine in Serum
[0166] The following outline describes the LC-MS-based assay
employed for the measurement of cocaine in plasma samples. Prior to
MS analysis, plasma samples, calibration standards and controls
were extracted using supported liquid extraction (SLE-ISOLUTE,
Biotage). Twenty-five microliters of samples (25 .mu.L) received
150 .mu.l of 5% Ammonium hydroxide and 25 .mu.l internal standard
solution (Cocaine-d3). After mixing and transfer to SLE plate,
samples were eluted with methylene chloride and evaporated to
dryness. After resuspension in 50 .mu.l of reconstitution
solution--5% ACN in 10 mM ammonium acetate in water, 10 .mu.l was
injected onto Thermo Hypercarb column at 0.5 ml/min. Mobile phase
was biphasic. Mass spectrophotometric detection was performed using
Api 4000, APCI positive interface and Multiple Reaction Monitoring
(MRM). Plasma concentrations of cocaine, benzoylecgonine and
ecgonine methyl ester were determined using this assay.
PK Analysis of AlbuBChE
[0167] AlbuBChE pharmacokinetic parameter values were determined
using serum concentration-time profiles for individual animals. The
computer software WinNonlin Professional (Version 4.0.1 Pharsight
Corporation, USA) was used. Specifically the model for
non-compartmental analysis with extravascular input was applied. If
there were fewer than 3 data points in the terminal phase of the
serum concentration curve, a terminal phase half-life and PK
parameters derived from the half-life were not calculated for that
profile. Parameters analyzed include t.sub.max, C.sub.max,
t.sub.1/2, AUC.sub.(0-t) and AUC.sub.(0-.infin.) were
calculated.
PK Analysis of Cocaine
[0168] When the data permits, pharmacokinetic parameter values for
cocaine and its metabolites were determined using plasma
concentration-time profiles for individual animals at all the study
days of cocaine administration. The computer software WinNonlin
Professional (Version 4.0.1 Pharsight Corporation, USA) was used.
Specifically the model for non-compartmental analysis with IV bolus
input for cocaine and extravascular input for the metabolites. If
there were fewer than 3 data points in the terminal phase of the
plasma concentration curve, a terminal phase half-life and PK
parameters derived from the half-life were not calculated for that
profile.
PK/PD Analysis
[0169] AlbuBChE PK/PD relationship was defined using the computer
software WinNonlin Professional (Version 4.0.1 Pharsight
Corporation, USA). Specifically the direct sigmoidal inhibitory
Effect E.sub.max model was use in which maximum effect (E.sub.max)
as measured by cocaine AUC was assumed at AlbuBChE concentration of
zero. The model equation can be described as follows:
Inhibitory Effect Sigmoid Emax, C=0 at E.sub.max, C=infinity at
E.sub.o
E=Emax-(Emax-E0)*(C**Gamma/(C**Gamma+EC50**Gamma))
[0170] The sigmoidal model was selected due to the higher activity
observed at the 48 hr timepoint compared to the 2 hr timepoint.
[0171] The PD parameter used in the analysis was cocaine AUC. PK
parameters used in the model were AlbuBChE plasma concentrations at
2, 48, 96, 120, and 240 hr post-AlbuBChE dose. Due to the fact that
AlbuBChE concentration was not measured at 2 hr post-AlbuBChE dose,
the concentration at 3 hr postdose was used in the analysis with
the assumptions that AlbuBChE concentration at 3 hr post AlbuBChE
dose may reflect AlbuBChE conc. at 2 hr post AlbuBChE dose.
Statistical Methods
[0172] Summary statistics of the concentration profiles and PK
parameter values by experimental group were calculated using the
descriptive statistics function in WinNonlin. Statistical
parameters reported are N, mean, SD and percent coefficient of
variation (% CV).
Results
AlbuBChE Concentration Profile
[0173] AlbuBChE concentration-time data and summary statistics are
listed in Tables 3 and 4. Individual animal AlbuBChE serum
concentration-time profiles following a single AlbuBChE IM
injection of 0.2, 1 or 5 mg/kg are shown in FIG. 2. Mean serum
concentration-time profiles for the three AlbuBChE dose groups are
shown in FIG. 3.
[0174] Following IM injection, all animals had measurable AlbuBChE
concentrations. Inter-animals variability per dose group appears to
be reasonable as indicated by % CV that ranged from 8.2 to 78.4 for
all time points. AlbuBChE serum concentration increased with
increasing dose.
TABLE-US-00008 TABLE 3 Individual and Mean AlbuBChE Serum
Concentration (ng/mL) vs. time (hr) Profile in Cynomolgus Monkeys
following a single IM administration of 0.2, 1 or 5 mg/kg/animal
AlbuBChE dose. Time AlbuBChE Dose: 0.2 mg/kg IM Mean (hr) 17691
17692 17693 (ng/mL) SD % CV 0 <LLOQ <LLOQ <LLOQ <LLOQ
-- -- 1 37.1 235.4 111.0 128 100 78.4 3 176.1 278.5 280.7 245 59.8
24.4 6 144.0 212.1 272.4 210 64.2 30.7 12 88.4 143.1 197.9 143 54.8
38.3 24 52.3 95.2 135.7 94.4 41.7 44.2 36 29.8 68.3 87.4 61.8 29.3
47.4 48 <LLOQ 66.4 80.7 73.6 n = 2 -- 72 <LLOQ 44.5 39.5 42.0
n = 2 -- 96 <LLOQ 30.1 <LLOQ <LLOQ -- -- 128 <LLOQ
<LLOQ <LLOQ <LLOQ -- -- 144 <LLOQ <LLOQ <LLOQ
<LLOQ -- -- 168 <LLOQ <LLOQ <LLOQ <LLOQ -- -- 216
<LLOQ <LLOQ <LLOQ <LLOQ -- -- 264 <LLOQ <LLOQ
<LLOQ <LLOQ -- -- Time AlbuBChE Dose: 1.0 mg/kg IM Mean (hr)
17694 17695 17696 (ng/mL) SD % CV 0 <LLOQ <LLOQ <LLOQ
<LLOQ -- -- 1 1058.9 462.4 1598.6 1040 568 54.6 3 1665.5 1374.0
1956.0 1665 291.0 17.5 6 1269.3 1266.3 1596.1 1377 189.6 13.8 12
884.3 968.1 1049.5 967 82.6 8.5 24 630.1 673.8 572.1 625 51.0 8.2
36 373.4 471.7 348.2 398 65.3 16.4 48 289.4 362.7 264.4 306 51.1
16.7 72 122.1 235.0 133.0 163 62.3 38.1 96 87.0 131.7 75.3 98.0
29.8 30.4 128 36.2 72.0 38.1 48.8 20.1 41.3 144 27.4 47.7 31.5 35.5
10.7 30.2 168 <LLOQ 39.5 <LLOQ <LLOQ -- -- 216 <LLOQ
<LLOQ <LLOQ <LLOQ -- -- 264 <LLOQ <LLOQ <LLOQ
<LLOQ -- -- Time 5.0 mg/kg IM Mean (hr) 17697 17698 17699
(ng/mL) SD % CV 0 <LLOQ <LLOQ <LLOQ <LLOQ -- -- 1
2580.0 6570.9 3821.0 4324 2042 47.2 3 4899.0 12683.9 6024.0 7869
4207.6 53.5 6 6016.0 17686.8 6530.3 10078 6594.7 65.4 12 4178.0
11105.8 5091.0 6792 3764.0 55.4 24 2836.0 5034.0 3325.0 3732 1154.1
30.9 36 1572.0 2929.0 1901.0 2134 707.9 33.2 48 1329.7 2644.4
1604.9 1860 693.4 37.3 72 637.7 1190.8 723.6 851 297.7 35.0 96
415.1 732.6 454.7 534.1 173.0 32.4 128 217.2 325.9 224.3 255.8 60.8
23.8 144 158.4 254.7 163.3 192.1 54.2 28.2 168 118.9 165.5 130.4
138.3 24.3 17.6 216 73.7 91.6 79.4 81.6 9.14 11.2 264 41.9 50.9
47.2 46.7 4.52 9.69 -- Not applicable LLOQ 27.2 ng/ml
TABLE-US-00009 TABLE 4 Summary of Mean AlbuBChE Serum Concentration
(ng/ml) vs. time (hr) in Cynomolgus Monkeys following a single IM
administration of 0.2, 1, or 5 mg/kg/animal AlbuBChE dose. Time
(hr) 0.2 mg/kg 1 mg/kg 5 mg/kg 1 128 1040 4324 3 245 1665 7869 6
210 1377 10078 12 143 967 6792 24 94.4 625 3732 36 61.8 398 2134 48
73.6 306 1860 72 42.0 163 851 96 -- 98.0 534 128 -- 48.8 256 144 --
35.5 192 168 -- -- 138 216 -- -- 81.6 264 -- -- 46.7 -- Not
applicable LLOQ 27.2 ng/ml
Concentration Profile of Cocaine, Benzoylecgonine and Ecgonine
Methyl Ester
[0175] LC/MS analysis was performed on all samples to analyze for
cocaine, benzoylecgonine and ecgonine methyl ester levels in monkey
plasma. Mean plasma concentration-time profiles in control animals
and following 0.2, 1, or 5 mg/kg can be found in Tables 5, 6, and 7
and FIGS. 4, 5 and 6 for cocaine, benzoylecgonine and ecgonine
methyl ester, respectively.
[0176] In general, cocaine plasma concentrations appear to decrease
as a function of AlbuBChE dose and increase as a function of time
post-AlbuBChE administration. Cocaine concentrations at 240 hr
post-AlbuBChE administration appears to be in the same range as Day
1 cocaine control group (FIG. 7). This suggests that AlbuBChE was
not active at 240 hr post-AlbuBChE administration of 0.2, 1 or 5
mg/kg. This also indicates that cocaine kinetics do not show time
dependent kinetics in cynomolgus monkeys making the comparison of
Day 1 single IV dose cocaine control to the multiple dose cocaine
profile following AlbuBChE administration feasible.
[0177] As one might expect based on cocaine metabolic pathway (FIG.
9), benzoylecgonine plasma concentrations appear to decrease as a
function of AlbuBChE dose and increase as a function of time
post-AlbuBChE administration.
[0178] Theoretically, plasma concentration of ecgonine methyl ester
should increase following AlbuBChE administration (FIG. 9). This
increase was only evident at the cocaine dose administered at 2 hr
post-AlbuBChE administration for all three AlbuBChE dose levels.
Ecgonine methyl ester plasma concentration could not be easily
distinguished from control following cocaine administration at 48,
96, 120 or 240 hr post-AlbuBChE administration.
TABLE-US-00010 TABLE 5 Mean Cocaine Plasma Concentration (ng/mL)
vs. time (min) in Cynomolgus monkeys following cocaine IV dose of 1
mg/kg in control animals (n = 2) or at 2, 48, 72, 96, 120 and 240
hr post-AlbuBChE IM dose of 0.2, 1, or 5 mg/kg (n = 3 per dose
group). Time Control (min) Day 1 2 hr 48 hr 96 hr 120 hr 240 hr
Control and 0.2 mg/kg AlbuBChE Dose 5 371 481 235 360 349 453 10
393 370 203 301 312 379 15 305 273 187 255 267 297 20 251 227 146
189 218 254 30 226 67.0 121 165 160 189 40 188 115 77.8 118 141 133
60 138 58.6 43.2 60.5 64.7 89.5 Control and 1 mg/kg AlbuBChE Dose 5
371 308 176 265 356 397 10 393 235 140 245 263 340 15 305 179 152
218 233 300 20 251 73.7 128 208 202 259 30 226 102 87.0 162 161 218
40 188 96.9 62.3 123 141 181 60 138 19.7 29.1 64.8 79.7 117 Control
and 5 mg/kg AlbuBChE Dose 5 371 145 126 176 298 346 10 393 77.9
89.9 166 217 283 15 305 43.5 82.7 174 216 283 20 251 12.8 49.5 136
161 226 30 226 110 36.7 99.0 139 211 40 188 4.96 18.9 80.6 113 161
60 138 2.26 5.74 35.2 49.5 111
TABLE-US-00011 TABLE 6 Mean Benzoylecgonine Plasma Concentration
(ng/mL) vs. time (min) in Cynomolgus monkeys following cocaine IV
dose of 1 mg/kg in control animals (n = 2) or at 2, 48, 72, 96, 120
and 240 hr post-AlbuBChE IM dose of 0.2, 1, or 5 mg/kg (n = 3 per
dose group). Day 1 Time (min) Control 2 hr 48 hr 96 hr 120 hr 240
hr Control and 0.2 mg/kg AlbuBChE Dose 5 47.6 50.6 27.3 49.7 55.1
53.5 10 65.4 49.7 29.4 47.7 46.9 53.3 15 63.0 50.4 29.0 52.6 53.1
62.9 20 72.0 65.1 28.2 55.1 55.9 63.7 30 82.4 28.6 37.1 56.5 64.2
69.8 40 87.2 58.0 36.9 55.2 61.4 74.5 60 96.9 60.5 35.6 57.8 66.2
80.9 Control and 1 mg/kg AlbuBChE Dose 5 47.6 30.1 24.0 43.9 37.2
40.0 10 65.4 33.2 23.7 41.2 38.9 47.2 15 63.0 32.5 24.9 40.0 42.2
55.0 20 72.0 32.5 26.7 43.7 40.2 56.7 30 82.4 28.9 29.8 47.4 51.3
64.6 40 87.2 38.4 29.6 46.7 50.9 68.3 60 96.9 29.6 29.8 49.6 54.4
75.2 Control and 5 mg/kg AlbuBChE Dose 5 47.6 22.3 20.0 44.2 44.6
44.9 10 65.4 23.7 21.0 43.5 47.5 56.4 15 63.0 22.0 24.4 49.7 50.3
71.9 20 72.0 22.9 23.7 51.1 57.3 83.4 30 82.4 31.1 25.9 56.1 63.6
88.4 40 87.2 21.3 25.5 57.7 69.5 101 60 96.9 20.1 24.4 60.5 69.2
108
TABLE-US-00012 TABLE 7 Ecgonine Methyl Ester Plasma Concentration
(ng/mL) vs. time (min) in Cynomolgus monkeys following cocaine IV
dose of 1 mg/kg in control animals (n = 2) or at 2, 48, 72, 96, 120
and 240 hr post-AlbuBChE IM dose of 0.2, 1, or 5 mg/kg (n = 3 per
dose group). Time Day 1 (min) Control 2 hr 48 hr 96 hr 120 hr 240
hr Control and 0.2 mg/kg AlbuBChE Dose 5 24.4 51.6 21.1 26.4 18.9
20.1 10 34.5 81.9 19.5 39.4 28.9 23.1 15 57.4 83.2 23.9 24.5 27.6
25.1 20 39.0 154 25.8 29.8 34.5 28.3 30 77.6 261 33.9 49.8 136 33.9
40 60.8 120 50.7 50.2 98 38.8 60 80.9 120 37.0 43.4 82.9 41.4
Control and 1 mg/kg AlbuBChE Dose 5 24.4 235 201 138 42.7 92.7 10
34.5 227 32.2 39.8 27.4 21.9 15 57.4 276 48.2 42.9 37.8 25.9 20
39.0 301 56.2 47.3 31.8 29.5 30 77.6 291 63.8 57.3 104 41.3 40 60.8
202 64.9 64.4 68.6 51.8 60 80.9 189 57.3 59.9 76.8 54.3 Control and
5 mg/kg AlbuBChE Dose 5 24.4 337 119 100 45.5 127 10 34.5 321 68.9
60.9 46.0 26.1 15 57.4 363 102 82.2 96.5 30.7 20 39.0 331 107 104
60.1 35.1 30 77.6 266 108 111 79.9 49.6 40 60.8 308 106 126 122
53.1 60 80.9 248 108 127 187 58.0
Pharmacokinetic Analysis of AlbuBChE
[0179] AlbuBChE pharmacokinetic parameter values following a single
IM dose of 0.2, 1 or 5 mg/kg AlbuBChE dose in individual animals
and descriptive statistics per group can be found in Table 8. Mean
PK parameter values for AlbuBChE following IM dosing at 0.2, 1 or 5
mg/kg are summarized in Table 9.
[0180] In general absorption from IM site of administration was
rapid with measureable concentrations observed at the first sample
collected (1 hr postdose). Maximum concentration was observed at 3
hr for the 0.2 and 1 mg/kg dose level. T.sub.max values was
slightly longer (6 hr) for the 5 mg/kg dose.
[0181] AlbuBChE exposure increased with increasing dose. Dose
normalized C.sub.max and AUC values appear to increase as a
function of dose suggesting a more than proportional increase in
exposure as a function of dose. This was also accompanied with a
increase in terminal elimination t1/2 as a function of dose
particularly between 1 and 5 mg/kg dose levels where t1/2 value
almost doubled.
TABLE-US-00013 TABLE 8 Individual and Mean AlbuBChE Pharmacokinetic
Parameter Values in Cynomolgus Monkeys following a single IM
administration of 0.2, 1 or 5 mg/kg/animal AlbuBChE dose. No.
Points t1/2 T.sub.max C.sub.max AUC.sub.(0-t) AUC.sub.(0-.infin.)
AUC Animal # R.sub.sq Lambda-z (hr) (hr) (ng/mL) (hr * ng/mL) (hr *
ng/mL) % Extrap 0.2 mg/kg AlbuBChE Dose 17691 1.00 3 15.3 3 176
2746 3404 19.3 17692 1.00 3 42.1 3 279 7878 9704 18.8 17693 0.980 7
24.4 3 281 8479 9867 14.1 Mean 27.2 245 6368 7658 17.4 SD 13.6 59.8
3151 3686 2.90 % CV 50.0 24.4 49.5 48.1 16.7 1 mg/kg AlbuBChE Dose
17694 0.990 6 28.4 3 1666 43128 44251 2.54 17695 0.992 8 34.3 3
1374 51298 53255 3.67 17696 0.992 6 30.3 3 1956 46185 47564 2.90
Mean 31.0 1665 46871 48357 3.04 SD 3.03 291 4128 4554 0.581 % CV
9.76 17.5 8.81 9.42 19.1 5 mg/kg AlbuBChE Dose 17697 0.999 4 63.4 6
6016 201754 205584 1.86 17698 1.00 3 56.4 6 17687 437848 441992
0.938 17699 1.00 3 65.5 6 6530 235983 240442 1.85 Mean 61.8 10078
291862 296006 1.55 SD 4.73 6595 127581 127623 0.532 % CV 7.66 65.4
43.7 43.1 34.3
TABLE-US-00014 TABLE 9 Mean Pharmacokinetic parameter values for
AlbuBChE following a single IM injection in cynomolgus monkeys at
0.2, 1 or 5 mg/kg. AlbuBChE Dose 5.0 mg/kg 0.2 mg/kg IM 1.0 mg/kg
IM IM t1/2 (hr) 27.2 31.0 61.8 t.sub.max (hr) 3 3 6 C.sub.max
(ng/mL) 245 1665 10078 AUC.sub.(0-t) (hr * ng/mL) 6368 46871 291862
AUC.sub.(0-.infin.) (hr * ng/mL) 7658 48357 296006 AUC % Extrap
17.4 3.04 1.55 Dose Normalized C.sub.max 1226 1665 2016 Dose
Normalized AUC.sub.(0-.infin.) 38292 48357 59201
Pharmacokinetic Analysis of Cocaine and Metabolites
[0182] Summary table of mean cocaine pharmacokinetic values per
dose group are presented in Table 10.
[0183] Cocaine pharmacokinetic profile was well characterized for
all dose groups and time points with one exception: plasma
concentration-time profile for the cocaine dose administered at 2
hr post-AlbuBChE dose was variable for all dose groups. As such,
the terminal elimination t could not be accurately characterized in
most of the animals on this cocaine dosing occasion.
[0184] Cocaine AUC.sub.(0-t) appears to decrease as a function of
AlbuBChE dose and increase as a function of time post-AlbuBChE
administration. Similarly, cocaine systemic plasma clearance
appears to increase as a function of AlbuBChE dose and return to
control values as a function of time post-AlbuBChE dose. At 240 hr
post-AlbuBChE administration cocaine AUC and clearance appear to be
in the same range as Day 1 cocaine control group.
[0185] For all three AlbuBChE dose levels, maximum AlbuBChE effect
on cocaine clearance or AUC was observed at 48 hr postdose. The
duration of effect was related to AlbuBChE dose levels. Following
the 5 mg/kg dose, AlbuBChE effect on cocaine AUC and clearance was
evident up to 120 hr postdose. At the 1 mg/kg dose, AlbuBChE effect
on cocaine AUC or clearance may still be evident at 96 to 120 hr
post-AlbuBChE dose relative to control or the 240 hr values. As
AlbuBChE dose decreased to 0.2 mg/kg, so did the duration of effect
with 48 hr being the last time point with elevated cocaine
clearance. Based on this data, a once or twice weekly dosing
regimen of AlbuBChE is likely.
[0186] As can be observed in FIG. 5, the concentration-time profile
of benzoylecgonine did not did not include a terminal elimination
phase. As such, pharmacokinetic characterization of benzoylecgonine
was limited to characterization of AUC.sub.(0-t). Summary table of
mean AUC values as a function of AlbuBChE dose and time
post-AlbuBChE dose in comparison with cocaine control group can be
found in Table 11. Consistent with cocaine metabolic pathway (FIG.
9), benzoylecgonine AUC decreased as a function of AlbuBChE dose
and increase as a function of time post-AlbuBChE administration as
shown in FIG. 11.
[0187] As can be observed in FIG. 6, the concentration-time profile
of ecgonine methyl ester was flat and did not did not include a
terminal elimination phase. As such, no pharmacokinetic
characterization of ecgonine methyl ester was conducted.
Theoretically, ecgonine methyl ester AUC should increase following
AlbuBChE administration (FIG. 9). This increase was only evident at
the cocaine dose administered at 2 hr post-AlbuBChE administration
for all three AlbuBChE dose levels. AlbuBChE effect on ecgonine
methyl ester could not be easily distinguished from control
following cocaine administration at 48, 96, 120 or 240 hr
post-AlbuBChE administration.
TABLE-US-00015 TABLE 10 Mean cocaine pharmacokinetic parameter
values in Cynomolgus Monkeys following 1 mg/kg cocaine IV dose in
control animals (n = 2) or at 2, 48, 72, 96, 120 and 240 hr
post-AlbuBChE IM dose of 0.2, 1, or 5 mg/kg (n = 3 per dose group).
Study AlbuBChE Time Dose t1/2 C.sub.0 AUC.sub.(0-t)
AUC.sub.(0-.infin.) AUC Cl V.sub.ss (hr) (mg/kg) (min) (ng/mL) (min
* ng/mL) (min * ng/mL) % Extrap (mL/min/kg) (mL/kg) Day 1 Control
41.8 371 14604 23407 35.4 48.5 2679 2 0.2 NA 628 11878 NA NA NA NA
48 0.2 21.2 274 7713 9094 15.1 110 3503 96 0.2 21.1 451 11145 13008
14.9 78.1 2506 120 0.2 23.0 391 11611 14059 16.1 72.5 2338 240 0.2
29.2 549 13693 17650 21.6 57.4 2181 2 1 NA 422 7880 NA NA NA NA 48
1 19.0 226 5965 6764 11.9 150 4304 96 1 23.9 288 10044 12313 18.4
81.6 2940 120 1 28.6 491 11526 14838 22.4 67.4 2687 240 1 31.0 467
14364 19623 26.7 51.1 2288 2 5 NA 271 3289 NA NA NA NA 48 5 11.4
177 3015 3110 3.0 325 5729 96 5 21.3 190 6624 7729 14.2 129 4111
120 5 22.3 411 9378 11046 14.5 93.0 2857 240 5 33.8 424 12952 18578
29.2 55.6 2637 NA Not applicable or not determined because data did
not permit.
TABLE-US-00016 TABLE 11 Summary Table of Mean Benzoylecgonine
AUC.sub.(0-t) (min * ng/mL) in control animals and as a function of
time post-AlbuBChE dose. 2 hr 48 hr 96 hr 120 hr 240 hr Control
4641 -- -- -- -- 0.2 mg/kg 3136 1989 3270 3583 4089 1 mg/kg 1960
1668 2741 2809 3660 5 mg/kg 1405 1441 3222 3624 5088
PK/PD Analysis
[0188] Based on the mechanism of action of AlbuBChE it was
anticipated that a direct effect inhibitory Emax model would be
able to characterize the PK/PD relationship of AlbuBChE and
cocaine.
[0189] PK and PD data used in the analysis are shown in Table 12.
The fit resulting from the direct Sigmoidal inhibitory Effect Emax
model are shown in FIG. 8.
[0190] The data clearly shows the inverse relationship between
AlbuBChE serum concentration and cocaine exposure. AlbuBChE serum
concentration that may result in 50% decrease in cocaine
concentration (EC50) was estimated by the model to be .about.600
ng/mL. E.sub.max, E.sub.0, and Gamma values were 12909 (min*ng/mL),
1096 (min*ng/mL), and 0.708, respectively.
TABLE-US-00017 TABLE 12 AlbuBChE PK/PD analysis Cynomolgus Monkeys
following a single IM administration of 0.2, 1 or 5 mg/kg AlbuBChE
dose. Cocaine was administered IV at a dose of 1 mg/kg in control
animals or at 2, 48, 96, 120 and 240 hr post-AlbuBChE dose. PK
parameters: AlbuBChE plasma concentrations per animals at 3.sup.#,
48, 96, 120 and 240 hr post-AlbuBChE dose. PD parameter: cocaine
AUC.sub.(0-t) per animal at 2, 48, 96, 120 and 240 hr post-AlbuBChE
dose. AlbuBChE AlbuBChE Cocaine Animal IM Dose Time Conc.
AUC.sub.(0-t) ID (mg/kg) (hr) (ng/mL) (min * ng/mL) 17689 0 2 0
18335 17690 0 2 0 10872 17691 0.2 2 107 13317 17692 0.2 2 257 10812
17693 0.2 2 196 11504 17694 1 2 1362 6597 17695 1 2 918 9746 17696
1 2 1777 7297 17697 5 2 3740 4594 17698 5 2 9627 3039 17699 5 2
4923 2234 17691 0.2 48 0 8550 17692 0.2 48 66.4 7315 17693 0.2 48
80.7 7276 17694 1 48 389 6025 17695 1 48 363 5033 17696 1 48 264
6839 17697 5 48 1330 3323 17698 5 48 2644 2628 17699 5 48 1605 3095
17691 0.2 96 0 13936 17692 0.2 96 30 8978 17693 0.2 96 0 10522
17694 1 96 87 9166 17695 1 96 132 9925 17696 1 96 75 11042 17697 5
96 415 6428 17698 5 96 733 6887 17699 5 96 455 6556 17691 0.2 120 0
11293 17692 0.2 120 0 11428 17693 0.2 120 0 12111 17694 1 120 36
10451 17695 1 120 72 11561 17696 1 120 38 12568 17697 5 120 217
7954 17698 5 120 326 9620 17699 5 120 224 10560 17691 0.2 240 0
13669 17692 0.2 240 0 12762 17693 0.2 240 0 14649 17694 1 240 0
14533 17695 1 240 0 13998 17696 1 240 0 14560 17697 5 240 58 11173
17698 5 240 71 13745 17699 5 240 63 13937 .sup.#AlbuBChE
concentration was not measured at 2 hr post-AlbuBChE dose, the 3 hr
post-AlbuBChE dose was used in the analysis.
Animal Observations
[0191] The animals were observed throughout the study. There were
no deaths during the study, and there were no clinical or cageside
observations associated with administration of AlbuBChE at either
dose. Administration of cocaine at a dose of 1 mg/kg without
pre-treatment with the test article resulted in hyperactivity and
increased respiratory rate with abdominal breathing.
Cocaine-related observations were not observed for five days
following pre-treatment with the test article at AlbuBChE doses
ranging from 0.2 to 5 mg/kg but returned on study days 6 and
11.
Conclusions
[0192] AlbuBChE was well tolerated in cyomolgus monkeys following a
single IM dose of 0.2, 1 or 5 mg/kg.
[0193] AlbuBChE absorption from IM site of administration was
rapid. T.sub.max was observed at 3 hr for the 0.2 and 1 mg/kg and 6
hr for the 5 mg/kg dose. AlbuBChE exposure appears to increase in a
more than proportional manner as a function of dose. Terminal
elimination t1/2 increased from 31 to 62 hr between 1 and 5 mg/kg
dose levels.
[0194] Cocaine was administered to control animals on Day 1 and at
2, 48, 96, 120 and 240 hr post-AlbuBChE dose. After each cocaine
administration, multiple samples were drawn with the objective of
determining the pharmacokinetic profile in response to decreasing
serum levels of AlbuBChE. Cocaine AUC.sub.(0-t) decreased as a
function of AlbuBChE dose and increase as a function of time
post-AlbuBChE administration.
[0195] The duration of effect was related to AlbuBChE dose levels.
AlbuBChE effect on cocaine AUC and clearance was evident up to 120
hr for 5 mg/kg dose, 96-120 hr for 1 mg/kg dose and 48 hr for the
0.2 mg/kg dose. Based on this data, a once or twice weekly dosing
regimen of AlbuBChE is likely.
[0196] PK/PD relationship in cynomolgus monkeys appears to indicate
an inverse relationship between AlbuBChE serum concentration and
cocaine exposure. AlbuBChE serum concentration that may result in
50% decrease in cocaine concentration (EC.sub.50) was estimated by
the direct Sigmoidal Inhibitory Effect E.sub.max model model to be
.about.600 ng/mL.
Example 2
AlbuBChE Pharmacokinetic Pharmacodynamic (PK/PD) Study in Squirrel
Monkeys
Objective
[0197] To evaluate AlbuBChE pharmacokinetics and pharmacodynamics
following a single 5 mg/kg intramuscular injection of AlbuBChE to
Squirrel monkeys. AlbuBChE pharmacodynamic effect was measured
following cocaine IV administration at a dose of 1 mg/kg in control
animals or at 2, 72 and 96 hr post-AlbuBChE dose.
Study Design
[0198] Dose Level: AlbuBChE dose level of 5 mg/kg was selected
based upon prior studies in Cynomolgous monkeys in which this dose
level was found to be effective in decreasing cocaine exposure.
Cocaine IV dose of 1 mg/kg was based on literature reports that
such dose is sufficient to cause an effect in monkeys without
causing excessive hyperactivity. The IM route of administration for
AlbuBChE dosing was selected because this is an intended route of
administration to humans.
TABLE-US-00018 TABLE 13 Test System and Neat Materials Species
Primate -Squirrel - non-naive ~0.8-1.2 kg Wash out ~1 week Period
In Life 1 week, not including wash out Period Number of 3 males for
AlbuBChE dose group Animals/ 2 Control with vehicle Sex/Group
Number of 1 dose groups + Control Groups (including Control) Total
5 number of animals on study Test Article AlbuBChE stock
formulation was stored in a freezer (-75 .+-. 15.degree. C.) and
protected from light. Pharmaco- Cocaine hydrochloride was stored
under ambient dynamic conditions protected from light. Test Article
Formulation The AlbuBChE stock formulation was thawed at room
Procedures temperature and once thawed was inverted gently. The
stock was diluted with Formulation Buffer to achieve a
concentration of 20 mg/mL. The cocaine hydrochloride formulation
was prepared by adding the required amount of cocaine hydrochloride
to the formulation container, adding the required amount of saline
to the container, and stirring until a clear formulation was
observed. The final concentration was expressed as the
hydrochloride salt. The formulation was filtered through a
0.22-.mu.m PVDF syringe filter. The formulation was stored at -75
.+-. 15.degree. C. until needed.
TABLE-US-00019 TABLE 14 Group Designation and Dosage Levels Dose
Dose Dose Number Level Concentration Volume of Group Treatment
(mg/kg) (mg/mL) (mL/kg) Animals 1 Single IV dose of 1 1.0 1 2
Cocaine 2 Single IM dose of 5 20 0.25 3 AlbuBChE Single IV dose of
1 1.0 1 Cocaine at 2, 72 and 96 hours after AlbuBChE dose
administration
AlbuBChE Pharmacokinetic Samples
[0199] Blood samples (0.4 mL) were collected from the femoral vein
and placed into serum separator tubes (SST) at pre-dose, 24, 72, 96
& 336 hrs post-dose (samples collected at 336 hr (14 days)
post-dose were intended to evaluate immunogenicity. The samples
were also analyzed for AlbuBChE concentration). Tubes were
maintained at room temperature for at least 1 hour, but not to
exceed 4 hours prior to centrifugation. Samples were centrifuged
and at least 200 .mu.L of serum was harvested and maintained on dry
ice prior to storage at approximately -70.degree. C.
Cocaine Pharmacokinetic Samples
[0200] Approximately 0.4 mL of whole blood was collected from the
femoral vein after every cocaine dose at 5 and 30 minutes postdose.
Blood was placed into K.sub.2EDTA blood collection tubes with
esterase inhibitor (diisopropylfluorophosphate (DFP). Tubes were
inverted several times and placed on wet ice upon collection.
Samples were centrifuged at 2-8.degree. C. within 45 minutes of
collection. The resultant plasma will be recovered and a single 200
.mu.L aliquot of plasma was placed into polypropylene tubes. Plasma
samples was frozen over dry ice and stored at approximately
-70.degree. C.
AlbuBChE Sample Analysis
[0201] The following paragraph briefly describes the ELISA based
assay employed in the measurement of AlbuBChE concentrations in
serum samples.
[0202] Immulon 4 HBX plates are coated with 100 ul of anti-human
BChE mAb 002-01 (Abcam ab17246) at 1 .mu.g/mL in PBS, overnight at
4 C. Blocking is done with 2% casein in phosphate buffered saline
(PBS), 200 .mu.L/well, 2 hours at room temperature. After washing,
100 .mu.L of diluted serum samples are added to the plates along
with standards. Standards are generated through 2.6 fold, serial
dilution of AlbuBChE from 420 to 5.2 ng/mL. Serum samples and
standards are maintained at 10% serum by dilution with buffer
containing pooled cynomolgus monkey serum. A wash step precedes
detection with 100 .mu.l of anti-HSA mAb-6502-HRP at 0.04 .mu.g/mL
for 1 hour. Wells are washed again, prior to developing with 100
.mu.L of tetramethylbenzidine substrate. After 15 minutes the
reaction is terminated with 100 .mu.L/well of 1N H.sub.2SO.sub.4
and read on SpectraMax plate reader at 450/570 nm. Values for
unknown serum samples are calculated by interpolation of standard
curve generated by 5-parameter fit of AlbuBChE standards. Serum
samples collected at predose and on Day 14 postdose were also
analyzed for immunogenicity.
AlbuBChE Pharmacokinetic and PK/PD Analysis
[0203] AlbuBChE pharmacokinetic parameter values were determined
using serum concentration-time profiles for individual animals. The
computer software WinNonlin Professional (Version 4.0.1 Pharsight
Corporation, USA) was used. Specifically the model for
non-compartmental analysis with extravascular input was
applied.
[0204] In spite of the limited amount of data, an attempt was made
to characterize PK/PD relationship. The computer software WinNonlin
Professional (Version 4.0.1 Pharsight Corporation, USA) was used.
Specifically the direct Inhibitory Effect E.sub.max model was use
in which E.sub.max was assumed at AlbuBChE concentration of zero.
The model equation can be described as follows:
E=E.sub.max*(1-(C/(C+EC50)))
[0205] The PD parameter used in the analysis was cocaine plasma
concentration at 5 min post-cocaine dose. PK parameters used in the
model were AlbuBChE plasma concentrations at 2, 72 and 96 hr
post-AlbuBChE dose. Due to the fact that AlbuBChE concentration was
not measured at 2 hr post-AlbuBChE dose, the 1st time sample
collected was used in the analysis (24 hr post-AlbuBChE dose) with
the assumptions that AlbuBChE concentration at 24 hr post AlbuBChE
dose may reflect AlbuBChE concentration at 2 hr post AlbuBChE dose.
Due to the limited data and the assumptions used in the analysis,
parameter values from this PK/PD analysis need to be viewed as
approximate estimates.
Results
AlbuBChE
[0206] Table 15 and FIG. 12 provide AlbuBChE serum
concentration-time profiles in three male Squirrel monkeys
following a single intramuscular administration of 5 mg/kg AlbuBChE
dose. Table summarizes AlbuBChE Squirrel monkeys pharmacokinetic
parameter values.
[0207] AlbuBChE terminal elimination slope was well characterized
as indicated by Rsq values being higher than 0.9. Area under the
curve was also well characterized as indicated by % AUC
extrapolated being less than 20%. The initial absorption phase of
AlbuBChE concentration-time profile was not characterized since the
first sample was collected at 24 hr postdose. As such, T.sub.max
and C.sub.max reported are apparent since actual T.sub.max can be
expected to occur between 3 and 6 hr. AlbuBChE exposure was
consistent for the three animals, with differences in levels only
clearly evident after 2 weeks. For example, AUC values for the
three animals were in a tight range with % CV of .about.7%.
AlbuBChE terminal elimination t1/2 was estimated to range from 45.5
to 65.5 hr (average half-life of 56.6 hours).
TABLE-US-00020 TABLE 15 AlbuBChE Serum Concentration (ng/mL) vs.
time (hr) Profile in individual Squirrel Monkeys following a single
IM administration of 5 mg/kg/animal AlbuBChE dose. Time (hr) MKY
547 MKY 3434 MKY 53B 0 <LLOD <LLOD <LLOD 24 2340.2 2296.7
2036.7 72 436.9 517.7 433.7 96 247.5 247.5 233.5 336 23.4 19.0
7.0
TABLE-US-00021 TABLE 16 AlbuBChE Pharmacokinetic Parameter Values
in individual Squirrel Monkeys following a single IM administration
of 5 mg/kg/animal AlbuBChE dose. No. MKY Points t1/2 Tmax Cmax
AUC(0-t) AUC(0-.infin.) AUC ID Rsq Lambda-z (hr) (hr) (ng/mL) (hr *
ng/mL) (hr * ng/mL) % Extrap 547 0.990 3 65.5 24 2340 135454 137666
1.61 3434 0.983 3 58.8 24 2297 136268 137881 1.17 53B 0.997 3 45.5
24 2037 120596 121056 0.380 Mean 130773 132201 SD 8822 9652 % CV
6.75 7.30
Cocaine and Metabolites
[0208] Cocaine was administered IV at a dose of 1 mg/kg in control
animals (n=2) or at 2, 72 and 96 hr post-AlbuBChE dose (n=3). The
bioanalytical method measured the plasma concentration of cocaine
and two of its metabolites: ecgonine methyl ester and
benzoylecgonine. Cocaine metabolic pathway is shown in FIG. 9,
ecgonine methyl ester metabolite is formed directly from cocaine
through the butrylcholine esterase enzyme. As such, this metabolite
can be predicted to increase following AlbuBChE administration.
[0209] Individual and mean cocaine plasma concentration (ng/mL) vs.
time (hr) data in control and AlbuBChE treated Squirrel monkeys are
shown in Table 17. A summary of the mean cocaine concentrations vs.
time are shown in FIG. 13. Highest cocaine concentrations were
observed in the control animals. The lowest cocaine levels were
observed at 2 hr following AlbuBChE administration reaching values
that are approximately 7% of control. Cocaine concentrations
increased as a function of time post-AlbuBChE administration yet
even at 96 hr post-AlbuBChE administration, cocaine concentration
were still 60% of control animals.
[0210] Individual and mean ecgonine methyl ester plasma
concentration (ng/mL) vs. time (hr) data in control and AlbuBChE
treated Squirrel monkeys are shown in Table 18. A summary of the
mean ecgonine methyl ester concentrations are shown in FIG. 14. As
expected, ecgonine methyl ester concentration at 5 min after
cocaine administration were low in the control animals. The values
were .about.40 fold higher in the AlbuBChE treated animals with the
highest concentrations observed at 2 hr post-AlbuBChE dose.
Ecgonine methyl ester concentrations decreased as a function of
time post-AlbuBChE administration yet even at 96 hr post-AlbuBChE
administration, ecgonine methyl ester concentration were still
higher than in the control animals.
[0211] Individual and mean benzoylecgonine plasma concentration
(ng/mL) vs. time (hr) data in control and AlbuBChE treated Squirrel
monkeys are shown in Table 19. A summary of the mean
benzoylecgonine concentrations are shown in FIG. 15. As
illustrated, AlbuBChE effect on the cocaine metabolite
benzoylecgonine was less pronounced.
[0212] FIG. 16 shows a summary of the blood levels of cocaine and
the cocaine metabolites ecgonine methyl ester (EME) and
benzoylecgonine (BZ) at 5 and 30 min following the cocaine
injection. Compared to a control cocaine injection following
administration of vehicle, 2 hours following administration of
AlbuBChE cocaine blood levels were significantly reduced at both
the 5- and 30-min time points (F.sub.3,7=7.34, p<0.05 and
F.sub.3,7=14.4, p<0.005 respectively). Seventy-two hours
following AlbuBChE cocaine levels were still significantly below
control levels 30-min post cocaine. The effects of AlbuBChE were
not significant at either 5 or 30 min time point 96 hours following
administration. The effects of AlbuBChE on EME levels were similar,
but in the opposite direction to those of cocaine. Blood levels of
EME were elevated at both the 5- and 30 min time point 2 hr
following AlbuBChE administration (F.sub.3,7=5.6, p<0.05 and
F.sub.3,7=33.3, p<0.001). Blood levels of EME remained
significantly elevated at 72 hours following AlbuBChE for the 30
min time point. While blood levels of BZ appeared to be slightly
reduced 2 hr following AlbuBChE, these effects were not
significant.
TABLE-US-00022 TABLE 17 Cocaine Plasma Concentration (ng/mL) vs.
time (hr) in individual Squirrel Monkeys following a single IM
administration of 5 mg/kg AlbuBChE dose. Cocaine was administered
IV at a dose of 1 mg/kg in control animals (n = 2) or at 2, 72 and
96 hr post-AlbuBChE dose (n = 3). Time Post- AlbuBChE MKY MKY MKY
Dose 547 3434 53B Mean SD % CV Cocaine Concentration at 5 min
following Cocaine dose at 2, 72 and 96 hr post-AlbuBChE Dose 2
0.575 20.0 22.4 14.3 12.0 83.5 72 41.3 176 104 107 67.4 62.9 96
97.9 191 112 134 50.2 37.5 Cocaine Concentration at 30 min
following Cocaine dose at 2, 72 and 96 hr post-AlbuBChE Dose 2
0.581 0.115 0.261 0.319 0.238 74.7 72 65.0 57.9 44.9 55.9 10.2 18.2
96 62.9 75.6 67.1 68.5 6.47 9.44 Cocaine Concentration at 5 and 30
min following cocaine dose in control animals Time post cocaine
dose MKY 548 MKY 27B Mean 5 min 185 239 212 30 min 82.6 154 118
Summary Table of Mean Cocaine Concentration vs. time in control
animals and as a function of time post-AlbuBChE dose Time Post
Cocaine 2 hr Post- 72 hr Post- 96 hr Post- (min) Control AlbuBChE
AlbuBChE AlbuBChE 5 212 14.3 107 134 30 118 0.319 55.9 68.5
<LLOQ (1 ng/mL)
TABLE-US-00023 TABLE 18 Ecgonine Methyl Ester Plasma Concentration
(ng/mL) vs. time (hr) in individual Squirrel Monkeys following a
single IM administration of 5 mg/kg AlbuBChE dose. Cocaine was
administered IV at a dose of 1 mg/kg in control animals (n = 2) or
at 2, 72 and 96 hr post-AlbuBChE dose (n = 3). Time Post- AlbuBChE
MKY MKY MKY Dose 547 3434 53B Mean SD % CV Ecgonine Methyl Ester
Concentration at 5 min following Cocaine dose at 2, 72 and 96 hr
post-AlbuBChE Dose 2 109 417 333 286 159 55.6 72 29.3 101 73.2 67.8
36.2 53.3 96 40.5 72.3 35.8 49.5 19.9 40.1 Ecgonine Methyl Ester
Concentration at 30 min following Cocaine dose at 2, 72 and 96 hr
post-AlbuBChE Dose 2 252 314 218 261 48.7 18.6 72 61.3 112 81.7
85.0 25.5 30.0 96 25.7 76.8 45.2 49.2 25.8 52.4 Ecgonine Methyl
Ester Concentration at 5 and 30 min following cocaine dose in
control animals Time post cocaine dose MKY 548 MKY 27B Mean 5 min
1.61 12.8 7.21 30 min 1.27 1.34 1.31 Summary Table of Mean Ecgonine
Methyl Ester Concentration vs. time in control animals and as a
function of time post-AlbuBChE dose Time Post Cocaine 2 hr Post- 72
hr Post- 96 hr Post- (min) Control AlbuBChE AlbuBChE AlbuBChE 5
7.21 286 67.8 49.5 30 1.31 261 85.0 49.2 <LLOQ (1 ng/mL)
TABLE-US-00024 TABLE 19 Benzoylecgonine Plasma Concentration
(ng/mL) vs. time (hr) in individual Squirrel Monkeys following a
single IM administration of 5 mg/kg AlbuBChE dose. Cocaine was
administered IV at a dose of 1 mg/kg in control animals (n = 2) or
at 2, 72 and 96 hr post-AlbuBChE dose (n = 3). Time Post- AlbuBChE
MKY MKY MKY Dose 547 3434 53B Mean SD % CV Benzoylecgonine
Concentration at 5 min following Cocaine dose at 2, 72 and 96 hr
post-AlbuBChE Dose 2 4.83 23.3 20.1 16.1 9.87 61.4 72 9.93 41.7
28.8 26.8 16.0 59.6 96 15.5 36.2 26.3 26.0 10.4 39.8
Benzoylecgonine Concentration at 30 min following Cocaine dose at
2, 72 and 96 hr post-AlbuBChE Dose 2 11.1 20.8 10.9 14.3 5.66 39.7
72 20.7 33.4 21 25.0 7.25 29.0 96 18.9 37.6 21.5 26.0 10.1 39.0
Benzoylecgonine Concentration at 5 and 30 min following cocaine
dose in control animals Time post cocaine dose MKY 548 MKY 27B Mean
5 min 34.4 31.6 33.0 30 min 25.1 29.9 27.5 Summary Table of Mean
Benzoylecgonine Concentration vs. time in control animals and as a
function of time post-AlbuBChE dose Time Post Cocaine 2 hr Post- 72
hr Post- 96 hr Post- (min) Control AlbuBChE AlbuBChE AlbuBChE 5
33.0 16.1 26.8 26.0 30 27.5 14.3 25.0 26.0 <LLOQ (1 ng/mL)
AlbuBChE PK/PD Relationship
[0213] In spite of the limited number of time points and animals,
an attempt was made to characterize AlbuBChE PK/PD relationship in
male Squirrel monkeys. PK and PD data used in the analysis are
shown in Table 20. The individual data and the fit resulting from
the direct Inhibitory Effect E.sub.max model are shown in FIG. 17.
The data clearly shows the inverse relationship between AlbuBChE
serum concentration and cocaine levels. AlbuBChE serum
concentration that may result in 50% decrease in cocaine
concentration (EC.sub.50) was estimated by the model to be -400
ng/mL. E.sub.max was 213 ng/mL. Due to the limited data and the
assumptions used in the analysis, parameter values from this PK/PD
analysis need to be viewed as approximate estimates.
TABLE-US-00025 TABLE 20 AlbuBChE PK/PD analysis Squirrel Monkeys
following a single IM administration of 5 mg/kg AlbuBChE dose.
Cocaine was administered IV at a dose of 1 mg/kg in control animals
(n = 2) or at 2, 72 and 96 hr post-AlbuBChE dose (n = 3). PK
parameters: AlbuBChE plasma concentrations at 24.sup.#, 72 and 96
hr post-AlbuBChE dose PD parameter: cocaine plasma concentration at
5 min post-cocaine dose. PD Cocaine concentration Time Post- PK at
5 min post- AlbuBChE AlbuBChE cocaine dose MKY ID Dose (hr) (ng/mL)
(ng/mL) 548 (Control) 2 0 185 27B (Control) 2 0 239 547 2 2340.2
0.575 72 436.9 41.3 96 247.5 97.9 3434 2 2296.7 20.0 72 517.7 176
96 247.5 191 53B 2 2036.7 22.4 72 433.7 104 96 233.5 112 .sup.#Due
to the fact that AlbuBChE concentration was not measured at 2 hr
post-AlbuBChE dose, the 1.sup.st time sample collected was used in
the analysis (24 hr post-AlbuBChE dose) with the assumptions that
AlbuBChE concentration at 24 hr post AlbuBChE dose may reflect
AlbuBChE conc. at 2 hr post AlbuBChE dose.
Conclusions
[0214] AlbuBChE pharmacokinetic profile was characterized in three
male Squirrel monkeys following a single IM 5 mg/kg AlbuBChE dose.
Extent of variability in exposure was minimal (.about.7%). AlbuBChE
terminal elimination t1 was estimated to range from 45.5 to 65.5
hr.
[0215] Cocaine was administered IV at a dose of 1 mg/kg in control
animals (n=2) or at 2, 72 and 96 hr post-AlbuBChE dose (n=3).
AlbuBChE caused a decrease in cocaine exposure. The effect was most
pronounced at 2 hr post-AlbuBChE dose (<7% of control). Cocaine
exposure was .about.60% of control at 96 hr post-AlbuBChE dose.
[0216] Consistent with AlbuBChE mechanism of action, exposure to
the cocaine metabolite ecgonine methyl ester increased .about.40
fold at 2 hr post-AlbuBChE administration and decreased as a
function of time post-AlbuBChE dose.
[0217] AlbuBChE effect was less pronounced on the cocaine
metabolite, benzoylecgonine.
[0218] PK/PD relationship in Squirrel monkeys appears to indicate
an inverse relationship between AlbuBChE serum concentration and
cocaine levels. AlbuBChE serum concentration that may result in 50%
decrease in cocaine concentration (EC.sub.50) was estimated by the
direct Inhibitory Effect E.sub.max model to be .about.400 ng/mL.
Due to the limited data and the assumptions used in the analysis,
parameter values from this PK/PD analysis need to be viewed as
approximate estimates.
Example 3
AlbuBChE
A Cardiovascular Safety Pharmacology Study in Cynomolgus Monkeys
with or without Administration of Cocaine
Objective
[0219] To evaluate the cardiovascular and respiratory safety of
AlbuBChE administered by the intramuscular (IM) route in 3 male and
3 female cynomolgus monkeys. In addition, the effect of cocaine
(administered by the intravenous (IV) route), AlbuBChE and their
combination on cardiovascular safety was evaluated in another group
of 3 male and 3 female cynomolgus monkeys.
Study Design
[0220] Prior to the start of the study, the monkeys were surgically
instrumented with telemetry transmitters and vascular access ports.
The following CV parameters were monitored: systolic pressure,
diastolic pressure, mean arterial blood pressure, mean heart rate
and mean rate-pressure product. Body temperatures changes were also
monitored.
[0221] Study Group 1 (3 males and 3 females), served as a control
group for testing the effect of AlbuBChE alone compared to its
vehicle. Monkeys were administered (IM) with the control article
AlbuBChE formulation buffer on Study Days (SDs) 1 and 4. Three
hours post formulation buffer administration respiratory and
cardiovascular (CV) parameters were recorded, respectively.
[0222] A single IM dose of the test article (AlbuBChE at 15 mg/kg)
was administered on SD 8 and 11. Three hours after AlbuBChE dosing
(corresponding to Tmax of AlbuBChE) CV and respiratory parameters
were monitored, respectively.
[0223] The effect of pretreatment with AlbuBChE prior to cocaine
dose on CV parameters was tested on the second group (Group 2) of
animals (3 males and 3 females). First baseline CV parameters were
recorded on SD 15 after an IM administration of AlbuBChE
formulation buffer followed by a single IV administration of saline
(cocaine vehicle), 3 hours later. The effect of cocaine on CV
parameters was measured on SD 18, on which the animals received a
single IM injection of AlbuBChE formulation buffer followed by a
single IV dose of cocaine (1 mg/kg, IV), 3 hours later.
[0224] The effect of pretreatment with AlbuBChE on cocaine--induced
changes in CV parameters was monitored on SD 22. CV parameters were
recorded subsequent to a single IM dose of AlbuBChE (15 mg/kg)
followed by a single IV dose of cocaine (1 mg/kg), 3 hours later
(see Table 21).
[0225] All animals were observed at least twice a day for
morbidity, mortality, injury, and availability of food and water.
The physiological responses to test article administration,
including blood pressure, heart rate, body temperature, and the
electrocardiograph (ECG), were monitored.
TABLE-US-00026 TABLE 21 Group Designation and Dosage Levels Test
Article or Test Article or Number of Study Study Interaction
Article Dose Interaction Article Animals Group Day Treatment Level
(mg/kg) Dose Conc. (mg/ml) Male Female 1 1 Formulation 0 0 3 3
Buffer-CV 4 Formulation 0 0 Buffer- Respiration 8 AlbuBChE-CV 15 30
11 AlbuBChE- 15 30 Respiration 2 15 Formulation 0 0 3 3 Buffer-CV
Saline-CV 0 0 18 Formulation 0 0 Buffer-CV Cocaine-CV 1 5 22
AlbuBChE-CV 15 30 Cocaine-CV 1 5
Results
[0226] All animals survived to study termination.
Study Group 1
[0227] AlbuBChE at 15 mg/kg (IM)) did not produce any significant
changes in the recorded CV parameters, nor in body temperature
compared to baseline values determined with AlbuBChE formulation
buffer, as shown in FIGS. 18-26.
Study Group 2
[0228] Within two minutes post cocaine (1.0 mg/kg, IV)
administration mean arterial blood pressure (MAP) was increased by
about 35 mmHg above baseline value of 105 mmHg. Pre-treatment with
15 mg/kg AlbuBChE prior to cocaine dose showed that not only was
the maximum blood pressure was decreased by AlbuBChE but the time
to reversal of the effect was also truncated by 4.5 fold (17
minutes upon pretreatment with AlbuBChE prior to cocaine dose
compared to 77 minutes when cocaine was given alone), as shown in
FIG. 27.
[0229] Administration of cocaine (1.0 mg/kg, IV) elicited a rapid
increase in heart rate from a baseline value of 140 beats/min
reaching a peak value of 240 beats/min (70% increase) within about
3 minutes. This cocaine--induced elevation in heart rate was
sustained for about 15 min dissipating gradually and returning to
baseline value 2 hours after cocaine administration. Pre-treatment
with AlbuBChE blunted cocaine-induced rise in heart rate showing a
rapid but moderate increase of 30% to a peak value of 182 beats/min
within about 3 minutes. This mild increase in heart rate was
reversed completely returning to baseline value already 30 minutes
after cocaine dose, as shown in FIG. 30.
[0230] Administration of cocaine (1.0 mg/kg, IV) resulted in a mild
and step wise increase in body temperature with a peak value of
38.7.degree. C. at 45 minutes post-dose compared to base line
temperature of 38.degree. C. Pre-treatment with AlbuBChE (15 mg/kg,
IM) prior to cocaine dose caused only a subtle increase in body
temperature to a value of 38.3.degree. C., as shown in FIG. 32.
Example 4
AlbuBChE
A Cocaine Self-Administration and Reinstatement of Cocaine
Self-Administration Study in Squirrel Monkeys
Methods
Subjects
[0231] Adult male squirrel monkeys (Saimiri sciureus) weighing 0.8
to 1.2 kg were used as subjects. All monkeys were individually
housed in a humidity and temperature controlled room and had free
access to water. The monkeys were fed following any experimental
procedures an amount of food (Lab Diet 5045, PMI Nutrition
International, Richmond, Ind.; Banana Softies, Bio-Serv,
Frenchtown, N.J.) determined to maintain a stable weight. Fresh
fruit, vegetables and environmental enrichment were also provided
daily. The animal care facilities were fully accredited by AAALAC
and all experiments were approved by the NIDA Intramural Research
Program Animal Care and Use Committee.
Cocaine Self-Administration
[0232] Three monkeys were trained to self-administer 30
.mu.g/kg/inj i.v. cocaine. Details of the self-administration
training procedure can be found elsewhere (Justinova et al., 2003).
In brief, the monkeys were placed in a seated position in a
Plexiglas restraint chair. The chair was enclosed in a larger
acoustical chamber. On the front wall of the restraint chair there
was a response lever and stimulus lights. A green light signaled
the beginning of the session. The monkeys were trained to make 10
responses (fixed-ratio, FR, 10) to receive an i.v. injection of
cocaine. The injection of cocaine was accompanied by the green
light turning off and a yellow stimulus light being presented for 2
sec. Following each cocaine injection there was also a 60-sec
timeout. At the end of the timeout, the green light was turned on.
Sessions lasted for 1 hour. Occasionally, saline was substituted
for cocaine. Following the establishment of stable cocaine
self-administration and reliable extinction of responding following
saline substitution, AlbuBChE (5 mg/kg, i.m.) or its vehicle (i.m.)
were given 2 hours prior to a self-administration session.
Self-administration responding was then measured for 5 consecutive
days. Following each drug test or saline substitution, responding
for 30 .mu.g/kg/inj cocaine was reestablished for at least 5 days.
Following reinstatement testing (see below), the dose of cocaine
available for self-administration was lowered to 10 .mu.g/kg/inj
and the effect of AlbuBChE (5 mg/kg, i.m.) was again
determined.
Reinstatement of Cocaine Self-Administration
[0233] Saline was substituted for cocaine in monkeys that were
reliably self-administering 30 .mu.g/kg/inj i.v. cocaine.
Responding rapidly decreased and remained at that low level over a
number of days. The vehicle of AlbuBChE was then given 2 hrs before
cocaine (0.3 mg/kg, i.v.), which was given 5 min prior to a session
where saline was available for self-administration to determine
whether cocaine would reinstate self-administration responding. The
same dose of cocaine was also given prior to saline
self-administration sessions 48 and 96 hours later. Subsequently,
AlbuBChE (5 mg/kg, i.m.) was given 2 hours prior to a second
sequence of 3 reinstatement tests with 0.3 mg/kg i.v. cocaine.
Following these tests, the monkeys were returned to baseline where
30 .mu.g/kg/inj cocaine was available for self-administration.
Subsequently, saline was again substituted for cocaine and a second
series of reinstatement tests were conducted in a similar manner
except that 0.1 mg/kg i.v. cocaine was given prior to reinstatement
sessions.
Drugs
[0234] AlbuBChE was supplied by TEVA Pharmaceutical (Netanya,
Israel) in a frozen solution at a concentration of 30 mg/ml. Once
thawed the solution was diluted to 15 mg/ml with vehicle and then
given to the monkeys in a volume of 0.33 ml/kg. Cocaine (NIDA,
Baltimore, Md.) was prepared in sterile saline and given in a
volume of 0.2 ml for self-administration. When given as a
pretreatment for the reinstatement studies, cocaine was given in a
volume of 0.3 ml/kg.
Data Analysis
[0235] The blood level data were analyzed separately for each
metabolite and time point (5 or 30 min). A one-way
analysis-of-variance (ANOVA) was performed with follow-up Dunnett
tests using the results of monkeys treated with vehicle as the
control. The self-administration data were analyzed separately for
response rate or injections following either vehicle treatment or
AlbuBChE treatment. Control data were included in the analysis by
taking the average of the last 3 days for the immediately preceding
baseline condition. A within subjects ANOVA was then performed with
follow-up tests contrasting days following vehicle or AlbuBChE with
the control. For the reinstatement tests, a two-way ANOVA was
performed for both response rate and injections, with time post
vehicle or AlbuBChE as the within-subjects factor and pretreatment
(vehicle or AlbuBChE) as the between subjects factor. Follow-up
contrast compared vehicle and AlbuBChE at each time point.
Results
[0236] In monkeys self-administering 30 .mu.g/kg/inj cocaine,
AlbuBChE reduced self-administration. The panels of FIG. 33 show
response rate (top panel) and injections (bottom panel) over
consecutive days. The first five days show baseline responding.
Saline was then substituted for cocaine and both rate
(F.sub.5,10=37.0, p<0.001) and injections (F.sub.5,10=188.7,
p<0.001) were significantly reduced for the 5 days of
substitution when compared to the average of the last 3 days of the
preceding baseline period. Following a 5 day return to baseline,
monkeys were treated with vehicle and given 5 days of
self-administration training. When compared to the average of the
last 3 days of the preceding baseline period, vehicle treatment had
no effect on injections (F.sub.5,10=2.6, p=0.10). An overall effect
of days was observed for response rate (F.sub.5,10=4.6, p<0.05),
however follow-up contrasts failed to reveal any significant change
in responding from control on any of the days following vehicle
treatment. Following vehicle treatment, monkeys were given 5 mg/kg
AlbuBChE and cocaine self-administration was tracked for 5 days.
Significant changes from the average of the last 3 days of the
previous baseline were observed for both response rate
(F.sub.5,10=4.6, p<0.05) and injections (F.sub.5,10=14.1,
p<0.001). Follow-up contrast revealed that response rate was
different from control on days 1-4 following treatment and
injections were significantly different from control on day 1 and 4
following treatment.
[0237] Following reinstatement testing (see below), monkeys were
returned to self-administration at a lower cocaine dose (10
.mu.g/kg/inj). Decreasing the maintenance dose decreased rate of
responding and number of injections (FIG. 33 right panels).
Following baseline stability, monkeys were given a vehicle
injection and self-administration continued for 5 days.
[0238] Neither response rate nor injections significantly changed
following vehicle injection compared to the average of the last 3
days on baseline. AlbuBChE (5 mg/kg) was then given and cocaine
self-administration continued for an additional 5 days. Both
response rate and injections were decreased 2 hrs following the
AlbuBChE injection, but these effects failed to reach
significance.
[0239] In monkeys that were responding for 30 .mu.g/kg/inj cocaine,
substituting saline for cocaine lead to a rapid decrease in
responding. The AlbuBChE vehicle was then given i.m. Two, 48 and 96
hrs later 0.3 mg/kg cocaine i.v. was given 5 min before a saline
substitution session. The administration of cocaine led to a
reinstatement of cocaine self-administration responding as shown in
the black bars in the left panels of FIG. 34. When 5 mg/kg AlbuBChE
was given i.m., and 2 hrs, hrs and 96 hrs later 0.3 mg/kg cocaine
i.v. was given immediately before a saline substitution session,
reinstatement of responding was significantly blocked at the 2 hr
time point for both response rate (F.sub.1,4=16.9, p<0.05) and
injections (F.sub.1,4=34.9, p<0.001). Monkeys were subsequently
tested in an identical manner with 0.1 mg/kg cocaine as the
reinstatement dose. While reinstatement to the lower cocaine dose
was more variable, a similar pattern of results was observed with
the effect of 5 mg/kg AlbuBChE again significant at 2 hrs for both
responses (F.sub.1,4=15.2, p<0.05) and injections
(F.sub.1,4=129.7, p<0.001).
[0240] In 4 monkeys that were trained to discriminate 0.6 mg/kg
methamphetamine from saline, cocaine fully substituted for
methamphetamine at a dose of 0.3 mg/kg (FIG. 35, top panel). When 5
mg/kg AlbuBChE was given 2 hrs prior to the determination of the
cocaine dose-effect function, 0.3 mg/kg cocaine failed to
generalize to the methamphetamine cue. Partial substitution
(approximately 70%) was observed at a higher cocaine dose (1.0
mg/kg). Twenty-four hrs following AlbuBChE, the 0.3 mg/kg dose of
cocaine still failed to completely generalize to the
methamphetamine cue (approximately 70%), but 1.0 mg/kg now fully
generalized to methamphetamine. By 48 hrs following AlbuBChE, the
0.3 mg/kg dose fully generalized to the methamphetamine cue.
Pretreatment with AlbuBChE 2 hrs prior to the determination of the
methamphetamine dose-effect function did not affect generalization
of methamphetamine to the methamphetamine cue (FIG. 36, top panel).
Pretreatment with AlbuBChE did not affect the rate of responding
under either treatment condition (FIGS. 35 and 36, bottom
panels).
[0241] The monkeys trained to self-administer cocaine and also
tested on the reinstatement procedure received a total of 4
injections of AlbuBChE. Blood was taken for determination of
AlbuBChE antibodies after the first, second and fourth AlbuBChE
injections. Following the first 2 injections, antibody levels were
below the level of detection (titer level of 20). Following the
fourth injection, however, antibody levels appeared elevated. In 2
monkeys the antibody titer was slightly elevated over the limit of
detection (21 and 43). For the third monkey, antibody levels were
clearly elevated (643). The concentration of AlbuBChE in the blood
for this monkey one week following the AlbuBChE injection was also
reduced (27 ng/ml vs 55 and 53 ng/ml for the other 2 monkeys).
Discussion
[0242] The results of these studies clearly show that pretreatment
with a modified form of BChE is effective in antagonizing the
behavioral effects of cocaine in a non-human primate. This work
extends previous work with this compound in rodents, where it was
shown that AlbuBChE could antagonize the toxic effects of cocaine
in rats and also block cocaine-induced reinstatement of cocaine
self-administration (Brimijoin et al., 2008). Here, pretreatment
with 5 mg/kg AlbuBChE reduced both response rate and injections in
monkeys self-administering 30 .mu.g/kg/inj cocaine. In these same
monkeys tested for reinstatement of cocaine self-administration
following self-administration extinction, 5 mg/kg AlbuBChE was
capable of blocking reinstatement of responding by either 0.3 mg/kg
or 0.1 mg/kg cocaine. When the dose available for
self-administration was reduced to 10 .mu.g/kg/inj, AlbuBChE again
appeared to reduce the response rate and number of injections
received, although these effects with 10 .mu.g/kg/inj cocaine were
more variable and failed to reach significance. Finally, the 5
mg/kg dose of AlbuBChE was also able to antagonize the
generalization of cocaine to a methamphetamine discriminative
stimulus, but this effect was specific to cocaine as AlbuBChE was
not able to antagonize the discriminative stimulus effects of
methamphetamine.
[0243] When tested against 30 .mu.g/kg/inj cocaine
self-administration, the effects of AlbuBChE were still evident 3
days later, although at a reduced effectiveness. On reinstatement
and discrimination, however, the effects of AlbuBChE were not
evident 48 hours following treatment. The half-life of AlbuBChE was
shown to be around 56 hours and effects on cocaine metabolism were
still evident 72 hours after AlbuBChE administration, so it might
have been expected that some effect on these procedures would have
been evident at 72 hours. A number of factors could contribute to
this failure to see a reduction in reinstatement or cocaine
generalization at 72 hrs. During self-administration smaller doses
of cocaine are given over an extended period of time. In the other
procedures a relatively larger dose of cocaine was administered as
a bolus injection. It may be easier for AlbuBChE to metabolize the
cocaine before it reaches the brain under the self-administration
conditions than under the other conditions. When AlbuBChE
metabolizes cocaine under self-administration, its effect is to
institute extinction conditions. Behavioral recovery from
extinction may also prolong the effects of AlbuBChE treatment on
self-administration. In the reinstatement study, responding
produced saline and the stimulus previous associated with
reinforcement. When the monkeys reinstated responding following a
cocaine injection, the monkeys still received saline for
responding, but they also received the stimulus previously
associated with cocaine. The presentation of this stimulus may also
have facilitated continued responding under the reinstatement
conditions (Spealman et al., 1999), masking some of the effect of
AlbuBChE.
[0244] The effects of AlbuBChE were almost certainly related to its
effects on the metabolism of cocaine. The administration of
AlbuBChE decreased the amount of cocaine in the blood for at least
72 hours. The fact that EME levels were also increased for at least
72 hours following AlbuBChE suggests that AlbuBChE was metabolizing
cocaine similarly to native BChE (Jones, 1984). This time course
for cocaine metabolism is similar to that seen for the
self-administration experiment where the monkeys were reinforced
with 30 .mu.g/kg/inj cocaine. Further, AlbuBChE had no effect on
response rate in the discrimination study, suggested that it does
not produce a non-specific effect on operant responding independent
of the presence of cocaine in the blood.
[0245] The observation of relatively high levels of AlbuBChE
antibodies in one monkey following its fourth injection indicates
that AlbuBChE might lose some of its effectiveness over multiple
injections. While the results for that one particular monkey did
not appear to be different from the other 2 monkeys tested, the
observation of antibodies suggests that some of the AlbuBChE would
be bound by antibodies which might decrease its ability to
metabolize cocaine. Further work will be needed to determine how
prevalent the antibody production is in primates and whether the
levels of antibody observed are sufficient to decrease the
effectiveness of AlbuBChE in metabolizing cocaine.
[0246] Cocaine continues to show up in both emergency room mentions
and medical examiner reports in the DAWN surveys. As such, a drug
that could counteract the toxic effects of cocaine may be useful
adjunct to emergency room treatment for patients abusing cocaine.
An advantage of using a drug that metabolizes cocaine is that it
will be specific for cocaine and should have minimal side effects.
However, it would be necessary to confirm that a patient is using
cocaine as AlbuBChE would not be effective against toxicity
produced by other drugs of abuse, such as the amphetamines, that
might produce a similar spectrum of toxic effects.
[0247] In addition to treating the toxic effects of cocaine,
AlbuBChE might also be useful as an adjunct to other treatments for
cocaine abuse. A person with a sufficient blood level of AlbuBChE
who takes cocaine would be expected to experience a reduced
subjective effect of cocaine. In the context of the current study,
this translates to less cocaine self-administration, reduced
reinstatement of cocaine self-administration and the failure of
cocaine to generalize to the methamphetamine discriminative
stimulus. As relapse to cocaine abuse remains a problem in
treatment, the reduced subjective effects of cocaine would be
expected to reduce relapse. For someone undergoing treatment that
does relapse to use, the presence of AlbuBChE in the blood would
metabolize much of the cocaine and potentially reduce the
likelihood of continued cocaine use. This would require an
individual to continue taking AlbuBChE throughout the period of
time that relapse would be expected. Therefore, it will be critical
to determine how prevalent the development of antibodies to
AlbuBChE is and how that might effect its ability to metabolize
cocaine.
[0248] In conclusion, pretreatment of squirrel monkeys with
AlbuBChE was able to reduce the amount of cocaine in the blood
following a cocaine bolus treatment. Pretreatment with AlbuBChE was
also able to reduce cocaine self-administration in monkeys that had
been trained to self-administer 10 and 30 .mu.g/kg/inj cocaine.
AlbuBChE was able to block cocaine-induced reinstatement of cocaine
self-administration for 2 different doses of cocaine. Finally,
AlbuBChE was also able to antagonize the discriminative effects of
cocaine in squirrel monkeys. The finding that AlbuBChE was able to
antagonize the behavioral effects of cocaine in 3 different models
of cocaine's behavioral effects suggests that it should be
effective as a potential treatment of cocaine abuse.
Example 5
A Double-Blind, Placebo-Controled, Single Ascending Dose of
AlbuBChE Followed by Multiple Doses of Cocaine to Evaluate the
Pharmacokinetic and Pharmacodynamic Parameters of Cocaine after
AlbuBChE Administration in Cocaine Recreational Volunteers
Objective
[0249] To determine cocaine blood levels following multiple doses
subsequent to AlbuBChE administration, and to determine the
behavioral, psychological and safety effects of cocaine following
multiple doses subsequent to AlbuBChE administration. To evaluate
the pharmacokinetics, safety, and tolerability of AlbuBChE.
Study Design
[0250] This is a 5-arm, parallel-group, active and
placebo-controlled, double blind, randomized study, to compare
treatment with AlbuBChE with placebo as a negative control.
[0251] Forty (40) non-treatment seeking healthy adults who have
used cocaine are randomly assigned to five (5) treatment groups.
Each group will contain eight subjects.
Group A (low dose): Subjects receive a single, intramuscular
injection of 50 mg of AlbuBChE. Group B (intermediate dose):
Subjects receive a single, intramuscular injection of 100 mg of
AlbuBChE. Group C (high-intermediate dose): Subjects receive a
single, intramuscular injection of 150 mg of AlbuBChE. Group D
(high dose): Subjects receive a single, intramuscular injection of
300 mg of AlbuBChE.
[0252] Group E (negative control): Subjects receive a single,
intramuscular injection of placebo.
[0253] The study is divided to 5 cohorts of 8 subjects per cohort
with 2 to 3 days apart.
Inpatient Hospital Phase
[0254] Subjects remain resident in the clinic from the evening
before Day 1 (check in; Day -1) until the morning of Day 11.
Subjects return to the clinic for follow-up visit on Day
18.+-.2.
Day -1: On the morning of day -1, a 40 mg intravenous dose of
cocaine is administered. Day 1: A single, intramuscular dose of
AlbuBChE or placebo is administered. Day 1, 4, 8 and 10: On Day 1,
at AlbuBChE C.sub.max (3 hours post dose) and at the morning of
days 4 (72 hours), 8 (168 hours) and 10 (216 hours) at the
corresponding time of AlbuBChE/placebo administration, a 40 mg
intravenous challenge dose of cocaine is administered.
[0255] Saline infusions are included (randomized with cocaine
infusions, administered 2 hours apart) on each day of cocaine
administration, and have the infusion type order single
blinded.
Study Drug Administration
[0256] Planned AlbuBChE doses are as described above for groups
A-E. Each subject receives a single dose of AlbuBChE or placebo by
intramuscular (IM) injection. 40 mg of cocaine hydrochloride,
prepared as a 1 mg/ml saline solution is injected into a forearm
vein at the rate of 1 mg/second using a constant rate infusion
pump.
Study Duration
[0257] The duration of the study (from screening until the End of
Study (EOS) visit) for each subject is approximately 11 to 12
weeks.
Study Population
[0258] A total of Forty (40) male subjects aged 18-55 who are
recreational cocaine users. Subjects who discontinue the study for
any reason after dosing are not replaced.
Main Inclusion/Exclusion Criteria
Inclusion Criteria
[0259] 1. Male volunteers aged 18-55 years, inclusive; [0260] 2.
Body mass index (BMI) 18-33 kg/m.sup.2 and weight of at least 50
kg; [0261] 3. Healthy, as determined by no clinically significant
medical history, physical examination, electrocardiogram (ECG),
vital signs and laboratory results at screening; [0262] 4.
Currently use cocaine by smoking, intranasal or i.v. route; defined
as at least once in a month prior to screening, 6 times in the past
year and 20 times over lifetime. Current use is confirmed through a
positive Urine Drug Test (UDS) prior to in-patient period (i.e. at
screening or between screening and Day -1); [0263] 5. Able to
understand the nature of the trial and any hazards of participating
in it; [0264] 6. Able to communicate satisfactorily with the
Investigator and to participate in, and comply with the
requirements of the entire trial; and [0265] 7. Willingness to give
written consent to participate, after reading the information and
consent form and after having the opportunity to discuss the trial
with the Investigator or his delegate and sign the Informed
Consent.
Exclusion Criteria
[0265] [0266] 1. Current or history of alcohol or drug dependence
according to DSM-IV-TR (Diagnostic and Statistical Manual of Mental
Disorders--last version) criteria (excluding nicotine and
caffeine), or participation in rehabilitation program (except
smoking cessation); [0267] 2. History of severe allergic or
anaphylactic reactions; [0268] 3. Known allergy or hypersensitivity
to natural or recombinant butyryl cholinesterase (BChE), human
serum albumin (HSA) or any other component of the formulation;
[0269] 4. History of any clinically significant (as determined by
the Principal Investigator) cardiac, endocrine, haematological,
hepatic, immunological, metabolic, urological, pulmonary,
neurological, dermatological, psychiatric, renal, or other major
disease; [0270] 5. History of any malignant disease; [0271] 6.
Major trauma or surgery in the 2 months before screening or at any
time between screening and check-in; [0272] 7. Acute infection
within 2 weeks before screening or at any time between screening
and check-in; [0273] 8. Clinically significant abnormal ECG
findings at screening or check-in visits, as determined by the
Principal Investigator; [0274] 9. Blood pressure outside the ranges
90-140 mmHg systolic or 45-90 mmHg diastolic (measured after a rest
of at least 5 min) at screening or check-in. Blood pressure may be
re-tested in the supine position at intervals of 5-10 min. The
pressure elevation is considered sustained if either the systolic
or the diastolic pressure exceeds the stated limits after three
assessments, and thus the subject may not be randomized; [0275] 10.
Heart rate <40 beats/min (measured after a rest of at least 5
min) at screening or check-in; [0276] 11. Known history of or a
positive test result for human immunodeficiency virus (HIV) types 1
or 2 at screening; [0277] 12. Known history of, or a positive test
result for hepatitis B surface antigen (HBsAg) or hepatitis C virus
(HCV) at screening; [0278] 13. Use of prescription or
over-the-counter (OTC) medication (other than paracetamol
(acetaminophen), .ltoreq.2 g/day or ibuprofen, 650 mg/day),
investigational drugs, vitamins, or herbal remedies, within 2 weeks
before dosing or within 5 half-lives before dosing, whichever is
longer; [0279] 14. Participation in another clinical trial of a new
chemical entity or a prescription medicine within the 3 months
before dosing or within 5 half-lives before dosing, whichever is
longer; [0280] 15. Loss of >400 mL blood (e.g. as a blood donor)
within 2 months before dosing, or received any blood, plasma, or
platelet transfusions within 3 months before check-in, or who have
planned donations during the study or within 3 months after the
study; [0281] 16. Subject consumes greater than 20 cigarettes per
day on average, in the month prior to screening, or be unable to
abstain from smoking (or use of any nicotine-containing substance)
for at least 6 hours (so that all pre-dose and
post-dose/infusion-related assessments can be completed
uninterrupted); [0282] 17. Positive UDS for amphetamine, cocaine
(only on at Day -1), opioids, cannabinoids, and benzodiazepines.
Positive result for benzodiazepines and cannabinoids acceptable as
long as stable or decreasing due to long half-lives. Positive UDS
for other drugs will result in rescheduling at discretion of
investigator/designee; Positive breath alcohol test will also
result in rescheduling at discretion of investigator/designee;
[0283] 18. Exposure to pesticides or any other organophosphates
(e.g. agricultural workers); [0284] 19. Inability to participate or
successfully complete the study, in the opinion of their general
practitioner or the Investigator, because the volunteer is: [0285]
a. mentally or legally incapacitated, or unable to give consent for
any reason; [0286] b. in custody due to an administrative or a
legal decision, or under supervised parole, or being admitted to a
sanitarium or social institution; [0287] c. unable to be contacted
in case of emergency; or [0288] d. unlikely to cooperate or comply
with the clinical study protocol or is unsuitable for any other
reason.
Safety, Tolerability, and Immunogenicity Analysis
Safety
[0289] Vital signs, including blood pressure, heart rate (part of
the PD parameters) and body temperature are monitored. Physical
examinations, clinical laboratory tests, and 12-lead ECGs are
performed, with 12-lead ECGs after each cocaine infusion, at
pre-infusion, 30 minutes and 2 hours post-infusion, and 8 hour
post-first infusion. Telemetry is performed for 4 hours immediately
after each cocaine injection.
[0290] Samples for BChE and acetyl cholinesterase (AChE) activity
are collected but are not all necessarily assayed. The activity of
BChE and AChE is determined using a colorimetric method based on
the Ellman assay, which serves as a neutralization assay for the
endogenous enzymes. Sampling timepoints are at screening, pre-dose
and 168 hours (Day 8), 240 hours (Day 11) and follow-up (Day
18.+-.2) post-dose. The blood tests for Ellman assay take place
before administration of cocaine (where relevant).
[0291] Intravenous infusions of cocaine are immediately terminated
if any of the following occur:
1. Systolic BP of 180 mm Hg or greater; 2. Diastolic BP of 100 mm
Hg or greater; 3. HR of 130 bpm or greater; or 4. Behavioral
manifestations of psychostimulant toxicity (agitation, psychosis,
or inability to cooperate with study procedures). Subjects are
considered to have not tolerated IV cocaine infusions if any of the
following occur: 1. Acute chest pain or shortness of breath; 2.
Systolic BP of 180 mm Hg or greater; 3. Diastolic BP of 120 mm Hg
or greater; 4. HR of [220-age X 0.85] bpm or greater (i.e.,
>than 170 bpm for a 20 year old subject); 5. Neurologic or
psychiatric events (e.g., panic or psychosis); 6. Clinically
significant ECG abnormalities (including heart block, three or more
sustained ectopic ventricular beats, or tachyarrhythmias); 7.
Stopping criteria for further cocaine infusion do not return to
acceptable limits within appropriate time frames (e.g., 30 min); 8.
Stopping criteria for further cocaine infusion are met for a second
time within the protocol; or 9. Any condition that in the clinical
judgment of the PI that is of sufficient magnitude to present a
danger to the subject.
Tolerability of AlbuBChE IM Injection
[0292] Local tolerability and pain (evaluated by the visual
analogue scale (VAS)) at the injection site are evaluated during
the first 24 h after dosing. Timepoints are: 20 min, 1, 2, 4, 8 and
24 hours post AlbuBChE dose.
Adverse events (AEs) are monitored throughout the study.
Immunogenicity (IG)
[0293] Samples for immunogenic levels are collected but are not all
necessarily assayed. Titers for antibodies against HSA, human
butyryl cholinesterase (hBChE) and AlbuBChE are evaluated. Sampling
timepoints are: pre-dose and 168 hours (Day 8), 240 hours (Day 11)
and follow-up (Day 18.+-.2) post-dose. The blood tests for IG
assays take place before administration of cocaine (where
relevant).
Pharmacokinetic Variables and Sampling
[0294] To determine serum concentration of AlbuBChE, blood samples
are collected before dosing and at several time points after
dosing. Where the data permits, the following pharmacokinetic (PK)
parameters are calculated: [0295] C.sub.max maximum observed serum
concentration [0296] t.sub.max time to achieve C.sub.max [0297]
AUC.sub.(0-t) area under the serum concentration-time curve from 0
h to the time of the last quantifiable concentration [0298]
AUC.sub.(0-.infin.) area under the serum concentration-time curve
extrapolated to infinity [0299] AUC.sub.ext percentage of
AUC.sub.(0-.infin.) due to extrapolation from t.sub.last to
infinity [0300] V.sub.d/f apparent volume of distribution during
terminal phase [0301] CL/f apparent total body clearance [0302]
.lamda..sub.z terminal elimination rate constant, estimated by
linear regression on the terminal phase of the semi-logarithmic
concentration versus time curve [0303] t.sub.1/2 apparent terminal
elimination half-life [0304] MRT mean residence time
[0305] Additional parameters may be calculated if deemed necessary.
Sampling timepoints for PK of AlbuBChE in serum are as follows:
pre-dose and 20, 40 min, 1 hour, 1.5 hours, 2 hours, 3 hours, 4
hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours (Day 2), 36
hours (Day 2), 48 hours (Day 3), 72 hours (Day 4), 96 hours (Day
5), 120 hours (Day 6), 144 hours (Day 7), 168 hours (Day 8), 192
hours (Day 9), and 216 hours (Day 10) post-dose. Where applicable,
sampling timepoints for PK of AlbuBChE are taken just before
cocaine infusion.
Pharmacodynamics Variables and Sampling
[0306] Blood samples are collected before dosing and at several
time-points after each dosing of cocaine. In vivo cocaine levels
(and its metabolites benzoylecgonine and ecgonine methyl ester) are
determined by validated LC/MS-MS method and are correlated to
AlbuBChE blood levels. Cocaine exposure (C.sub.max, t.sub.max,
AUC.sub.(0-t), AUC.sub.(0-.infin.) and V.sub.d) and clearance (CL,
.lamda..sub.z, t.sub.1/2) are determined. PD sampling timepoints of
cocaine levels in serum after each cocaine dose are as follows:
pre-dose, 2, 5, 10, 15, 30, 45, 60, 90, and 120 minutes post
dose.
[0307] Blood pressure and heart rate are measured pre-infusion and
2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, and 300 minutes post
infusion.
Behavioral and Psychological Effects (Subjective Effects)
[0308] Subjective effects are measured using a computerized (21 CFR
11 validated) visual analogue scale (VAS). The time points for
collection of subjective effects match with those for cocaine blood
samples. The following set of VAS are: Drug Liking, Take Drug
Again, Overall Drug Liking, High, Good Effects, Bad Effects,
Feeling Rush, Desire for Cocaine, Feeling Anxious, Over-Stimulated,
and Any Drug Effects.
[0309] PD sampling timepoints of VAS parameters after each cocaine
dose are as follows: pre-dose, 2, 5, 10, 15, 30, 45, 60, 90, and
120 minutes post dose.
Urine Collection
[0310] Urine is collected for metabolic investigations over a 52
hour interval after cocaine administration on Day -1, Day 4 and Day
8. The collection ranges are: (i) immediately after cocaine dose,
followed by collection intervals of (ii) two intervals of three
hours, and (iii) nine intervals of six hours.
Statistical Analysis
Pharmacokinetics
[0311] PK parameters are derived from serum concentrations for each
dose level (number of subjects, mean, SD, geometric mean (for AUCs
and C.sub.max), coefficient of variation, minimum, median, and
maximum). Dose proportionality (for AlbuBChE only) using the power
model is evaluated for C.sub.max, AUC.sub.0-t, and
AUC.sub.0-.infin..
Pharmacodynamics
[0312] Time course of PD effects (subjective and objective) is
determined. PD parameters include: Emax (maximum effect), TEmax
(time to Emax), time averaged area under the effect curve (TA_AUE),
partial AUE for early time points, and cumulative AUE.
Safety
[0313] Safety data is determined using descriptive statistics and
change from Baseline, where appropriate, for each treatment group.
AEs are coded using the latest version of the Medical Dictionary
for Regulatory Activities (MedDRA) and summarized by system organ
class and preferred term.
Bioanalytical
[0314] Frozen serum samples for determining AlbuBChE concentrations
and antibodies are shipped on dry ice for analysis. Analyses of
cocaine and metabolite levels is performed using validated
methods.
Results
Safety, Tolerability and Immunogenicity
[0315] AlbuBChE is safe to administer to humans. No significant
safety issues are observed with intramuscular (IM) AlbuBChE doses
of mg to 300 mg. AlbuBChE is well tolerated, with no unacceptable
side effects. Local tolerability and pain at the IM injection site
are acceptable, with no significant adverse events. The
administration of 50 mg to 300 mg of AlbuBChE as a single dose does
not provoke a significant immune response. Titers for antibodies
agains HSA, human butyryl cholinesterase (hBChE) and AlbuBChE are
all within acceptable limits.
Pharmacokinetic Variables
[0316] Maximum observed serum concentration of AlbuBChE, C.sub.max,
increases with increasing dose of AlbuBChE. AlbuBChE absorption
from the IM site of administration is rapid, with a short time to
achieve C.sub.max. AUC.sub.(0-t) and AUC.sub.(0-.infin.) indicate
that AlbuBChE levels persist for a significant and clinically
useful amount of time. V.sub.d/f values indicate that the majority
of AlbuBChE is present in the circulation during the terminal
phase. CL/f values indicate that AlbuBChE is cleared from the body
at an acceptable rate. Apparent terminal elimination half-life
values indicate a weekly or twice weekly dosing of AlbuBChE to
maintain therapeutic levels. The mean residence time of AlbuBChE is
within clinically acceptable limits.
Pharmacodynamic Variables
[0317] Maximum observed serum concentration of cocaine, C.sub.max,
decreases with increasing dose of AlbuBChE. C.sub.max levels are
achieved very rapidly following a 40 mg intravenous dose of
cocaine, with a short t.sub.max. AUC.sub.(0-t) and
AUC.sub.(0-.infin.) of cocaine and its metabolites benzolyecgonine
and ecgonine methyl ester indicate that cocaine is more rapidly
metabolized in subjects receiving a dose of AlbuBChE prior to
cocaine exposure than subjects receiving placebo. Cocaine
AUC.sub.(0-t) decreases as a function of AlbuBChE dose and
increases as a function of time post-AlbuBChE administration.
V.sub.d values indicate that the majority of cocaine is present in
the circulation during the terminal phase. CL values indicate more
rapid cocaine clearance following administration of AlbuBChE
compared to placebo, with clearance rate decreasing as time post
AlbuBChE administration increases. Similarly, apparent terminal
elimination half-life values indicate more rapid cocaine clearance
following administration of AlbuBChE compared to placebo, with
clearance rate decreasing as time post AlbuBChE administration
increases.
Behavioral and Phychological Effects
[0318] Subjects administered AlbuBChE report significantly
different results on the visual analogue scale (VAS) compared to
subjects administered placebo. AlbuBChE administration decreases
cocaine liking. AlbuBChE administration decreases the desire to
take cocaine again. AlbuBChE administration decreases overall drug
liking. AlbuBChE administration decreases the "high" associated
with cocaine exposure. AlbuBChE administration decreases the
subjective feeling of "good effects" after cocaine exposure.
AlbuBChE administration decreases subjective feeling of "rush"
associated with cocaine exposure. AlbuBChE administration decreases
the desire for cocaine. AlbuBChE administration reduces feelings of
anxiousness following cocaine exposure. AlbuBChE administration
reduces feelings of being over-stimulated following cocaine
exposure. AlbuBChE administration reduces the reporting of "any
drug effects."
[0319] Subjects administered AlbuBChE prior to cocaine exposure do
not report significantly different "bad effects" following AlbuBChE
administration compared to subjects administered placebo. AlbuBChE
reduces the feeling of "bad effects" following cocaine
exposure.
Discussion
[0320] The present Example determines cocaine blood levels
following multiple doses of cocaine subsequent to AlbuBChE
administration, and determines the behavioral, psychological and
safety effects of cocaine following multiple doses subsequent to
AlbuBChE administration in an inpatient setting.
[0321] The primary measure of a successful treatment of cocaine
abuse and dependence in an outpatient setting is a period of
abstinence during and following treatment. The facilitation of a
period of abstinence, such as a two or three week period of
abstinence, indicates a successful treatment. Secondary outcome
measures include reduction in cocaine levels or amount of cocaine
intake, overall proportion of cocaine non-use days, proportion of
successful subjects, the largest number of consecutive cocaine
non-use days, and severity of cocaine dependence as evaluated, for
example, by the cocaine selective severity assessment (CCSA).
[0322] Previous studies have found that AlbuBChE has a half life of
approximately 8 hours in rats and have speculated that the
potential half-life of AlbuBChE would range from 1 to several days
in humans. (Brimijoin et al., 2008; Gao et al. 2009). Gao et al.
noted that the observed half-life of monomeric AlbuBChE in rats is
shorter than that of native tetrameric BChE, and speculated that
the half-life of AlbuBChE could be increased by post-translational
modifications such as polyethylene glycosylation.
[0323] The present Example determines that the half-life of
AlbuBChE when administered by intramuscular injection is dose
dependent, with half-life values increasing with increasing dose.
The half-life values at the specified dosages allow for a weekly or
twice weekly dosing schedule without the need for
post-translational modifications such as polyethylene
glycosylation.
[0324] Previous studies have also reported that intravenous
administration of AlbuBChE to rats can cause a modest increase in
blood pressure and mild lethargy. (Brimijoin et al. 2008). In
constrast, a significant increase in blood pressure is not observed
when AlbuBChE is administered to humans by intramuscular injection,
nor is lethargy reported in a significant number of subjects.
[0325] Human subjects administered AlbuBChE do not report a
significant increase in cocaine cravings. In contrast, desire to
use cocaine is significantly decreased following AlbuBChE dosing
and remains significantly depressed for up to one week following a
single administration of AlbuBChE.
[0326] The findings of the present Example indicate that
intramuscular administration of AlbuBChE to humans at the specified
dosages does not result in any unacceptable side effects and that
the specified dosages will allow for the successful treatment of
biological effects of cocaine exposure.
REFERENCES
[0327] Brimijoin, S., Gao, Y., Anker, J. J., Gliddon, L. A.,
LaFleur, D., Shah, R., Zhao, Q., Singh, M., and Carroll, M. E., "A
Cocaine Hydrolase Engineered from Human Butyrylcholinesterase
Selectively Blocks Cocaine Toxicity and Reinstatement of Drug
Seeking in Rats". Neuropsychopharmacology, 33: 2715-2725, 2008.
[0328] Brogan, W. C. 3rd, Kemp, P. M., Bost, R. O., Glamann, D. B.,
Lange, R. A., Hillis, L. D., "Collection and handling of clinical
blood samples to assure the accurate measurement of cocaine
concentration," J. Anal. Toxicol. 1992 May-June; 16(3):152-4.
[0329] Davies, B. and Morris, T., "Physiological parameters in
laboratory animals and humans," Pharm. Res., 10: 1093-1095, 1993.
[0330] Diagnostic and Statistical Manual of Mental Disorders,
American Psychiatric Publishing, Inc.; 4th edition (June 2000)
[0331] "Guidance for Industry: Estimating the Maximum Safe Starting
Dose in Initial Clinical Trials for Therapeutics in Adult Healthy
Volunteers," U.S. Department of Health and Human Services, Food and
Drug Administration Center for Drug Evaluation and Research (CDER),
July 2005. [0332] Pan, Y., Gao, D., Yang, W., Cho, H., Yahg, G.,
Tai, H., Zhan, C., "Computational redesign of human
butyrylcholinesterase for anticocaine medication," PNAS,
102(46):16656-61, 2005. [0333] Sun, H., Shen, M., Pang, Y.,
Lockridge, O., Brimijoin, S., "Cocaine Metabolism Accelerated by a
Re-Engineered Human Butyrylcholinesterase," Journal of Pharmacology
and Experimental Therapeutics, 302(2): 710-16, 2002. [0334] US
Patent Application Publication No. 2008/0194481, published Aug. 14,
2008 (U.S. Ser. No. 11/932,823, filed Oct. 31, 2007). [0335] Gao,
et al. (2009) "An Albumin-Bytyrylcholinesterase for Cocaine
Toxicity and Addiction: Catalytic and Pharmacokinetic Properties,"
NIH Public Access Author Manuscript, published in final edited form
as Chem. Biol. Interact. 2008 Sep. 25; 175(1-3): 83-87.
Sequence CWU 1
1
41529PRTHomo sapiens 1Glu Asp Asp Ile Ile Ile Ala Thr Lys Asn Gly
Lys Val Arg Gly Met 1 5 10 15 Asn Leu Thr Val Phe Gly Gly Thr Val
Thr Ala Phe Leu Gly Ile Pro 20 25 30 Tyr Ala Gln Pro Pro Leu Gly
Arg Leu Arg Phe Lys Lys Pro Gln Ser 35 40 45 Leu Thr Lys Trp Ser
Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn Ser 50 55 60 Cys Cys Gln
Asn Ile Asp Gln Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 75 80 Met
Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn 85 90
95 Val Trp Ile Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu Ile Trp
100 105 110 Ile Tyr Gly Gly Gly Phe Gln Thr Gly Thr Ser Ser Leu His
Val Tyr 115 120 125 Asp Gly Lys Phe Leu Ala Arg Val Glu Arg Val Ile
Val Val Ser Met 130 135 140 Asn Tyr Arg Val Gly Ala Leu Gly Phe Leu
Ala Leu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly
Leu Phe Asp Gln Gln Leu Ala Leu Gln 165 170 175 Trp Val Gln Lys Asn
Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Val 180 185 190 Thr Leu Phe
Gly Glu Ser Ser Gly Ala Ala Ser Val Ser Leu His Leu 195 200 205 Leu
Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser 210 215
220 Gly Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg
225 230 235 240 Asn Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser
Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Lys Cys Leu Arg Asn Lys
Asp Pro Gln Glu Ile 260 265 270 Leu Leu Asn Glu Ala Phe Val Val Pro
Tyr Gly Thr Pro Leu Gly Val 275 280 285 Asn Phe Gly Pro Thr Val Asp
Gly Asp Phe Leu Thr Asp Met Pro Asp 290 295 300 Ile Leu Leu Glu Leu
Gly Gln Phe Lys Lys Thr Gln Ile Leu Val Gly 305 310 315 320 Val Asn
Lys Asp Glu Gly Thr Trp Phe Leu Val Gly Gly Ala Pro Gly 325 330 335
Phe Ser Lys Asp Asn Asn Ser Ile Ile Thr Arg Lys Glu Phe Gln Glu 340
345 350 Gly Leu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu
Ser 355 360 365 Ile Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gln Arg
Pro Glu Asn 370 375 380 Tyr Arg Glu Ala Leu Gly Asp Val Val Gly Asp
Tyr Asn Phe Ile Cys 385 390 395 400 Pro Ala Leu Glu Phe Thr Lys Lys
Phe Ser Glu Trp Gly Asn Asn Ala 405 410 415 Phe Phe Tyr Tyr Phe Glu
His Arg Ser Ser Lys Leu Pro Trp Pro Glu 420 425 430 Trp Met Gly Val
Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Leu 435 440 445 Pro Leu
Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Leu Ser 450 455 460
Arg Ser Ile Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro 465
470 475 480 Asn Glu Thr Gln Asn Asn Ser Thr Ser Trp Pro Val Phe Lys
Ser Thr 485 490 495 Glu Gln Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr
Arg Ile Met Thr 500 505 510 Lys Leu Arg Ala Gln Gln Cys Arg Phe Trp
Thr Ser Phe Phe Pro Lys 515 520 525 Val 2585PRTHomo sapiens 2Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10
15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145
150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu
Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser
Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val
Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu
Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr
Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg
Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265
270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys
Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met
Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala
Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390
395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu
Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg
Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515
520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln
Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys
Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly
Leu 580 585 323PRTArtificial Sequencesignal peptide 3Met Arg Pro
Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu Ala 1 5 10 15 Leu
Trp Ala Pro Ala Arg Gly 20 41137PRTArtificial SequenceAlbuBChE 4Met
Arg Pro Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu Ala 1 5 10
15 Leu Trp Ala Pro Ala Arg Gly Glu Asp Asp Ile Ile Ile Ala Thr Lys
20 25 30 Asn Gly Lys Val Arg Gly Met Asn Leu Thr Val Phe Gly Gly
Thr Val 35 40 45 Thr Ala Phe Leu Gly Ile Pro Tyr Ala Gln Pro Pro
Leu Gly Arg Leu 50 55 60 Arg Phe Lys Lys Pro Gln Ser Leu Thr Lys
Trp Ser Asp Ile Trp Asn 65 70 75 80 Ala Thr Lys Tyr Ala Asn Ser Cys
Cys Gln Asn Ile Asp Gln Ser Phe 85 90 95 Pro Gly Phe His Gly Ser
Glu Met Trp Asn Pro Asn Thr Asp Leu Ser 100 105 110 Glu Asp Cys Leu
Tyr Leu Asn Val Trp Ile Pro Ala Pro Lys Pro Lys 115 120 125 Asn Ala
Thr Val Leu Ile Trp Ile Tyr Gly Gly Gly Phe Gln Thr Gly 130 135 140
Thr Ser Ser Leu His Val Tyr Asp Gly Lys Phe Leu Ala Arg Val Glu 145
150 155 160 Arg Val Ile Val Val Ser Met Asn Tyr Arg Val Gly Ala Leu
Gly Phe 165 170 175 Leu Ala Leu Pro Gly Asn Pro Glu Ala Pro Gly Asn
Met Gly Leu Phe 180 185 190 Asp Gln Gln Leu Ala Leu Gln Trp Val Gln
Lys Asn Ile Ala Ala Phe 195 200 205 Gly Gly Asn Pro Lys Ser Val Thr
Leu Phe Gly Glu Ser Ser Gly Ala 210 215 220 Ala Ser Val Ser Leu His
Leu Leu Ser Pro Gly Ser His Ser Leu Phe 225 230 235 240 Thr Arg Ala
Ile Leu Gln Ser Gly Ser Phe Asn Ala Pro Trp Ala Val 245 250 255 Thr
Ser Leu Tyr Glu Ala Arg Asn Arg Thr Leu Asn Leu Ala Lys Leu 260 265
270 Thr Gly Cys Ser Arg Glu Asn Glu Thr Glu Ile Ile Lys Cys Leu Arg
275 280 285 Asn Lys Asp Pro Gln Glu Ile Leu Leu Asn Glu Ala Phe Val
Val Pro 290 295 300 Tyr Gly Thr Pro Leu Gly Val Asn Phe Gly Pro Thr
Val Asp Gly Asp 305 310 315 320 Phe Leu Thr Asp Met Pro Asp Ile Leu
Leu Glu Leu Gly Gln Phe Lys 325 330 335 Lys Thr Gln Ile Leu Val Gly
Val Asn Lys Asp Glu Gly Thr Trp Phe 340 345 350 Leu Val Gly Gly Ala
Pro Gly Phe Ser Lys Asp Asn Asn Ser Ile Ile 355 360 365 Thr Arg Lys
Glu Phe Gln Glu Gly Leu Lys Ile Phe Phe Pro Gly Val 370 375 380 Ser
Glu Phe Gly Lys Glu Ser Ile Leu Phe His Tyr Thr Asp Trp Val 385 390
395 400 Asp Asp Gln Arg Pro Glu Asn Tyr Arg Glu Ala Leu Gly Asp Val
Val 405 410 415 Gly Asp Tyr Asn Phe Ile Cys Pro Ala Leu Glu Phe Thr
Lys Lys Phe 420 425 430 Ser Glu Trp Gly Asn Asn Ala Phe Phe Tyr Tyr
Phe Glu His Arg Ser 435 440 445 Ser Lys Leu Pro Trp Pro Glu Trp Met
Gly Val Met His Gly Tyr Glu 450 455 460 Ile Glu Phe Val Phe Gly Leu
Pro Leu Glu Arg Arg Asp Asn Tyr Thr 465 470 475 480 Lys Ala Glu Glu
Ile Leu Ser Arg Ser Ile Val Lys Arg Trp Ala Asn 485 490 495 Phe Ala
Lys Tyr Gly Asn Pro Asn Glu Thr Gln Asn Asn Ser Thr Ser 500 505 510
Trp Pro Val Phe Lys Ser Thr Glu Gln Lys Tyr Leu Thr Leu Asn Thr 515
520 525 Glu Ser Thr Arg Ile Met Thr Lys Leu Arg Ala Gln Gln Cys Arg
Phe 530 535 540 Trp Thr Ser Phe Phe Pro Lys Val Asp Ala His Lys Ser
Glu Val Ala 545 550 555 560 His Arg Phe Lys Asp Leu Gly Glu Glu Asn
Phe Lys Ala Leu Val Leu 565 570 575 Ile Ala Phe Ala Gln Tyr Leu Gln
Gln Cys Pro Phe Glu Asp His Val 580 585 590 Lys Leu Val Asn Glu Val
Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 595 600 605 Glu Ser Ala Glu
Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 610 615 620 Lys Leu
Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 625 630 635
640 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln
645 650 655 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro
Glu Val 660 665 670 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu
Thr Phe Leu Lys 675 680 685 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His
Pro Tyr Phe Tyr Ala Pro 690 695 700 Glu Leu Leu Phe Phe Ala Lys Arg
Tyr Lys Ala Ala Phe Thr Glu Cys 705 710 715 720 Cys Gln Ala Ala Asp
Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 725 730 735 Leu Arg Asp
Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 740 745 750 Ala
Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 755 760
765 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser
770 775 780 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys
His Gly 785 790 795 800 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp
Leu Ala Lys Tyr Ile 805 810 815 Cys Glu Asn Gln Asp Ser Ile Ser Ser
Lys Leu Lys Glu Cys Cys Glu 820 825 830 Lys Pro Leu Leu Glu Lys Ser
His Cys Ile Ala Glu Val Glu Asn Asp 835 840 845 Glu Met Pro Ala Asp
Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 850 855 860 Lys Asp Val
Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 865 870 875 880
Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 885
890 895 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys
Cys 900 905 910 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val
Phe Asp Glu 915 920 925 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu
Ile Lys Gln Asn Cys 930 935 940 Glu Leu Phe Glu Gln Leu Gly Glu Tyr
Lys Phe Gln Asn Ala Leu Leu 945 950 955 960 Val Arg Tyr Thr Lys Lys
Val Pro Gln Val Ser Thr Pro Thr Leu Val 965 970 975 Glu Val Ser Arg
Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 980 985 990 Pro Glu
Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 995 1000
1005 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp
1010 1015 1020 Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg
Arg Pro 1025 1030 1035 Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr
Val Pro Lys Glu 1040 1045 1050 Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp Ile Cys Thr Leu 1055 1060 1065 Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala Leu Val Glu 1070 1075 1080 Leu Val Lys His Lys
Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala 1085 1090 1095 Val Met Asp
Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala 1100 1105 1110 Asp
Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 1115 1120
1125 Ala Ala Ser Gln Ala Ala Leu Gly Leu 1130 1135
* * * * *