U.S. patent application number 14/410389 was filed with the patent office on 2016-03-17 for beta-lactamase targeted photosensitizer for pesticide and pest detection.
The applicant listed for this patent is The General Hospital Corporation, New England Biolabs. Invention is credited to Jeremy Foster, Tayyaba Hasan, Ulysses W. Sallum, Barton Slatko.
Application Number | 20160073634 14/410389 |
Document ID | / |
Family ID | 49769424 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160073634 |
Kind Code |
A1 |
Hasan; Tayyaba ; et
al. |
March 17, 2016 |
BETA-LACTAMASE TARGETED PHOTOSENSITIZER FOR PESTICIDE AND PEST
DETECTION
Abstract
Photoactivatable pesticide compounds and methods for the use
thereof in the elimination and detection of pests are provided.
Inventors: |
Hasan; Tayyaba; (Arlington,
MA) ; Sallum; Ulysses W.; (Arlington, MA) ;
Slatko; Barton; (Ipswich, MA) ; Foster; Jeremy;
(Beverly, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation
New England Biolabs |
Boston
Ipswich |
MA
MA |
US
US |
|
|
Family ID: |
49769424 |
Appl. No.: |
14/410389 |
Filed: |
June 21, 2013 |
PCT Filed: |
June 21, 2013 |
PCT NO: |
PCT/US13/47045 |
371 Date: |
December 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61663410 |
Jun 22, 2012 |
|
|
|
Current U.S.
Class: |
514/205 ;
250/200; 250/459.1; 540/226 |
Current CPC
Class: |
A01N 25/00 20130101;
G01N 21/6428 20130101; A01N 43/90 20130101; G01N 2021/6432
20130101; C07D 501/16 20130101; G01N 2021/6439 20130101; A01N 25/00
20130101; A01N 43/90 20130101 |
International
Class: |
A01N 43/90 20060101
A01N043/90; A01N 25/00 20060101 A01N025/00; G01N 21/64 20060101
G01N021/64; C07D 501/16 20060101 C07D501/16 |
Claims
1. A pesticidal composition comprising a pesticidally effective
amount of one or more photosensitizers that are linked by one or
more moieties cleavable by a .beta.-lactamase expressed by a pest
wherein said linked moieties are present in an amount sufficient to
quench photoactivation of said photosensitizers and wherein said
one or more photosenitizers are capable of generating a phototoxic
species upon dequenching and light-activation.
2. The pesticidal composition of claim 1, wherein the one or more
moieties cleavable by the .beta.-lactamase comprise: a
cephalosporin, a penicillin, a penem, a carbapenem, a monocyclic
mobactem, or a fragment thereof.
3. The photosensitizer composition of claim 2, wherein the one or
more moieties cleavable by the .beta.-lactamase comprises a
cephalosporin, a penicillin, or a fragment thereof.
4. The pesticidal composition of claim 3, wherein the cephalosporin
or penicillin fragment comprises a beta-lactam ring.
5. The pesticidal composition of claim 3, wherein the one or more
moieties cleavable by .beta.-lactamase is a cephalosporin.
6. The pesticidal composition of claim 5, wherein at least one
photosensitizer is bound at the 3' position of a cephalosporin.
7. The pesticidal composition of claim 1, wherein the
photosensitizer is a porphyrin.
8. The pesticidal composition of claim 7, wherein the porphyrin is
selected from the group consisting of a porfimer sodium,
hematoporphyrin IX, hematoporphyrin ester, dihematoporphyrin ester,
synthetic diporphyrin, O-substituted tetraphenyl porphyrin,
3,1-meso tetrakis porphyrin, hydroporphyrin, benzoporphyrin
derivative, benzoporphyrin monoacid derivative, monoacid ring
derivative, tetracyanoethylene adduct of benzoporphyrin, dimethyl
acetylenedicarboxylate adduct of benzoporphyrin,
.delta.-aminolevulinic acid, benzonaphthoporphyrazine, naturally
occurring porphyrin, ALA-induced protoporphyrin IX, synthetic
dichlorin, bacteriochlorin tetra(hydroxyphenyl) porphyrin,
purpurin, octaethylpurpurin derivative, etiopurpurin,
tin-etio-purpurin, porphycene, chlorin, chlorin e.sub.6,
mono-1-aspartyl derivative of chlorin e.sub.6, di-1-aspartyl
derivative of chlorin e.sub.6, tin(IV) chlorin e.sub.6,
meta-tetrahydroxyphenylchlorin, chlorin e.sub.6 monoethylendiamine
monamide, verdin, zinc methyl pyroverdin, copro II verdin trimethyl
ester, deuteroverdin methyl ester, pheophorbide derivative,
pyropheophorbide, texaphyrin, lutetium (III) texaphyrin, and
gadolinium(III) texaphyrin.
9. The pesticidal composition of claim 1, wherein the
photosensitizer is a photoactive dye.
10. The pesticidal composition of claim 9, wherein the photoactive
dye is selected from the group consisting of a merocyanine,
phthalocyanine, chloroaluminum phthalocyanine, sulfonated aluminum
PC, ring-substituted cationic PC, sulfonated AlPc, disulfonated or
tetrasulfonated derivative, sulfonated aluminum naphthalocyanine,
naphthalocyanine, tetracyanoethylene adduct, crystal violet, azure
.beta. chloride, benzophenothiazinium, benzophenothiazinium
chloride (EtNBS), phenothiazine derivative, rose Bengal, toluidine
blue derviatives, toluidine blue O (TBO), methylene blue (MB), new
methylene blue N (NMMB), new methylene blue BB, new methylene blue
FR, 1,9-dimethylmethylene blue chloride (DMMB), methylene blue
derivatives, methylene green, methylene violet Bernthsen, methylene
violet 3RAX, Nile blue, Nile blue derivatives, malachite green,
Azure blue A, Azure blue B, Azure blue C, safranine O, neutral red,
5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,
5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,
thiopyronine, and thionine.
11. The pesticidal composition of claim 1, wherein the
photosensitizer is selected from the group consisting of a
Diels-Alder adduct, dimethyl acetylene dicarboxylate adduct,
anthracenedione, anthrapyrazole, aminoanthraquinone, phenoxazine
dye, chalcogenapyrylium dye, cationic selena, tellurapyrylium
derivative, cationic imminium salt and tetracycline.
12. The composition of claim 1, wherein the composition comprises a
one or more of the same photosensitizer.
13. The composition of claim 1, wherein the .beta.-lactamase
comprises SEQ ID NO:1 or a fragment thereof.
14. The composition of claim 1, wherein the .beta.-lactamase
comprises a protein domain having at least about 50%, 55%, 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, identity to SEQ ID
NO:1.
15. The composition of claim 1, wherein the .beta.-lactamase
comprises SEQ ID NO: 2 or a fragment thereof.
16. The composition of claim 1, wherein the .beta.-lactamase
comprises a protein domain having at least about 50%, 55%, 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, identity to SEQ ID
NO:2.
17. A pesticidal composition comprising a pesticidally effective
amount of a backbone coupled to one or more photosensitizers and
one or more binders effective to quench photoactivation, wherein
the binders are connected to the backbone through a one or more
moieties cleavable by a .beta.-lactamase expressed by a pest and
wherein said one or more photosenitizers are capable of generating
a phototoxic species upon dequenching and light-activation.
18. The pesticidal composition of claim 1, further comprising a
pesticidally acceptable carrier or excipient.
19. A method for eliminating a pest from a host, said method
comprising the steps of: contacting the pest with an effective
amount of a pesticidal composition of claim 1; cleaving one or more
moieties cleavable by .beta.-lactamase to dequench the
photosensitizer composition; and light-activating the composition
to produce a phototoxic species, thereby eliminating the pest from
said host.
20. The method of claim 19, wherein the phototoxic species is also
fluorescent.
21. The method of claim 19, wherein the pest is an arthropod, a
nematode, or an insect and the host is a plant.
22. The method of claim 21, wherein the arthropod or insect is
selected from the group consisting of nematodes, grubs, weevils,
borers, aphids, moths, mosquitoes, flies, ticks, termites, beetles,
caterpillar, cutworms, earworms, armyworms, and budworms.
23. The method of claim 21, wherein the plant is selected from the
group consisting of corn, maize, wheat, tobacco, cotton, rice,
soybean, peanut, sugarcane, hay, sorghum, lettuces, kales,
cabbages, fruit trees, and horitcultural flowers.
24. The method of claim 19, wherein the pest is a parasite and the
host is an animal.
25. The method of claim 24, wherein the parasite is selected from
the group consisting of ticks, lice, mites, ascarids, filarias,
hookworms, pinworms, whipworms, strongyles, Trichinella spiralis,
Dirofilaria immitis, Haemonchus contortus, Brugia malayi and
Myrmeconema neotropicum.
26. The method of claim 24, wherein the host is a human.
27. A method for eliminating a pest from an industrial material,
said method comprising the steps of: contacting the pest with a
pesticidal composition of claim 1; cleaving one or more moieties
cleavable by .beta.-lactamase to dequench the photosensitizer
composition; and light-activating the composition to produce a
phototoxic species, thereby eliminating the pest from said
host.
28. The method of claim 27, wherein the pest is selected from the
group consisting of beetles, termites, and hymenopterons.
29. The method of claim 27, wherein the industrial material is
selected from the group consisting of plastics, adhesives, sizes,
paper and card, leather, wood and processed wood products.
30. A method for eliminating a pest from an enclosed space, said
method comprising the steps of: contacting the pest with a
pesticidal composition of claim 1; cleaving one or more moieties
cleavable by .beta.-lactamase to dequench the photosensitizer
composition; and light-activating the composition to produce a
phototoxic species, thereby eliminating the pest from said
space.
31. The method of claim 30, wherein the pest is selected from the
group consisting of scorpions, spiders, woodlice, pillbugs,
bedbugs, millipedes, centipedes, caterpillars, moths, silverfish,
cockroaches, grasshoppers, locusts, flies and mosquitoes.
32. A method for detecting a pest, said method comprising the steps
of: contacting the pest with a quenched photosensitizer composition
comprising a plurality of photosensitizers that are linked by one
or more moieties cleavable by a .beta.-lactamase expressed by the
pest, wherein said linked photosensitizers are present in an amount
sufficient to quench photoactivation of said photosensitizers;
cleaving one or more moieties cleavable by the .beta.-lactamase to
dequench the photosensitizer composition; and light-activating the
composition to produce a fluorescent species, and detecting the
pest by observing the fluorescence, thereby detecting the presence
of the pest.
33. The method of claim 32, wherein the florescent species is also
phototoxic.
34. A method for controlling an insect pest, the method comprising
contacting the pest with an effective amount of a pesticidal
composition of claim 1; cleaving one or more moieties cleavable by
.beta.-lactamase to dequench the photosensitizer composition; and
light-activating the composition to produce a phototoxic species,
thereby controlling the insect pest.
35. The method of claim 34, wherein the insect is Aedes
albopictus.
36. A method for controlling a filarial nematode, the method
comprising contacting the with worm with an effective amount of a
pesticidal composition of claim 1; cleaving one or more moieties
cleavable by .beta.-lactamase to dequench the photosensitizer
composition; and light-activating the composition to produce a
phototoxic species, thereby controlling the worm.
37. The method of claim 36, wherein the filarial nematode is
Wuchereria bancrofti, Brugia malayi, or B. timori.
38. A method for ameliorating filariais in a subject, the method
comprising administering to the subject an effective amount of a
composition of claim 1; cleaving one or more moieties cleavable by
.beta.-lactamase to dequench the photosensitizer composition; and
light-activating the composition to produce a phototoxic species,
thereby ameliorating the filariasis.
39. The method of claim 38, wherein the method reduces the filarial
load in the subject by at least about 10-25% or more.
40. The method of claim 38, wherein the filariasis is associated
with Wuchereria bancrofti, Brugia malayi, and/or B. timori.
41. A method for controlling a fouling pest on an object in contact
with saltwater or brackish water, the method comprising contacting
the pest with an effective amount of a pesticidal composition of
claim 1; cleaving one or more moieties cleavable by
.beta.-lactamase to dequench the photosensitizer composition; and
light-activating the composition to produce a phototoxic species,
thereby controlling the pest.
42. The method of claim 41, wherein the fouling pest is a goose
barnacle, an acorn barnacle or a sessile Oligochaeta.
43. The method of claim 1 wherein the light-activation is from
exposure to sunlight.
44. The method of claim 1 wherein the light-activation is from
administration of LED lighting.
45. The method of claim 1 wherein the light-activation is from
administration of laser lighting.
46. The method of claim 1, wherein the composition comprises a
beta-lactamase enzyme-activated-photosensitizer (.beta.-LEAP).
47. A kit for eliminating a pest comprising the pesticidal
composition of claim 1 and instructions for using the pesticidal
composition to eliminate the pest.
48. A kit for detecting a pest comprising a photosensitizer
composition comprising one or more photosensitizers that are linked
by one or more moieties cleavable by a .beta.-lactamase expressed
by the pest, wherein said linked moieties are present in an amount
sufficient to quench photoactivation of said photosensitizers and
wherein said one or more photosenitizers are capable of generating
a fluorescent species upon dequenching and light-activation, and
instructions for using the photosensitizer composition to detect
the pest.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/663,410, filed on Jun. 22, 2012, the entire
contents of which are incorporated herein by reference.
RELATED DISCLOSURES
[0002] The subject matter disclosed in this application may be
related to the subject matter disclosed in U.S. patent application
publication no. US 2010-0016208 A1, published on Jan. 21, 2010, and
U.S. patent application publication no. US 2011-0112059 A1,
published on May 12, 2011, each of which is hereby expressly
incorporated herein in its entirety by reference.
INCORPORATION BY REFERENCE
[0003] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
paragraphing priority from any of these applications and patents,
and each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein in their entireties by reference. More generally, documents
or references are cited in this text, either in a Reference List
before the paragraphs, or in the text itself; and, each of these
documents or references ("herein-cited references"), as well as
each document or reference cited in each of the herein-cited
references (including any manufacturer's specifications,
instructions, etc.), are hereby expressly incorporated herein in
their entireties by reference.
BACKGROUND OF THE INVENTION
[0004] Human filariasis is a major global health problem. The
diseases are caused by infections with parasitic filarial nematode
worms leading_two related disorders. The nematode species
responsible for human lymphatic filariasis (LF) are Wuchereria
bancrofti, Brugia malayi and to a less extent, B. timori, while
human onchocerciasis is caused by the related filarial nematode,
Onchocerca volvulus.
[0005] Onchocerciasis is cutaneous, wherein adult worms reside in
palpable fibrous nodules, which are a result of inflammation to
dead microfilaria F1 generation released by adult females after
mating). One other aspect of the malady is blindness caused by
microfilaria migration and invasion of the cornea, keratitis,
retinal lesions and degeneration of the optic nerve. Lymphatic
filariasis, on the other hand, is a disease associated with
dysfunction of lymphatic tissue and lymphodema, leading to a
disease state known as elephantiasis, including hydrocoele.
[0006] More than 150 million individuals in 80 countries are
infected with parasitic filarial nematode worms responsible for
lymphatic filariasis (LF) and onchocerciasis (river blindness),
with an estimated 1 billion people at risk of infection, ranking
filariasis as one of the major causes of global morbidity.
[0007] The life cycle of these parasites requires an arthropod
vector; LF is transmitted by both anopheline and culicine
mosquitoes whereas O. volvulus is transmitted by blackflies
(Simulium species) (Blacklock, 1926; Mullen and Durden, 2009).
Adult worms can live for 10 years or more and the microfilaria
released into the blood are picked up by the insect vector during a
blood meal, undergo several molts and then are delivered to the
human host in a subsequent insect bite wound. The infective larvae
undergo several molts and develop into male or female adults, mate
and give rise to the microfilaria to continue life cycle. There are
several challenges associated with attempts to eliminate
filariasis: (1.) there are no macrofiliaricidal (adulticide) drugs
available. Control programs rely on sustained delivery of
antiparasitic drugs, such as DEC (diethylcarbamazine), albendazole,
and ivermectin, which have been the mainline drugs of choice for
filariasis control. However, these drugs are not effective
adulticides and repeated community-wide doses (as part of MDA, mass
drug administration) are required to suppress microfilarial
production and reduce transmission.
[0008] Likewise, Over time insects have become both more numerous
and more destructive to plants, both agriculturally and
domestically. A host of small insects attack grasses and forage
crops, many of them being so small that they are unnoticed though
their aggregate injury is enormous. Larger pests, such as worms,
grubs, grasshoppers, flies, boll weevils, bollworms, and ticks are
equally dangerous to plant life. In total, the damage to crops and
other plants from insect attack represents losses of billions of
dollars annually.
[0009] Photosensitizers (PS) are light-sensitive compounds which
undergo a photochemical reaction after the absorption of light
quantum. Such photodynamic compounds have been successfully used
for antibacterial photodynamic therapies acting throughout the
body. A strength of such photodynamic therapy (PDT) is the broad
range of targets hit by the reactive molecular species it produces.
Targeting the photoreactivity of PSs through catalysis by
parasite-specific enzymes maintains this advantage. Still, the
current state of PDT has focused on the targeting of
parasite-specific enzymes within the human body.
[0010] As such, photodynamic compounds having improved specificity
for arthropod, nematode, insect and parasite-specific enzymes
capable of targeting and killing an insect (or other unwanted
organism that produces an enzyme capable of hydrolyzing the
construct) such as mosquitoes, biting flies, fruit flies, sand
flies, barnacles, crustacea, and cockroaches, outside of the human
body, would be desirable.
SUMMARY OF THE INVENTION
[0011] The invention provides, inter alia, novel methods to control
and/or kill arthropods, nematodes, insects and parasites in plant
and animal (e.g., human) hosts via the targeted release of free
photosensitizer from a quenched to an unquenched and active state
by .beta.-lactamases that are produced by the insect. The invention
is based, at least in part, on the discovery that when a pest,
e.g., an insect, ingests an enzyme-cleavable .beta.-lactamase
specific construct, the construct is cleaved by .beta.-lactamases
that are produced by the insect, resulting in the release of free
photosensitizer within the insect. When the insect is then exposed
to light, the free photosensitizer is converted to a phototoxic
species that kills the insect. In certain advantageous embodiments,
the insect also fluoresces as a result of the cleavage allowing for
insect detection via fluorescence emission.
[0012] Thus, in one aspect, the invention provides a pesticidal
composition comprising a pesticidally effective amount of one or
more photosensitizers that are linked by one or more moieties
cleavable by .beta.-lactamase, wherein the linked photosensitizers
are present in an amount sufficient to quench photoactivation of
the photosensitizers and wherein said one or more photosenitizers
are capable of generating a phototoxic species upon dequenching and
light-activation.
[0013] In yet another aspect, the invention provides a pesticidal
composition comprising pesticidally effective amount of one or more
photosensitizers and one or more binders effective to quench
photoactivation, wherein the photosensitizers are connected to the
binder through one or more moieties cleavable by a .beta.-lactamase
expressed by a pest and wherein said one or more photosenitizers
are capable of generating a phototoxic species upon dequenching and
light-activation. In a further embodiment, the binder is a
fluorophore.
[0014] In yet another aspect, the invention provides a pesticidal
composition comprising a backbone coupled to one or more
photosensitizers and one or more binders effective to quench
photoactivation, wherein the binders are connected to the backbone
through one or more moieties cleavable by a .beta.-lactamase
expressed by a pest and wherein said one or more photosenitizers
are capable of generating a phototoxic species upon dequenching and
light-activation.
[0015] In yet another aspect, the invention provides a pesticidal
composition comprising a backbone coupled to a plurality of
photosensitizers and one or more binders effective to quench
photoactivation, wherein the photosensitizers are connected to the
backbone through one or more moieties cleavable by
.beta.-lactamase.
[0016] In accordance with the invention, the pest is an animal that
expresses .beta.-lactamase. In particular embodiments, the pest is
an animal that expresses a .beta.-lactamase comprising the protein
domain sequence:
TABLE-US-00001 (SEQ ID NO: 1)
ILTEKRKILVDCGDPWNGTQIIQALSKYSLNCDDITDLIITHGHSDHCGN
LSLFQQAKIYMGDDMAKDGIYEGIWTLDDFVKIRPTPGHTDRSIIVLDTE
YGTVAIVGDIFEEENDDDSWKENSKYPEEQQKSRKIILKEADWIIPGH (GenBANK protein
sequence XP_001891895) or a fragment thereof.
[0017] In certain embodiments, the pest expresses a
.beta.-lactamase comprising a protein domain sequence having at
least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, identity (e.g., when compared to the overall length of the
protein sequence) to SEQ ID NO:1 or a fragment thereof.
[0018] In still other embodiments, the pest is an animal that
expresses a .beta.-lactamase comprising the protein domain
sequence:
TABLE-US-00002 (SEQ ID NO: 2)
TNTYIIGTGKRRILLDAGDENVPEYIGHLKKVISDERILINDIIVSHWH
HDHIGGVDEVLDIIENKDSCKVWKFPRADAPDGTIRNANINHLKHGQKF
NIEGATLEVLHTPGHTTDHVVLVLHEDNSLFSADCILGEGSTVEEDLYE
YTKSLQAIQDAKPSVIYPG (GenBANK protein sequence XP_001656361) or a
fragment thereof.
[0019] In certain embodiments, the pest expresses a
.beta.-lactamase comprising a protein domain sequence having at
least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, identity (e.g., when compared to the overall length of the
protein sequence) to SEQ ID NO:2 or a fragment thereof.
[0020] In certain embodiments, the host is an animal, e.g.,
human.
[0021] In other embodiments the host is a plant. Plants in
accordance with the invention include, but are not limited to corn,
maize, wheat, tobacco, cotton, rice, soybean, peanut, sugarcane,
hay, sorghum, lettuces, kales, cabbages, fruit trees, or
horitcultural flowers.
[0022] In embodiment embodiments, the pests targeted in plants
include, but are not limited to, nematodes, grubs, weevils, borers,
aphids, moths, mosquitoes, flies, ticks, termites, beetles,
caterpillar, cutworms, earworms, armyworms, or budworms.
[0023] In certain embodiments, the photosensitizer is a porphyrin.
The porphyrin can be, but is not limited to, a porfimer sodium,
hematoporphyrin IX, hematoporphyrin ester, dihematoporphyrin ester,
synthetic diporphyrin, O-substituted tetraphenyl porphyrin,
3,1-meso tetrakis porphyrin, hydroporphyrin, benzoporphyrin
derivative, benzoporphyrin monoacid derivative, monoacid ring
derivative, tetracyanoethylene adduct of benzoporphyrin, dimethyl
acetylenedicarboxylate adduct of benzoporphyrin,
.delta.-aminolevulinic acid, benzonaphthoporphyrazine, naturally
occurring porphyrin, ALA-induced protoporphyrin IX, synthetic
dichlorin, bacteriochlorin tetra(hydroxyphenyl) porphyrin,
purpurin, octaethylpurpurin derivative, etiopurpurin,
tin-etio-purpurin, porphycene, chlorin, chlorin e.sub.6,
mono-1-aspartyl derivative of chlorin e.sub.6, di-1-aspartyl
derivative of chlorin e.sub.6, tin(IV) chlorin e.sub.6,
meta-tetrahydroxyphenylchlorin, chlorin e.sub.6 monoethylendiamine
monamide, verdin, zinc methyl pyroverdin, copro II verdin trimethyl
ester, deuteroverdin methyl ester, pheophorbide derivative,
pyropheophorbide, texaphyrin, lutetium (III) texaphyrin, or
gadolinium(III) texaphyrin.
[0024] In other embodiments, the photosensitizer is a photoactive
dye. The photoactive dye includes, but is not limited to, a
merocyanine, phthalocyanine, chloroaluminum phthalocyanine,
sulfonated aluminum PC, ring-substituted cationic PC, sulfonated
AlPc, disulfonated or tetrasulfonated derivative, sulfonated
aluminum naphthalocyanine, naphthalocyanine, tetracyanoethylene
adduct, crystal violet, azure .beta. chloride,
benzophenothiazinium, benzophenothiazinium chloride (EtNBS),
phenothiazine derivative, phenothiaziniums such as rose Bengal,
toluidine blue derivatives, toluidine blue O (TBO), methylene blue
(MB), new methylene blue N (NMMB), new methylene blue BB, new
methylene blue FR, 1,9-dimethylmethylene blue chloride (DMMB),
methylene blue derivatives, methylene green, methylene violet
Bernthsen, methylene violet 3RAX, Nile blue, Nile blue derivatives,
malachite green, Azure blue A, Azure blue B, Azure blue C, safranin
O, neutral red, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium
chloride, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium
chloride, thiopyronine, or thionine.
[0025] In still other embodiments, the photosensitizer includes,
but is not limited to, a Diels-Alder adduct, dimethyl acetylene
dicarboxylate adduct, anthracenedione, anthrapyrazole,
aminoanthraquinone, phenoxazine dye, chalcogenapyrylium dye,
cationic selena, tellurapyrylium derivative, cationic imminium
salt, or tetracycline.
[0026] In yet another embodiment, the photosensitizer composition
comprises a plurality of the same photosensitizer.
[0027] In one embodiment, the moiety cleavable by .beta.-lactamase
of the photosensitizer composition comprises a cephalosporin, a
penicillin, a penem, a carbapenem, a monocyclic monobactem, or a
fragment thereof. In a further embodiment, the moiety cleavable by
.beta.-lactamase of the photosensitizer composition comprises a
cephalosporin, a penicillin, or a fragment thereof. The
cephalosporin or penicillin fragment can comprise a beta-lactam
ring, and the enzyme cleavage site can be cleaved by a lactamase.
In another embodiment, the moiety cleavable by .beta.-lactamase is
a cephalosporin. At least one photosensitizer can be bound at the
3' position of the cephalosporin.
[0028] In yet another embodiment, a binder is present and connected
to the photosensitizer by one or more moieties cleavable by
.beta.-lactamase. In specific embodiments, binder can be a
fluorophore or an other photosensitizer.
[0029] In yet another aspect, the invention provides a
pharmaceutical composition comprising a pesticidally effective
amount of a photosensitizer composition of the invention and a
pharmaceutically acceptable excipient or carrier.
[0030] In yet another aspect, the invention provides a method of
eliminating a pest from a host, the method comprising the steps of:
contacting the pest with an effective amount of a pesticidal
composition comprising one or more photosensitizers that are linked
by one or more moieties cleavable by a .beta.-lactamase expressed
by the pest, wherein the one or more moieties cleavable by the
.beta.-lactamase comprises an enzyme cleavage site for an enzyme of
a pathogen, and optionally one or more binders, and wherein the
linked photosensitizers are present in an amount sufficient to
quench photoactivation of the photosensitizers; cleaving one or
more moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a phototoxic species, thereby eliminating the pest from the
host.
[0031] In certain embodiments, phototoxic species is also
fluorescent.
[0032] In other embodiments, the pest is an arthropod, a nematode
or an insect and the host is a plant. In some embodiments, the pest
is selected from the group consisting of nematodes, grubs, weevils,
borers, aphids, moths, mosquitoes, flies, ticks, termites, beetles,
caterpillar, cutworms, earworms, armyworms, and budworms. In other
embodiments, the plant is selected from the group consisting of
corn, maize, wheat, tobacco, cotton, rice, soybean, peanut,
sugarcane, hay, sorghum, lettuces, kales, cabbages, fruit trees,
and horitcultural flowers.
[0033] In still other embodiments, the pest is a parasite and the
host is an animal or human. In some embodiments, the parasite is
selected from the group consisting of ticks, lice, mites, ascarids,
filarias, hookworms, pinworms, whipworms, strongyles, Trichinella
spiralis, Dirofilaria immitis, Haemonchus contortus, Brugia malayi
and Myrmeconema neotropicum.
[0034] In another aspect, the invention provides a method for
eliminating a pest from an industrial material, the method
comprising the steps of: contacting the pest with an effective
amount of a pesticidal composition comprising one or more
photosensitizers that are linked by one or more moieties cleavable
by a .beta.-lactamase expressed by the pest, wherein the one or
more moieties cleavable by the .beta.-lactamase comprises an enzyme
cleavage site for an enzyme of a pathogen, and optionally one or
more binders, and wherein the linked photosensitizers are present
in an amount sufficient to quench photoactivation of the
photosensitizers; cleaving one or more moieties cleavable by the
.beta.-lactamase to dequench the photosensitizer composition and
light-activating the composition to produce a phototoxic species,
thereby eliminating the pest from said host.
[0035] In certain embodiments, the pest is selected from the group
consisting of beetles, termites, and hymenopterons.
[0036] In other embodiments, the industrial material is selected
from the group consisting of plastics, adhesives, sizes, paper and
card, leather, wood and processed wood products.
[0037] In another aspect, the invention provides a method for
eliminating a pest from an enclosed space, the method comprising
the steps of: contacting the pest with an effective amount of a
pesticidal composition comprising one or more photosensitizers that
are linked by one or more moieties cleavable by a .beta.-lactamase
expressed by the pest, wherein the one or more moieties cleavable
by the .beta.-lactamase comprises an enzyme cleavage site for an
enzyme of a pathogen, and optionally one or more binders, and
wherein the linked photosensitizers are present in an amount
sufficient to quench photoactivation of the photosensitizers;
cleaving one or more moieties cleavable by the .beta.-lactamase to
dequench the photosensitizer composition and light-activating the
composition to produce a phototoxic species, thereby eliminating
the pest from said space.
[0038] In some embodiments, the pest is selected from the group
consisting of scorpions, spiders, woodlice, pillbugs, bedbugs,
millipedes, centipedes, caterpillars, moths, silverfish,
cockroaches, grasshoppers, locusts, flies and mosquitoes.
[0039] In another aspect, the invention provides a method for
detecting a pest, said method comprising the steps of: contacting
the pest with a quenched photosensitizer composition comprising a
plurality of photosensitizers that are linked by one or more
moieties cleavable by a .beta.-lactamase expressed by the pest,
wherein said linked photosensitizers are present in an amount
sufficient to quench photoactivation of said photosensitizers;
cleaving one or more moieties cleavable by the .beta.-lactamase to
dequench the photosensitizer composition; and light-activating the
composition to produce a fluorescent species, and detecting the
pest by observing the fluorescence, thereby detecting the presence
of the pest.
[0040] In some embodiments, the florescent species is also
phototoxic.
[0041] In another aspect, the invention provides a method for
controlling an insect pest, the method comprising the steps of:
contacting the pest with an effective amount of a pesticidal
composition comprising one or more photosensitizers that are linked
by one or more moieties cleavable by a .beta.-lactamase expressed
by the pest, wherein the one or more moieties cleavable by the
.beta.-lactamase comprises an enzyme cleavage site for an enzyme of
a pathogen, and optionally one or more binders, and wherein the
linked photosensitizers are present in an amount sufficient to
quench photoactivation of the photo sensitizers; cleaving one or
more moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a phototoxic species, thereby controlling said insect
pest.
[0042] In certain embodiments, the insect is Aedes albopictus.
[0043] In another aspect, the invention provides a method for
controlling a filarial nematode, the method comprising the steps
of: contacting the worm with an effective amount of a pesticidal
composition comprising one or more photosensitizers that are linked
by one or more moieties cleavable by a .beta.-lactamase expressed
by the pest, wherein the one or more moieties cleavable by the
.beta.-lactamase comprises an enzyme cleavage site for an enzyme of
a pathogen, and optionally one or more binders, and wherein the
linked photosensitizers are present in an amount sufficient to
quench photoactivation of the photosensitizers; cleaving one or
more moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a phototoxic species, thereby controlling said worm.
[0044] In certain embodiments, the filarial nematode is Wuchereria
bancrofti, Brugia malayi, or B. timori.
[0045] In another aspect, the invention provides a method for
ameliorating filariasis in a subject, the method comprising the
steps of: administering to the subject an effective amount of a
composition comprising one or more photosensitizers that are linked
by one or more moieties cleavable by a .beta.-lactamase expressed
by the pest, wherein the one or more moieties cleavable by the
.beta.-lactamase comprises an enzyme cleavage site for an enzyme of
a pathogen, and optionally one or more binders, and wherein the
linked photosensitizers are present in an amount sufficient to
quench photoactivation of the photo sensitizers; cleaving one or
more moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a phototoxic species, thereby ameliorating filariasis.
[0046] In some embodiments, the filariasis is associated with
Wuchereria bancrofti, Brugia malayi, and/or B. timori.
[0047] In certain embodiments, the method reduces the filarial load
in the subject by at least about 10-25% or more.
[0048] In another aspect, the invention provides a method for
controlling a fouling pest on an object in contact with saltwater
or brackish water, the method comprising the steps of: contacting
the pest with an effective amount of a pesticidal composition
comprising one or more photosensitizers that are linked by one or
more moieties cleavable by a .beta.-lactamase expressed by the
pest, wherein the one or more moieties cleavable by the
.beta.-lactamase comprises an enzyme cleavage site for an enzyme of
a pathogen, and optionally one or more binders, and wherein the
linked photosensitizers are present in an amount sufficient to
quench photoactivation of the photosensitizers; cleaving one or
more moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a phototoxic species, thereby controlling the pest.
[0049] In certain embodiments, the fouling pest is a goose
barnacle, an acorn barnacle or a sessile Oligochaeta.
[0050] Methods of the invention may further comprise the step of
obtaining the photosensitizer composition, linker or binder.
[0051] The light-activation in the methods of the invention may be
from exposure to sunlight, administration of LED lighting, or
administration of laser lighting.
[0052] In yet another aspect, the invention provides a kit for
eliminating a pest the comprising a pesticidal composition of the
invention and instructions for using the pesticidal composition to
eliminate the pest in accordance with the methods of the
invention.
[0053] In yet another aspect, the invention provides a kit for
detecting a pest the a photosensitizer composition comprising one
or more photosensitizers that are linked by one or more moieties
cleavable by a .beta.-lactamase expressed by the pest, wherein said
linked moieties are present in an amount sufficient to quench
photoactivation of said photosensitizers and wherein said one or
more photosenitizers are capable of generating a fluorescent
species upon dequenching and light-activation, and instructions for
using the photosensitizer composition to detect the pest.
[0054] Other aspects of the invention are described in the
following disclosure, and are within the ambit of the
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0055] Various advantageous features and embodiments of the present
invention are described below with reference to the accompanying
drawings in which:
[0056] FIG. 1 schematically depicts the development of a
carbamate-linked photosensitizer (PS) that is inactive (with or
without light) while linked and is light-activatable only when
released by the .beta.-lactamase enzyme-mediated cleavage.
[0057] FIG. 2a shows .sup.1H NMR spectra obtained for
7-[(2-phenylacetyl)amino]cephalosporanic acid in CDCl.sub.3 as a
solvent. FIG. 2b shows .sup.1H NMR spectrum obtained for
7-[(2-phenylacetyl)amino]3-hydrodxymethy cephalosporanic acid in
DMSO-d.sub.6 as a solvent. Major proton peaks are marked on the
spectra.
[0058] FIG. 3 shows MS spectra obtained for (a)
7-[(2-phenylacetyl)amino]3-hydrodxymethy cephalosporanic acid; and
(b) cephalosporanic acid-toluidine blue O prodrug.
[0059] FIG. 4 shows UV-visible spectra obtained for the
photosensitizer (TBO) (black line) vs. the Cephalosporanic
acid-photosensitizer prodrug (red line) in ethanol at a
concentration of 2.0.times.10.sup.-5 M.
[0060] FIG. 5 shows fluorescence emission spectra obtained for the
photosensitizer (TBO) (black line) vs. the Cephalosporanic
acid-photosensitizer prodrug (red line) in ethanol at 635 nm
excitation.
[0061] FIG. 6 shows plots of (a) fluorescence emission vs.
wavelength and (b) flurorescence emission vs. time for the
Cephalosporanic acid-photosensitizer prodrug, depicting the
enzyme-mediated cleavage of the prodrug.
[0062] FIG. 7 shows the synthesis of .beta.-LEAP
[0063] FIG. 8 shows the increasing relative fluorescence resulting
from activation of .beta.-LEAP by enzymatic cleavage in Brugia
malayi adults and 1st stage larvae (microfilariae).
[0064] FIG. 9 shows a confocal laser scanning micrograph of Brugia
malayi adults and 1st stage larvae following exposure to
.beta.-LEAP and 650 nm light.
[0065] FIG. 10 shows a confocal laser scanning microscopy of an
adult Brugia malayi female following exposure to .beta.-LEAP and
650 nm light.
[0066] FIG. 11 shows the effects of photodynamic therapy with the
photosensitizer EtNBS to kill adult Brugia malayi.
[0067] FIG. 12 shows the increasing relative fluorescence resulting
from activation of .beta.-LEAP by enzymatic cleavage in Aedes
albopictus cells.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the foregoing
drawings and the following non-limiting definitions that are given
by way of example to facilitate understanding of the invention.
I. DEFINITIONS
[0069] The term "photosensitizer" refers to a photoactivatable
compound, or a biological precursor thereof, that produces a
reactive species (e.g., oxygen) having a photochemical (e.g., cross
linking) or phototoxic effect on a cell, cellular component or
biomolecule. As used herein, a photosensitizer refers to a
substance which, upon irradiation with electromagnetic energy of
the appropriate wavelength (e.g., light), produces a cytotoxic
effect.
[0070] As used herein, the term "fluorescent dye" refers to dyes
that are fluorescent when illuminated with light but do not produce
reactive species that are phototoxic or otherwise capable of
reacting with biomolecules. A photosensitizer will fluoresce when
illuminated with a certain wavelength and power of light and also
produce reactive species that is phototoxic under the same or
different wavelength and power of light. The term "photoactive
dye," as used herein, means that the illuminated photosensitizer
produces a fluorescent species, but not necessarily a reactive
species in phototoxic amounts (i.e., a phototoxic species).
Depending on the wavelength and power of light administered, a
photosensitizer can be activated to fluoresce and, therefore, act
as a photoactive dye, but not produce a phototoxic species. The
wavelength and power of light can be adapted by methods known to
those skilled in the art to bring about a phototoxic effect where
desired.
[0071] As used herein, the term "backbone" refers to an agent that
functions to couple one or more components of a photosensitizer
composition of the invention, such as, for example, a polyamino
acid or like agent that is linked to one or more photosensitizers
and/or one or more binders and/or one or more targeting moieties
The backbone itself additionally can be a targeting moiety, e.g.
polylysine. A "backbone" as used herein is as a moiety higher in
molecular weight and capable of loading more photoactive molecules
than a `linker`. Backbone can be a polymeric structure which
provides a base to add multiple units (more than three). Examples
of backbones that can be used according to the invention, include,
but are not limited to polyethylene glycol and polyproline.
[0072] As used herein, the term "linker" or "moiety cleavable by
.beta.-lactamase" refers to an agent capable of linking two
components of the photosensitizer composition together (e.g., a
photosensitizer to another photosensitizer, a photosensitizer to a
binder, a photosensitizer to a backbone, or a binder to a
backbone).
[0073] As used herein, the term "binder" refers to an agent that
absorbs energy from an adjacent, activated photosensitizer or
otherwise inactivates the photosensitizer, and, thus, quenches the
photosensitizer.
[0074] The term "nucleic acid" is intended to include nucleic acid
molecules, e.g., polynucleotides which include an open reading
frame encoding a polypeptide, and can further include non-coding
regulatory sequences, and introns. In addition, the terms are
intended to include one or more genes that map to a functional
locus. In addition, the terms are intended to include a specific
gene for a selected purpose. Accordingly, the term is intended to
include any gene encoding a .beta.-lactamase.
[0075] As used herein, the terms "peptide", "polypeptide", and
"protein" are, unless specified otherwise, used interchangeably.
Peptides, polypeptides, and proteins used in methods and
compositions described herein can be recombinant, purified from
natural sources, or chemically synthesized. For example, reference
to the use of a bacterial protein or a protein from bacteria,
includes the use of recombinantly produced molecules, molecules
purified from natural sources, or chemically synthesized
molecules.
[0076] The term "plurality" refers to at least two, preferably at
least about 10 and even more preferably, at least about 20 or more
photosensitizer or binder molecules present in a composition of the
invention.
[0077] The term "host" is used herein to include both living hosts
and non-living/inanimate hosts. Examples of living hosts include
plants and animals. e.g., humans. Non-living/inanimate hosts
include industrial sites, public areas, and man-made surfaces
including household surfaces (kitchen surfaces, floors, walls,
ceilings, etc.), patios and the like.
[0078] The term "animal" is used herein to refer to a living
animal, including a human, that carries an unwanted organism, the
unwanted organism being the target of the methods described
herein.
[0079] The term "plant" is used herein to refer to any plant
including, but not limited to agricultural crops, fruit trees, nut
trees, domestic crops, and flowers. Such plants and crops include,
but are not limited to, corn, maize, wheat, tobacco, cotton, rice,
soybean, peanut, sugarcane, hay, sorghum, lettuces, kales,
cabbages, apples, oranges, pears, pumpkins, tomatoes, fruit trees,
or horitcultural flowers.
[0080] As used herein, "pest" or "target organism" means an animal,
e.g., insect, parasite or otherwise, that expresses a
.beta.-lactamase capable of cleaving the moieties which bind the
photosensitizers in the compositions of the inventions. Such pests
include any pest that adversely impacts on the health and
productivity of plants or other animals, or compromises the
integrity of an industrial material or dwelling. Specific pests
include, but are not limited to, nematodes, grubs, weevils, borers,
aphids, moths, mosquitoes, flies, ticks, termites, beetles,
caterpillar, cutworms, earworms, armyworms, or budworms. In one
embodiment, the mosquito is Ades albopictus.
[0081] As used herein, the term "parasite" includes an animal
organism that lives in or on another and takes its nourishment from
that other organism. Parasites in accordance with the invention
include, e.g., protozoa, nematodes, helminths and arthropods.
Parasites in accordance with the invention further include
symbiotic animal organisms. In one embodiment, the parasite is
Brugia malayi. Methods of the invention include controlling or
eliminating Brugia malayi from a human or insect host (e.g., Aedes
albopictus).
[0082] As used herein, the term "pesticidally effective amount"
refers to that amount of a photosensitizer composition that, when
administered to or ingested by a pest, is sufficient to eliminate,
terminate or otherwise control the pest. Thus, e.g., a pesticidally
effective amount of a photosensitizer composition as described
herein is a quantity sufficient to result in the death of a pest so
that the adverse effects of the pest are reduced or alleviated. In
one embodiment, a composition of the invention comprises an
effective amount of a prodrug construct with a photosensitizer and
a quencher linked by a beta-lactam ring, resulting in a diminished
phototoxicity. This construct is refered to as beta-lactamase
enzyme-activated-photosensitizer (.beta.-LEAP). The synthesis of is
provided at FIG. 7.
[0083] As used herein, the term "control" is meant reducing the
survival, proliferation, or reproduction of a pest. Such reduction
may be by at least about 10%, 25%, 50%, 75% or more. In one
embodiment, a composition of the invention ameliorates or controls
a parasite infestation (for example, as in filariasis) by reducing
the parasitic load in a subject by at least about 5, 10, 50, 75 or
100%.
[0084] As used herein, a "peptide antibiotic" is a linear or cyclic
oligopeptide, or an active fragment, or analog thereof, which
possesses antibiotic activity against bacterial or fungal species,
and which is synthesized enzymatically on a multi-protein complex
to which it is attached by a thioether bond. A peptide antibiotic
may include non-ribosomal amino acids such as D amino acids, and
may include non-amino acid residues such as esters of lactic acid
or valeric acid.
[0085] The term "obtaining" as in "obtaining" the "photosensitizer
composition," "linker" or "binder," is intended to include
purchasing, synthesizing or otherwise acquiring the elements of the
invention.
[0086] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the like;
"consisting essentially of" or "consists essentially" likewise has
the meaning ascribed in U.S. patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited is
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0087] Other definitions appear in context throughout this
disclosure.
II. COMPOSITIONS OF THE INVENTION
[0088] A. Photosensitizers
[0089] Photosensitizers known in the art are typically selected for
use according to: 1) efficacy in delivery, 2) proper localization
in target tissues, 3) wavelengths of absorbance, 4) proper
excitatory wavelength, 5) purity, and 6) in vivo effects on
pharmacokinetics, metabolism, and reduced toxicity.
[0090] A photosensitizer for clinical use is optimally amphiphilic,
meaning that it shares the opposing properties of being
water-soluble, yet hydrophobic. The photosensitizer should be
water-soluble in order to pass through the bloodstream
systemically, however it should also be hydrophobic enough to pass
across cell membranes. Modifications, such as attaching polar
residues (amino acids, sugars, and nucleosides) to the hydrophobic
porphyrin ring, can alter polarity and partition coefficients to
desired levels. Such methods of modification are well known in the
art.
[0091] In specific embodiments, photosensitizers of the present
invention absorb light at a relatively long wavelength, thereby
absorbing at low energy. Low-energy light can travel further
through tissue than high-energy light, which becomes scattered.
Optimal tissue penetration by light occurs between about 650 and
about 800 nm. Porphyrins found in red blood cells typically absorb
at about 630 nm, and new, modified porphyrins have optical spectra
that have been "red-shifted", in other words, absorbs lower energy
light. Other naturally occurring compounds have optical spectra
that is red-shifted with respect to porphyrin, such as chlorins
found in chlorophyll (about 640 to about 670 nm) or
bacteriochlorins found in photosynthetic bacteria (about 750 to
about 820 nm).
[0092] Photosensitizers of the invention can be any known in the
art, and optionally coupled to molecular carriers.
[0093] i) Porphyrins and Hydroporphyrins
[0094] Porphyrins and hydroporphyrins can include, but are not
limited to, Photofrin.RTM. (porfimer sodium), hematoporphyrin IX,
hematoporphyrin esters, dihematoporphyrin ester, synthetic
diporphyrins, O-substituted tetraphenyl porphyrins (picket fence
porphyrins), 3,1-meso tetrakis (o-propionamido phenyl) porphyrin,
hydroporphyrins, benzoporphyrin derivatives, benzoporphyrin
monoacid derivatives (BPD-MA), monoacid ring "a" derivatives,
tetracyanoethylene adducts of benzoporphyrin, dimethyl
acetylenedicarboxylate adducts of benzoporphyrin, endogenous
metabolic precursors, .delta.-aminolevulinic acid,
benzonaphthoporphyrazines, naturally occurring porphyrins,
ALA-induced protoporphyrin IX, synthetic dichlorins,
bacteriochlorins of the tetra(hydroxyphenyl) porphyrin series,
purpurins, tin and zinc derivatives of octaethylpurpurin,
etiopurpurin, tin-etio-purpurin, porphycenes, chlorins, chlorin
e.sub.6, mono-1-aspartyl derivative of chlorin e.sub.6,
di-1-aspartyl derivative of chlorin e.sub.6, tin(IV) chlorin
e.sub.6, meta-tetrahydroxyphenylchlorin, chlorin e.sub.6
monoethylendiamine monamide, verdins such as, but not limited to
zinc methyl pyroverdin (ZNMPV), copro II verdin trimethyl ester
(CVTME) and deuteroverdin methyl ester (DVME), pheophorbide
derivatives, and pyropheophorbide compounds, texaphyrins with or
without substituted lanthanides or metals, lutetium (III)
texaphyrin, and gadolinium(III) texaphyrin.
[0095] Porphyrins, hydroporphyrins, benzoporphyrins, and
derivatives are all related in structure to hematoporphyrin, a
molecule that is a biosynthetic precursor of heme, which is the
primary constituent of hemoglobin, found in erythrocytes.
First-generation and naturally occurring porphyrins are excited at
about 630 nm and have an overall low fluorescent quantum yield and
low efficiency in generating reactive oxygen species. Light at
about 630 nm can only penetrate tissues to a depth of about 3 mm,
however there are derivatives that have been `red-shifted` to
absorb at longer wavelengths, such as the benzoporphyrins BPD-MA
(Verteporfin). Thus, these `red-shifted` derivatives show less
collateral toxicity compared to first-generation porphyrins.
[0096] Chlorins and bacteriochlorins are also porphyrin
derivatives, however these have the unique property of hydrogenated
exo-pyrrole double bonds on the porphyrin ring backbone, allowing
for absorption at wavelengths greater than about 650 nm Chlorins
are derived from chlorophyll, and modified chlorins such as
meta-tetra hydroxyphenylchlorin (mTHPC) have functional groups to
increase solubility. Bacteriochlorins are derived from
photosynthetic bacteria and are further red-shifted to about 740
nm. A specific embodiment of the invention uses chlorin.sub.e6.
[0097] Purpurins, porphycenes, and verdins are also porphyrin
derivatives that have efficacies similar to or exceeding
hematoporphyrin. Purpurins contain the basic porphyrin macrocycle,
but are red-shifted to about 715 nm Porphycenes have similar
activation wavelengths to hematoporphyrin (about 635 nm), but have
higher fluorescence quantum yields. Verdins contain a cyclohexanone
ring fused to one of the pyrroles of the porphyrin ring. Phorbides
and pheophorbides are derived from chlorophylls and have 20 times
the effectiveness of hematoporphyrin. Texaphyrins are new
metal-coordinating expanded porphyrins. The unique feature of
texaphyrins is the presence of five, instead of four, coordinating
nitrogens within the pyrrole rings. This allows for coordination of
larger metal cations, such as trivalent lanthanides. Gadolinium and
lutetium are used as the coordinating metals. In a specific
embodiment, the photosensitizer can be Antrin.RTM., otherwise known
as motexafin lutetium.
[0098] 5-aminolevulinic acid (ALA) is a precursor in the heme
biosynthetic pathway, and exogenous administration of this compound
causes a shift in equilibrium of downstream reactions in the
pathway. In other words, the formation of the immediate precursor
to heme, protoporphyrin IX, is dependent on the rate of
5-aminolevulinic acid synthesis, governed in a negative-feedback
manner by concentration of free heme. Conversion of protoporphyrin
IX is slow, and where desired, administration of exogenous ALA can
bypass the negative-feedback mechanism and result in accumulation
of phototoxic levels of ALA-induced protoporphyrin IX. ALA is
rapidly cleared from the body, but like hematoporphyrin, has an
absorption wavelength of about 630 nm.
[0099] First-generation photosensitizers are exemplified by the
porphyrin derivative Photofrin.RTM., also known as porfimer sodium.
Photofrin.RTM. is derived from hematoporphyrin-IX by acid treatment
and has been approved by the Food and Drug Administration for use
in PDT. Photofrin.RTM. is characterized as a complex and
inseparable mixture of monomers, dimers, and higher oligomers.
There has been substantial effort in the field to develop pure
substances that can be used as successful photosensitizers. Thus,
in a specific embodiment, the photosensitizer is a benzoporphyrin
derivative ("BPD"), such as BPD-MA, also commercially known as
Verteporfin. U.S. Pat. No. 4,883,790 describes BPDs. Verteporfin
has been thoroughly characterized (Richter et al., 1987; Aveline et
al., 1994; Levy, 1994) and it has been found to be a highly potent
photosensitizer for PDT. Verteporfin has been used in PDT treatment
of certain types of macular degeneration, and is thought to
specifically target sites of new blood vessel growth, or
angiogenesis, such as those observed in "wet" macular degeneration.
Verteporfin is typically administered intravenously, with an
optimal incubation time range from 1.5 to 6 hours. Verteporfin
absorbs at 690 nm, and is activated with commonly available light
sources. One tetrapyrrole-based photosensitizer having recent
success in the clinic is MV0633 (Miravant).
[0100] In specific embodiments, the photosensitizer has a chemical
structure that includes multiple conjugated rings that allow for
light absorption and photoactivation. Such specific embodiments
include motexafin lutetium (Antrin.RTM.) and chlorin.sub.e6.
[0101] ii) Cyanine and Other Photoactive Dyes
[0102] Cyanine and other dyes include but are not limited to a
merocyanine, phthalocyanine, chloroaluminum phthalocyanine,
sulfonated aluminum PC, ring-substituted cationic PC, sulfonated
AlPc, disulfonated or tetrasulfonated derivative, sulfonated
aluminum naphthalocyanine, naphthalocyanine, tetracyanoethylene
adduct, crystal violet, azure .beta. chloride,
benzophenothiazinium, benzophenothiazinium chloride (EtNBS),
phenothiazine derivative, phenothiaziniums such as rose Bengal,
toluidine blue derivatives, toluidine blue O (TBO), methylene blue
(MB), new methylene blue N (NMMB), new methylene blue BB, new
methylene blue FR, 1,9-dimethylmethylene blue chloride (DMMB),
methylene blue derivatives, methylene green, methylene violet
Bernthsen, methylene violet 3RAX, Nile blue, Nile blue derivatives,
malachite green, Azure blue A, Azure blue B, Azure blue C, safranin
O, neutral red, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium
chloride, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium
chloride, thiopyronine, or thionine.
[0103] Cyanines are deep blue or purple compounds that are similar
in structure to porphyrins. However, these dyes are much more
stable to heat, light, and strong acids and bases than porphyrin
molecules. Cyanines, phthalocyanines, and naphthalocyanines are
chemically pure compounds that absorb light of longer wavelengths
than hematoporphyrin derivatives with absorption maxima at about
680 nm. Phthalocyanines, belonging to a new generation of
substances for PDT are chelated with a variety of diamagnetic
metals, chiefly aluminum and zinc, which enhance their
phototoxicity. A ring substitution of the phthalocyanines with
sulfonated groups will increase solubility and affect the cellular
uptake. Less sulfonated compounds, which are more lipophilic, show
the best membrane-penetrating properties and highest biological
activity. The kinetics are much more rapid than those of HPD,
where, for example, high tumor to tissue ratios (8:1) were observed
after 1-3 hours. The cyanines are eliminated rapidly and almost no
fluorescence can be seen in the tissue of interest after 24
hours.
[0104] Other photoactive dyes such as methylene blue and rose
bengal, are also used for photodynamic therapy. Methylene blue is a
phenothiazine cationic dye that is exemplified by its ability to
specifically target mitochondrial membrane potential. Rose-bengal
and fluorescein are xanthene dyes that are well documented in the
art for use in photodynamic therapy. Rose bengal diacetate is an
efficient, cell-permeant generator of singlet oxygen. It is an
iodinated xanthene derivative that has been chemically modified by
the introduction of acetate groups. These modifications inactivate
both its fluorescence and photosensitization properties, while
increasing its ability to cross cell membranes. Once inside the
cell, esterases remove the acetate groups and restore rose bengal
to its native structure. This intracellular localization allows
rose bengal diacetate to be a very effective photosensitizer.
[0105] iii) Other Photosensitizers
[0106] Diels-Alder adducts, dimethyl acetylene dicarboxylate
adducts, anthracenediones, anthrapyrazoles, aminoanthraquinone,
phenoxazine dyes, chalcogenapyrylium dyes such as cationic selena
and tellurapyrylium derivatives, cationic imminium salts, and
tetracyclines are other compounds that also exhibit photoactive
properties and can be used advantageously in photodynamic therapy.
Other photosensitizers that do not fall in either of the
aforementioned categories have other uses besides photodynamic
therapy, but are also photoactive. For example, anthracenediones,
anthrapyrazoles, aminoanthraquinone compounds are often used as
anticancer therapies (i.e. mitoxantrone, doxorubicin).
Chalcogenapyrylium dyes such as cationic selena- and
tellurapyrylium derivatives have also been found to exhibit
photoactive properties in the range of about 600 to about 900 nm
range, more preferably from about 775 to about 850 nm. In addition,
antibiotics such as tetracyclines and fluoroquinolone compounds
have demonstrated photoactive properties.
[0107] B. Linkers/Moieties Cleavable by .beta.-Lactamase
[0108] Linkers of the invention are capable of linking two
components of the photosensitizer composition together (e.g., a
photosensitizer to another photosensitizer, a photosensitizer to a
binder, a photosensitizer to a backbone, or a binder to a
backbone). Any bond which is capable of linking the components such
that they are stable under physiological conditions for the time
needed for administration, ingestion by the pest and activation
within the pest is suitable, but covalent linkages are preferred.
The link between two components may be direct, e.g., where a
photosensitizer is linked directly to another photosensitizer, or
indirect, e.g., where a photosensitizer is linked to an
intermediate, e.g., linked to a backbone, and that intermediate is
linked to another photosensitizer. A linker should function under
conditions of temperature, pH, salt, solvent system, and other
reactants that substantially retain the chemical stability of the
photosensitizer and the backbone (if present).
[0109] Linkers according to the invention comprise moieties
cleavable by a .beta.-lactamase enzyme. In one aspect of the
invention, linker cleavage by a .beta.-lactamase causes reduction
of the quenching that results from the conformation adopted by the
multiple photosensitizers linked to one another. In another aspect,
linker cleavage by .beta.-lactamase causes reduction of the
quenching that results from inclusion of a binder.
[0110] A linker can link components without the addition to the
linked components of elements of the linker. Other linkers result
in the addition of elements of the linker to the linked components.
For example, linkers can be cross-linking agents that are homo- or
hetero-bifunctional, and wherein one or more atomic components of
the agent can be retained in the composition.
[0111] Many linkers react with an amine and a carboxylate, to form
an amide, or an alcohol and a carboxylate to form an ester. Linkers
are known in the art, see, e.g., M. Bodansky, "Principles of
Peptide Synthesis", 2nd ed., referenced herein, and T. Greene and
P. Wuts, "Protective Groups in Organic Synthesis," 2nd Ed, 1991,
John Wiley, NY. Linkers should link component moieties stably, but
such that there is only minimal or no denaturation or deactivation
of the photosensitizer or other linked component.
[0112] The pesticidal compositions of the invention can be prepared
by linking the photosensitizers to one another or to other
components using methods known in the art. A variety of linkers,
including cross-linking agents, can be used for covalent
conjugation. Examples of cross-linking agents include
N,N'-dicyclohexylcarbodiimide (DCC),
N-succinimidyl-S-acetyl-thioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
ortho-phenylenedimaleimide (o-PDM), and sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC)
(Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M A et al.
(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include
those described by Paulus and Behring (1985) Ins. Mitt.,
78:118-132; Brennan et al. (1985) Science 229:81-83 and Glennie et
al., (1987) J. Immunol, 139:2367-2375. A large number of linkers
for peptides and proteins, along with buffers, solvents, and
methods of use, are described in the Pierce Chemical Co. catalog,
pages T-155 to T-200, 1994 (3747 N. Meridian Rd., Rockford Ill.,
61105, U.S.A.; Pierce Europe B.V., P.O. Box 1512, 3260 BA Oud
Beijerland, The Netherlands), the contents of which are hereby
incorporated by reference.
[0113] DCC is a useful linker (Pierce #20320; Rockland, Ill.). DCC
(N,N'-dicyclohexylcarbodiimide) is a carboxy-reactive cross-linker
commonly used as a linker in peptide synthesis. Another useful
cross-linking agent is SPDP (Pierce #21557), a heterobifunctional
cross-linker for use with primary amines and sulfhydryl groups.
SPDP produces cleavable cross-linking such that, upon further
reaction, the agent is eliminated, so the photosensitizer can be
linked directly to a backbone or molecular carrier. Other useful
linking agents are SATA (Pierce #26102) for introduction of blocked
SH groups for two-step cross-linking (Pierce #26103), and
sulfo-SMCC (Pierce #22322), reactive towards amines and
sulfhydryls. Other cross-linking and coupling agents are also
available from Pierce Chemical Co. (Rockford, Ill.).
[0114] Additional useful linking agents are hydrazines or hydrazine
derivatives, compounds that are very soluble in water and soluble
in alcohol. Hydrazines are corrosive and strong reducing agents,
though they constitute weaker bases than ammonia. Hydrazines are
dibasic and form many salts, e.g., mono- and di-hydrochlorides,
mono- and di-nitrates, and two sulfates. The hydrazine resin has
been found to be a novel and highly useful platform for polyamide
synthesis. The hydrazine resin is stable to elevated coupling
temperatures, yet is cleaved rapidly at moderate temperatures by a
wide range of nucleophiles following a mild and selective oxidation
protocol.
[0115] Additional compounds and processes, particularly those
involving a Schiff base as an intermediate, for conjugation of
proteins to other proteins or to other compositions, for example,
to reporter groups or to chelators for metal ion labeling of a
protein, are disclosed in EP 243,929 A2 (published Nov. 4,
1987).
[0116] Photosensitizers which contain carboxyl groups can be joined
to lysine s-amino groups in target polypeptides either by preformed
reactive esters (such as N-hydroxy succinimide ester) or esters
conjugated in situ by a carbodiimide-mediated reaction. The same
applies to photosensitizers that contain sulfonic acid groups,
which can be transformed to sulfonyl chlorides, which react with
amino groups. Photosensitizers that have carboxyl groups can be
joined to amino groups on the polypeptide by an in situ
carbodiimide method or by hydrazine or hydrazine derivatives.
Photosensitizers can also be attached to hydroxyl groups, of serine
or threonine residues or to sulfhydryl groups, of serine or
threonine residues or to sulfhydryl groups of cysteine
residues.
[0117] Methods of joining components of a composition can use
heterobifunctional cross linking reagents. These agents bind a
functional group in one chain and a different functional group in a
second chain. These functional groups typically are amino,
carboxyl, sulfhydryl, and aldehyde. There are many permutations of
appropriate moieties that will react with these groups and with
differently formulated structures, to join them together (described
in the Pierce Catalog and Merrifield et al. (1994) Ciba Found Symp.
186:5-20).
[0118] Generally, the photosensitizer compositions of the invention
can be prepared by linking the photosensitizer to another
photosensitizer, a binder and/or a backbone using methods described
in the following Examples or by methods known in the art. A variety
of linkers can be used for covalent conjugation.
[0119] Yield from linking reactions can be assessed by spectroscopy
of product eluting from a chromatographic fractionation in the
final step of purification. The presence of unlinked
photosensitizer and reaction products containing the
photosensitizer can be followed by the physical property that the
photosensitizer absorbs light at a characteristic wavelength and
extinction coefficient, so incorporation into products can be
monitored by absorbance at that wavelength or a similar wavelength.
Linking of one or more photosensitizer molecules to another or to a
binder or to a backbone shifts the peak of absorbance in the
elution profile in fractions eluted using sizing gel
chromatography, e.g., with the appropriate choice of Sephadex G50,
G100, or G200 or other such matrices (Pharmacia-Biotech, Piscataway
N.J.). Choice of appropriate sizing gel, for example Sephadex gel,
can be determined by that gel in which the photosensitizer elutes
in a fraction beyond the excluded volume of material too large to
interact with the bead, i.e., the uncoupled starting
photosensitizer composition interacts to some extent with the
fractionation bead and is concomitantly retarded to some
extent.
[0120] The correct useful gel can be predicted from the molecular
weight of the uncoupled photosensitizer. The successful reaction
products of photosensitizer compositions coupled to additional
moieties generally have characteristic higher molecular weights,
causing them to interact with the chromatographic bead to a lesser
extent, and thus appear in fractions eluting earlier than fractions
containing the uncoupled photosensitizer substrate. Unreacted
photosensitizer substrate generally appears in fractions
characteristic of the starting material, and the yield from each
reaction can thus be assessed both from size of the peak of larger
molecular weight material, and the decrease in the peak of
characteristic starting material. The area under the peak of the
product fractions is converted to the size of the yield using the
molar extinction coefficient.
[0121] The product can be analyzed using NMR, integrating areas of
appropriate product peaks, to determine relative yields with
different linkers. A red shift in absorption of a photosensitizer
of several nm has often been observed following coupling to a
polyamino acid. Linking to a larger moiety such as a protein might
produces a comparable shift, as linking to an antibody resulted in
a shift of about 3-5 nm in that direction compared to absorption of
the free photosensitizer. Relevant absorption maxima and extinction
coefficients in 0.1M NaOH/1% SDS are, for chlorin e6, 400 nm and
150,000 M.sup.-1, cm.sup.-1, and for benzoporphyrin derivative, 430
nm and 61,000 M.sup.-1, cm.sup.-1.
[0122] C. Binders
[0123] The binder may, without limitation, be a peptide, a cyclic
peptide, a polypeptide, a peptidomimetic, a protein, a fusion
protein, a hybrid molecule or a dimer, multimer, or a conjugate of
the above that binds or quenches, and, thus, may inhibit, suppress,
neutralize, or decrease activity of, the photosensitizer. The
binder may include, without limitation, a naturally occurring
inhibitor, a receptor, a soluble receptor, an antibody, a
polyclonal antibody, a monoclonal antibody, a bispecific antibody,
an antibody fragment, a single chain antibody, anti-idiotype
antibodies, a peptabody, a peptide, an oligopeptides, an
oligonucleotide, a cyclic peptide (i.e., a peptide that is circular
in nature), a peptide-lipid conjugate, a hormone, an antigen, an
epitope, a receptor, a chemokine, a nucleic acid, a ligand or a
dimer, multimer, or a conjugate of the above. Naturally occurring
binders are binders that quench the photosensitizer and are found
in nature.
[0124] In one aspect, the binder is a fluorophore. The property
that renders a fluorophore (or any other binder) a suitable
quencher is the capability of absorbing energy from the activated
photosensitizer.
[0125] Fluorophores of the present invention can be any known in
the art, including photosensitizers, fluorescent dyes, and
photoactive dyes.
[0126] Photosensitizers can be any known in the art, as previously
described. For example, hematoporphyrin derivatives have been used
as fluorescent probes to investigate the development of human
atherosclerotic plaques (Spokojny (1986) J. Am. Coll. Cardiol.
8:1387-1392). Ideally, the photosensitizer acting as a binder has a
different excitation wavelength than the photosensitizer acting to
produce a cytotoxic effect on the pathogen or host cell infected
with the pathogen.
[0127] Fluorescent dyes of the present invention can be any known
in the art, including, but not limited to
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl
ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein;
6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein;
5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether,
-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein
succinimidyl ester; 6-carboxyfluorescein succinimidyl ester;
5-(and-6)-carboxyfluorescein succinimidyl ester;
5-(4,6-dichlorotriazinyl) aminofluorescein;
2',7'-difluorofluorescein; eosin-5-isothiocyanate;
erythrosin-5-isothiocyanate; 6-(fluorescein-5-carboxamido) hexanoic
acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido)
hexanoic acid or succinimidyl ester; fluorescein-5-EX succinimidyl
ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate;
Oregon Green.RTM. 488 carboxylic acid, or succinimidyl ester;
Oregon Green.RTM. 488 isothiocyanate; Oregon Green.RTM. 488-X
succinimidyl ester; Oregon Green.RTM. 500 carboxylic acid; Oregon
Green.RTM. 500 carboxylic acid, succinimidyl ester or
triethylammonium salt; Oregon Green.RTM. 514 carboxylic acid;
Oregon Green.RTM. 514 carboxylic acid or succinimidyl ester;
Rhodamine Green.TM. carboxylic acid, succinimidyl ester or
hydrochloride; Rhodamine Green.TM. carboxylic acid,
trifluoroacetamide or succinimidyl ester; Rhodamine Green.TM.-X
succinimidyl ester or hydrochloride; Rhodol Green.TM. carboxylic
acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester;
bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl
ester); 5-(and-6)-carboxynaphthofluorescein,
5-(and-6)-carboxynaphthofluorescein succinimidyl ester;
5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G
hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester;
6-carboxyrhodamine 6G succinimidyl ester;
5-(and-6)-carboxyrhodamine 6G succinimidyl ester;
5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl
ester or bis-(diisopropylethylammonium) salt;
5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine;
5-(and-6)-carboxytetramethylrhodamine;
5-carboxytetramethylrhodamine succinimidyl ester;
6-carboxytetramethylrhodamine succinimidyl ester;
5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;
6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;
6-carboxy-X-rhodamine succinimidyl ester;
5-(and-6)-carboxy-X-rhodamine succinimidyl ester;
5-carboxy-X-rhodamine triethylammonium salt; Lissamine.TM.
rhodamine B sulfonyl chloride; malachite green isothiocyanate;
NANOGOLD.RTM. mono(sulfosuccinimidyl ester); QSY.RTM. 21 carboxylic
acid or succinimidyl ester; QSY.RTM. 7 carboxylic acid or
succinimidyl ester; Rhodamine Red.TM.-X succinimidyl ester;
6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid
succinimidyl ester; tetramethylrhodamine-5-isothiocyanate;
tetramethylrhodamine-6-isothiocyanate;
tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red.RTM.
sulfonyl; Texas Red.RTM. sulfonyl chloride; Texas Red.RTM.-X STP
ester or sodium salt; Texas Red.RTM.-X succinimidyl ester; Texas
Red.RTM.-X succinimidyl ester; and
X-rhodamine-5-(and-6)-isothiocyanate.
[0128] Fluorescent dyes of the present invention can also be, for
example, bodipy dyes commercially available from Molecular Probes,
including, but not limited to BODIPY.RTM. FL; BODIPY.RTM. TMR STP
ester; BODIPY.RTM. TR-X STP ester; BODIPY.RTM. 630/650-X STP ester;
BODIPY.RTM. 650/665-X STP ester;
6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propi-
onic acid succinimidyl ester;
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid succinimidyl ester;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid sulfosuccinimidyl ester or sodium salt;
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino)hexanoic acid;
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino)hexanoic acid or succinimidyl ester;
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cy-
steic acid, succinimidyl ester or triethylammonium salt;
6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino-
)hexanoic acid or succinimidyl ester;
4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3--
propionic acid succinimidyl ester;
4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styry-
loxy)acetyl)aminohexanoic acid or succinimidyl ester;
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propioni-
c acid;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-p-
ropionic acid succinimidyl ester;
4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza
s-indacene-3-yl)phenoxy)acetyl) amino)hexanoic acid or succinimidyl
ester; and
6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryl-
oxy)acetyl)aminohexanoic acid or succinimidyl ester.
[0129] Fluorescent dyes the present invention can also be, for
example, alexa fluor dyes commercially available from Molecular
Probes, including but not limited to Alexa Fluor.RTM. 350
carboxylic acid; Alexa Fluor.RTM. 430 carboxylic acid; Alexa
Fluor.RTM. 488 carboxylic acid; Alexa Fluor.RTM. 532 carboxylic
acid; Alexa Fluor.RTM. 546 carboxylic acid; Alexa Fluor.RTM. 555
carboxylic acid; Alexa Fluor.RTM. 568 carboxylic acid; Alexa
Fluor.RTM. 594 carboxylic acid; Alexa Fluor.RTM. 633 carboxylic
acid; Alexa Fluor.RTM. 647 carboxylic acid; Alexa Fluor.RTM. 660
carboxylic acid; and Alexa Fluor.RTM. 680 carboxylic acid.
[0130] Fluorescent dyes the present invention can also be, for
example, cy dyes commercially available from Amersham-Pharmacia
Biotech, including, but not limited to Cy3 NHS ester; Cy 5 NHS
ester; Cy5.5 NHS ester; and Cy 7 NHS ester.
[0131] Photoactive dyes of the present invention can be any
photosensitizer known in the art which will fluoresce but not
necessarily produce a reactive species in phototoxic amounts when
illuminated. Depending on the wavelength and power of light
administered, a photosensitizer can be activated to fluoresce and,
therefore, act as a photoactive dye, but not produce a phototoxic
effect unless, in some cases, the wavelength and power of light is
suitably adapted to induce a phototoxic effect.
[0132] Throughout this specification, any reference to a binder
should be construed to refer to each of the binders identified and
contemplated herein and to each biologically equivalent molecule.
"Biologically equivalent" means compositions of the present
invention which are capable of preventing action of the
photosensitizer in a similar fashion, but not necessarily to the
same degree.
[0133] D. Backbones
[0134] Pesticidal compositions according to the invention include
those in which a "backbone" moiety, such as a polyamino acid, is
linked to a photosensitizer and/or to a binder.
[0135] Inclusion of a backbone in a composition with a
photosensitizer and/or binder can provide a number of advantages,
including the provision of greater stoichiometric ranges of
photosensitizers and/or binders and/or targeting moieties coupled
per backbone. If the backbone possesses intrinsic affinity for a
target pest, the affinity of the composition can be enhanced by
coupling to the backbone.
[0136] Peptides useful in the methods and compounds of the
invention for design and characterization of backbone moieties
include poly-amino acids which can be homo- and hetero-polymers of
L-, D-, racemic DL- or mixed L- and D-amino acid composition, and
which can be of defined or random mixed composition and sequence.
Examples of naturally-occurring peptides with mixed D and L amino
acid residues include bacitracin and tyrocidin. These peptides may
be modeled after particular natural peptides, and optimized by the
technique of phage display and selection for enhanced binding to a
chosen target, so that the selected peptide of highest affinity is
characterized and then produced synthetically.
[0137] Further modifications of functional groups can be introduced
for purposes, for example, of increased solubility, decreased
aggregation, and altered extent of hydrophobicity. Examples of
non-peptide backbones include nucleic acids and derivatives of
nucleic acids such as DNA, RNA and peptide nucleic acids;
polysaccharides and derivatives such as starch, pectin, chitins,
celluloses and hemi-methylated celluloses; lipids such as
triglyceride derivatives and cerebrosides; synthetic polymers such
as polyethylene glycols (PEGs) and PEG star polymers; dextran
derivatives, polyvinyl alcohols, N-(2-hydroxypropyl)-methacrylamide
copolymers, poly (DL-glycolic acid-lactic acid); and compositions
containing elements of any of these classes of compounds.
III. PESTS AND ADMINISTRATION OF THE PESTICIDAL COMPOSITIONS OF THE
INVENTION
[0138] A) Plant and Plant Organs
[0139] The pesticidal compositions are suitable for protecting
plants and plant organs, for increasing the harvest yields, for
improving the quality of the harvested goods and for controlling
animal pests, in particular insects, arachnids and nematodes, which
are encountered in agriculture, in forests, in gardens and leisure
facilities, in the protection of stored products and of materials,
and in the hygiene sector, and have good plant tolerance, favorable
toxicity to warm-blooded animals and good environmental
compatibility. They may preferably be employed as crop protection
agents.
[0140] In certain embodiments, the pesticidal compositions
according to the invention can be used, for example, and without
limitation, to treat propagation material such as tubers or
rhizomes, but also seeds, seedlings or seedlings pricking out and
plants or plants pricking out. These methods of treatment can also
be useful to treat roots. The methods of treatment according to the
invention can also be useful to treat the overground parts of the
plant such as trunks, stems or stalks, leaves, flowers and fruit of
the concerned plant.
[0141] Plants in accordance with the invention include, but are not
limited to corn; tobacco; cotton; soybean; sugarcane; hay; sorghum;
kales; cabbages; flax; vine; fruit or vegetable crops such as
Rosaceae sp. (for instance pip fruit such as apples and pears, but
also stone fruit such as apricots, almonds and peaches),
Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae
sp., Fagaceae sp., Moraceae sp., Oleaceae sp., Actimidaceae sp.,
Lauraceae sp., Musaceae sp. (for instance banana trees and
plantains), Rubiaceae sp., Theaceae sp., Sterculiceae sp., Rutaceae
sp. (for instance lemons, oranges and grapefruit); Solanaceae sp.
(for instance tomatoes), Liliaceae sp., Asteraceae sp. (for
instance lettuces), Umbelliferae sp., Cruciferae sp.,
Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp. (for
instance peas), Rosaceae sp. (for instance strawberries); major
crops such as Graminae sp. (for instance maize, lawn or cereals
such as wheat, rice, barley and triticale), Asteraceae sp. (for
instance sunflower), Cruciferae sp. (for instance colza), Fabacae
sp. (for instance peanuts), Papilionaceae sp. (for instance
soybean), Solanaceae sp. (for instance potatoes), Chenopodiaceae
sp. (for instance beetroots); horticultural and forest crops,
including flowers; as well as genetically modified homologues of
these crops.
[0142] In certain embodiments, the pesticidal compositions
according to the invention can be also used, for example, and
without limitation, in connection with the following plants: [0143]
Dicotyledonous weeds of the genera: Abutilon, Amaranthus, Ambrosia,
Anoda, Anthemis, Aphanes, Atriplex, Bellis, Bidens, Capsella,
Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolvulus,
Datura, Desmodium, Emex, Erysimum, Euphorbia, Galeopsis, Galinsoga,
Galium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia,
Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver,
Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus,
Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis,
Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi,
Trifolium, Urtica, Veronica, Viola, Xanthium. Dicotyledonous crops
of the genera: Arachis, Beta, Brassica, Cucumis, Cucurbita,
Helianthus, Daucus, Glycine, Gossypium, Ipomoea, Lactuca, Linum,
Lycopersicon, Nicotiana, Phaseolus, Pisum, Solanum, Vicia. [0144]
Monocotyledonous weeds of the genera: Aegilops, Agropyron,
Agrostis, Alopecurus, Apera, Avena, Brachiaria, Bromus, Cenchrus,
Commelina, Cynodon, Cyperus, Dactyloctenium, Digitaria,
Echinochloa, Eleocharis, Eleusine, Eragrostis, Eriochloa, Festuca,
Fimbristylis, Heteranthera, Imperata, Ischaemum, Leptochloa,
Lolium, Monochoria, Panicum, Paspalum, Phalaris, Phleum, Poa,
Rottboellia, Sagittaria, Scirpus, Setaria, Sorghum. [0145]
Monocotyledonous crops of the genera: Allium, Ananas, Asparagus,
Avena, Hordeum, Oryza, Panicum, Saccharum, Secale, Sorghum,
Triticale, Triticum, Zea.
[0146] B) Formulation
[0147] i) For Use in Plants
[0148] The pesticidal compositions of the invention can be used as
such, in the form of their formulations or in the use forms
prepared therefrom by further dilution, such as ready-to-use
solutions, suspensions, emulsions, powders, pastes and granules.
They are used in a customary manner, for example by watering,
spraying, atomizing or broadcasting.
[0149] The pesticidal compositions of the invention can be applied
both before and after emergence of the plants. They can also be
incorporated into the soil before sowing.
[0150] All plants and plant parts can be treated in accordance with
the invention. Plants are to be understood as meaning in the
present context all plants and plant populations such as desired
and undesired wild plants or crop plants (including naturally
occurring crop plants). Crop plants can be plants which can be
obtained by conventional plant breeding and optimization methods or
by biotechnological and recombinant methods or by combinations of
these methods, including the transgenic plants and inclusive of the
plant cultivars protectable or not protectable by plant breeders'
rights. Plant parts are to be understood as meaning all parts and
organs of plants above and below the ground, such as shoot, leaf,
flower and root, examples which may be mentioned being leaves,
needles, stalks, stems, flowers, fruit bodies, fruits, seeds,
roots, tubers and rhizomes. The plant parts also include harvested
material, and vegetative and generative propagation material, for
example cuttings, tubers, rhizomes, offsets and seeds.
[0151] Treatment according to the invention of the plants and plant
parts with the pesticidal compositions is carried out directly or
by allowing the compounds to act on their surroundings, environment
or storage space by the customary treatment methods, for example by
immersion, spraying, evaporation, fogging, scattering, painting on
and, in the case of propagation material, in particular in the case
of seeds, also by applying one or more coats.
[0152] The pesticidal compositions can be converted to the
customary formulations, such as solutions, emulsions, wettable
powders, suspensions, powders, dusts, pastes, soluble powders,
granules, suspension-emulsion concentrates, natural and synthetic
materials impregnated with active compound and microencapsulations
in polymeric substances.
[0153] These formulations are produced in a known manner, for
example by mixing the pesticidal compositions with extenders, that
is, liquid solvents, and/or solid carriers, optionally with the use
of surfactants, that is emulsifiers and/or dispersants, and/or
foam-formers.
[0154] If the extender used is water, it is also possible to employ
for example organic solvents as auxiliary solvents. Essentially,
suitable liquid solvents are: aromatics such as xylene, toluene or
alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic
hydrocarbons such as chlorobenzenes, chloroethylenes or methylene
chloride, aliphatic hydrocarbons such as cyclohexane or paraffins,
for example petroleum fractions, mineral and vegetable oils,
alcohols such as butanol or glycol and also their ethers and
esters, ketones such as acetone, methyl ethyl ketone, methyl
isobutyl ketone or cyclohexanone, strongly polar solvents such as
dimethylformamide and dimethyl sulphoxide, and also water.
[0155] Suitable Solid Carriers are: for example ammonium salts and
ground natural minerals such as kaolins, clays, talc, chalk,
quartz, attapulgite, montmorillonite or diatomaceous earth, and
ground synthetic minerals, such as highly disperse silica, alumina
and silicates; suitable solid carriers for granules are: for
example crushed and fractionated natural rocks such as calcite,
marble, pumice, sepiolite and dolomite, and also synthetic granules
of inorganic and organic meals, and granules of organic material
such as sawdust, coconut shells, maize cobs and tobacco stalks;
suitable emulsifiers and/or foam-formers are: for example nonionic
and anionic emulsifiers, such as polyoxyethylene fatty acid esters,
polyoxyethylene fatty alcohol ethers, for example alkylaryl
polyglycol ethers, alkylsulphonates, alkyl sulphates,
arylsulphonates and also protein hydrolysates; suitable dispersants
are: for example lignin-sulphite waste liquors and
methylcellulose.
[0156] Tackifiers such as carboxymethylcellulose and natural and
synthetic polymers in the form of powders, granules or latices,
such as gum arabic, polyvinyl alcohol and polyvinyl acetate, as
well as natural phospholipids such as cephalins and lecithins, and
synthetic phospholipids, can be used in the formulations. Other
possible additives are mineral and vegetable oils.
[0157] It is possible to use colorants such as inorganic pigments,
for example iron oxide, titanium oxide and Prussian Blue, and
organic dyestuffs, such as alizarin dyestuffs, azo dyestuffs and
metal phthalocyanine dyestuffs, and trace nutrients such as salts
of iron, manganese, boron, copper, cobalt, molybdenum and zinc.
[0158] The pesticidal compositions of the invention can be used in
the form of concentrates or generally customary formulations, such
as powders, granules, solutions, suspensions, emulsions or
pastes.
[0159] The formulations mentioned can be prepared in a manner known
per se, for example by mixing the active compounds with at least
one solvent or diluent, emulsifier, dispersing agent and/or binder
or fixing agent, a water repellent, if appropriate siccatives and
UV stabilizers and if appropriate dyestuffs and pigments, and also
other processing auxiliaries.
[0160] ii.) For Household or Industrial Use
[0161] In the field of household or industrial use, the pesticidal
compositions of the invention are used alone or in combination with
other suitable active compounds, such as phosphoric acid esters,
carbamates, pyrethroids, neonicotinoides, growth regulators or
active compounds from other known classes of pesticides.
[0162] The pesticidal compositions of the invention can be used in
aerosols, pressure-free spray products, for example pump and
atomizer sprays, automatic fogging systems, foggers, foams, gels,
evaporator products with evaporator tablets made of cellulose or
polymer, liquid evaporators, gel and membrane evaporators,
propeller-driven evaporators, energy-free or passive evaporation
systems, moth papers, moth bags and moth gels, as granules or
dusts, in baits for spreading or in bait stations.
[0163] iii.) For Antifouling Use
[0164] Using the compounds according to the invention as an
antifouling agent, alone or in combination with other active
compounds, allows the use of heavy metals such as, for example, in
bis-(trialkyltin) sulphides, tri-n-butyltin laurate, tri-n-butyltin
chloride, copper(I) oxide, triethyltin chloride,
tri-n-butyl(2-phenyl-4-chlorophenoxy)tin, tributyltin oxide,
molybdenum disulphide, antimony oxide, polymeric butyl titanate,
phenyl-(bispyndine)-bismuth chloride, tri-n-butyltin fluoride,
manganese ethylenebisthio-carbamate, zinc dimethyldithiocarbamate,
zinc ethylenebisthiocarbamate, zinc salts and copper salts of
2-pyridinethiol 1-oxide, bisdimethyldithiocarbamoylzinc
ethylene-bisthiocarbamate, zinc oxide, copper(I)
ethylene-bisdithiocarbamate, copper thiocyanate, copper naphthenate
and tributyltin halides to be dispensed with, or the concentration
of these compounds substantially reduced.
[0165] The pesticidal compositions can be used to control fouling
by mixing the compositions into ready-to-use antifouling paints If
appropriate, the ready-to-use antifouling paints can additionally
comprise other active compounds, preferably algicides, fungicides,
herbicides, molluscicides, or other antifouling active
compounds.
The antifouling compositions used comprise the active compound
according to the invention of the compounds according to the
invention in a concentration of 0.001 to 50% by weight, in
particular 0.01 to 20% by weight. Moreover, the antifouling
compositions according to the invention comprise the customary
components such as, for example, those described in Ungerer, Chem.
Ind. 1985, 37, 730-732 and Williams, Antifouling Marine Coatings,
Noyes, Park Ridge, 1973.
[0166] Antifouling paints can further comprise, in particular,
binders. Examples of recognized binders are polyvinyl chloride in a
solvent system, chlorinated rubber in a solvent system, acrylic
resins in a solvent system, in particular in an aqueous system,
vinyl chloride/vinyl acetate copolymer systems in the form of
aqueous dispersions or in the form of organic solvent systems,
butadiene/styrene/acrylonitrile rubbers, drying oils such as
linseed oil, resin esters or modified hardened resins in
combination with tar or bitumens, asphalt and epoxy compounds,
small amounts of chlorine rubber, chlorinated polypropylene and
vinyl resins.
[0167] iv.) For Veterinary Use
[0168] In the field of veterinary uses, the pesticidal compositions
of the invention can be used in a known manner by enteral
administration in the form of, for example, tablets, capsules,
potions, drenches, granules, pastes, boluses, the feed-through
process and suppositories, by parenteral administration, such as,
for example, by injection (intramuscular, subcutaneous,
intravenous, intraperitoneal and the like), implants, by nasal
administration, by dermal use in the form, for example, of dipping
or bathing, spraying, pouring on and spotting on, washing and
powdering, and also with the aid of moulded articles containing the
active compound, such as collars, ear marks, tail marks, limb
bands, halters, marking devices and the like.
[0169] v.) For Use in Humans
[0170] In humans, a therapeutically effective amount of a
composition of the invention can be administered in one or more
doses. An effective amount is an amount that is sufficient to
palliate, ameliorate, reduce, stabilize, reverse or slow the
progression of a pest infestation, such as a parasite infestation,
or tick infestation. A therapeutically effective amount can be
provided in one or a series of administrations. The effective
amount is generally determined by the physician on a case-by-case
basis and is within the skill of one in the art.
[0171] As a rule, the dosage for in vivo therapeutics or
diagnostics will vary. Several factors are typically taken into
account when determining an appropriate dosage. These factors
include age, sex and weight of the patient, the condition being
treated, the severity of the condition and the form of the
nanoparticle being administered.
[0172] Compositions of the present invention may be administered by
a mode appropriate for the form of composition. Available routes of
administration include subcutaneous, intramuscular,
intraperitoneal, intradermal, oral, intranasal, intrapulmonary
(i.e., by aerosol), intravenously, intramuscularly, subcutaneously,
intracavity, intrathecally or transdermally, alone or in
combination with nanoparticle compositions. Therapeutic
nanoparticle compositions (e.g., a nanoparticle containing a
photosensitizer core, a polymer shell, and a targeting aptamer
fixed to the surface of the shell in an appropriate excipient) are
often administered by injection or by gradual perfusion.
[0173] Compositions for oral, intranasal, or topical administration
can be supplied in solid, semi-solid or liquid forms, including
tablets, capsules, powders, liquids, and suspensions. Compositions
for injection can be supplied as liquid solutions or suspensions,
as emulsions, or as solid forms suitable for dissolution or
suspension in liquid prior to injection. For administration via the
respiratory tract, a preferred composition is one that provides a
solid, powder, or liquid aerosol when used with an appropriate
aerosolizer device. Although not required, compositions are
preferably supplied in unit dosage form suitable for administration
of a precise amount. Also contemplated by this invention are slow
release or sustained release forms, whereby a relatively consistent
level of the active compound are provided over an extended
period.
[0174] Another method of administration is intralesionally, for
instance by direct injection directly into the site of pest
infestation. Intralesional administration of various forms are
useful in that they do not cause the toxicity seen with systemic
administration of immunologic agents (Fletcher and Goldstein,
1987), (Rabinowich et al., 1987), (Rosenberg et al., 1986), (Pizza
et al., 1984).
[0175] C) Pests
[0176] i. Plant Pests
[0177] The pesticidal compositions are active against normally
sensitive and resistant species and against all or some stages of
development. The abovementioned pests include, but are not limited
to: [0178] From the order of the Isopoda, for example, Oniscus
asellus, Armadillidium vulgare and Porcellio scaber. [0179] From
the order of the Diplopoda, for example, Blaniulus guttulatus.
[0180] From the order of the Chilopoda, for example, Geophilus
carpophagus and Scutigera spp. [0181] From the order of the
Symphyla, for example, Scutigerella immaculata. [0182] From the
order of the Thysanura, for example, Lepisma saccharina. [0183]
From the order of the Collembola, for example, Onychiurus armatus.
[0184] From the order of the Orthoptera, for example, Acheta
domesticus, Gryllotalpa spp., Locusta migratoria migratorioides,
Melanoplus spp. and Schistocerca gregaria. [0185] From the order of
the Blattaria, for example, Blatta orientalis, Periplaneta
americana, Leucophaea maderae, Blattella germanica. [0186] From the
order of the Dermaptera, for example, Forficula auricularia. [0187]
From the order of the Isoptera, for example, Reticulitermes spp.
[0188] From the order of the Phthiraptera, for example, Pediculus
humanus corporis, Haematopinus spp., Linognathus spp., Trichodectes
spp. and Damalinia spp. [0189] From the order of the Thysanoptera,
for example, Hercinothrips femoralis, Thrips tabaci, Thrips palmi
and Frankliniella accidentalis. [0190] From the order of the
Heteroptera, for example, Eurygaster spp., Dysdercus intermedius,
Piesma quadrata, Cimex lectularius, Rhodnius prolixus and Triatoma
spp. [0191] From the order of the Homoptera, for example, Aleurodes
brassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis
gossypii, Brevicoryne brassicae, Cryptomyzus ribis, Aphis fabae,
Aphis pomi, Eriosoma lanigerum, Hyalopterus arundinis, Phylloxera
vastatrix, Pemphigus spp., Macrosiphum avenae, Myzus spp., Phorodon
humuli, Rhopalosiphum padi, Empoasca spp., Euscelis bilobatus,
Nephotettix cincticeps, Lecanium corni, Saissetia oleae, Laodelphax
striatellus, Nilaparvata lugens, Aonidiella aurantii, Aspidiotus
hederae, Pseudococcus spp. and Psylla spp. [0192] From the order of
the Lepidoptera, for example, Pectinophora gossypiella, Bupalus
piniarius, Chematobia brumata, Lithocolletis blancardella,
Hyponomeuta padella, Plutella xylostella, Malacosoma neustria,
Euproctis chrysorrhoea, Lymantria spp., Bucculatrix thurberiella,
Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltia spp.,
Earias insulana, Heliothis spp., Mamestra brassicae, Panolis
flammea, Spodoptera spp., Trichoplusia ni, Carpocapsa pomonella,
Pieris spp., Chilo spp., Pyrausta nubilalis, Ephestia kuehniella,
Galleria mellonella, Tineola bisselliella, Tinea pellionella,
Hofmannophila pseudospretella, Cacoecia podana, Capua reticulana,
Choristoneura fumiferana, Clysia ambiguella, Homona magnanima,
Tortrix viridana, Cnaphalocerus spp., Oulema oryzae. [0193] From
the order of the Coleoptera, for example, Anobium punctatum,
Rhizopertha dominica, Bruchidius obtectus, Acanthoscelides
obtectus, Hylotrupes bajulus, Agelastica alni, Leptinotarsa
decemlineata, Phaedon cochleariae, Diabrotica spp., Psylliodes
chrysocephala, Epilachna varivestis, Atomaria spp., Oryzaephilus
surinamensis, Anthonomus spp., Sitophilus spp., Otiorrhynchus
sulcatus, Cosmopolites sordidus, Ceuthorrhynchus assimilis, Hypera
postica, Dermestes spp., Trogoderma spp., Anthrenus spp., Attagenus
spp., Lyctus spp., Meligethes aeneus, Ptinus spp., Niptus
Hololeucus, Gibbium Psylloides, Tribolium Spp., Tenebrio molitor,
Agriotes spp., Conoderus spp., Melolontha melolontha, Amphimallon
solstitialis, Costelytra zealandica and Lissorhoptrus oryzophilus.
[0194] From the order of the Hymenoptera, for example, Diprion
spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis and Vespa
spp. [0195] From the order of the Diptera, for example, Aedes spp.,
Anopheles spp., Culex spp., Drosophila melanogaster, Musca spp.,
Fannia spp., Calliphora erythrocephala, Lucilia spp., Chrysomyia
spp., Cuterebra spp., Gastrophilus spp., Hyppobosca spp., Stomoxys
spp., Oestrus spp., Hypoderma spp., Tabanus spp., Tannia spp.,
Bibio hortulanus, Oscinella frit, Phorbia spp., Pegomyia hyoscyami,
Ceratitis capitata, Dacus oleae, Tipula paludosa, Hylemyia spp. and
Liriomyza spp. [0196] From the order of the Siphonaptera, for
example, Xenopsylla cheopis and Ceratophyllus spp. [0197] From the
class of the Arachnida, for example, Scorpio maurus, Latrodectus
mactans, Acarus siro, Argas spp., Ornithodoros spp., Dermanyssus
gallinae, Eriophyes ribis, Phyllocoptruta oleivora, Boophilus spp.,
Rhipicephalus spp., Amblyomma spp., Hyalomma spp., Ixodes spp.,
Psoroptes spp., Chorioptes spp., Sarcoptes spp., Tarsonemus spp.,
Bryobia praetiosa, Panonychus spp., Tetranychus spp.,
Hemitarsonemus spp., Brevipalpus spp.
[0198] ii. Phytoparasitic Nematodes
[0199] The pesticidal compositions of the invention are active act
against phytoparasitic nematodes including, but are not limited
to:
[0200] Pratylenchus spp., Radopholus similis, Ditylenchus dipsaci,
Tylenchulus semipenetrans, Heterodera spp., Globodera spp.,
Meloidogyne spp., Aphelenchoides spp., Longidorus spp., Xiphinema
spp., Trichodorus spp., Bursaphelenchus spp.
[0201] iii. Veterinary and Livestock Pests
[0202] The pesticidal compositions of the invention are also useful
in the veterinary medicine sector against animal parasites
(ectoparasites), such as hard ticks, soft ticks, mange mites,
harvest mites, flies (biting and licking), parasitic fly larvae,
lice, hair lice, feather lice and fleas. These parasites include,
but are not limited to: [0203] From the order of the Anoplurida,
for example, Haematopinus spp., Linognathus spp., Pediculus spp.,
Phtirus spp. and Solenopotes spp. [0204] From the order of the
Mallophagida and the suborders Amblycerina and Ischnocerina, for
example, Trimenopon spp., Menopon spp., Trinoton spp., Bovicola
spp., Werneckiella spp., Lepikentron spp., Damalina spp.,
Trichodectes spp. and Felicola spp. [0205] From the order of the
Diptera and the suborders Nematocerina and Brachycerina, for
example, Aedes spp., Anopheles spp., Culex spp., Simulium spp.,
Eusimulium spp., Phlebotomus spp., Lutzomyia spp., Culicoides spp.,
Chrysops spp., Hybomitra spp., Atylotus spp., Tabanus spp.,
Haematopota spp., Philipomyia spp., Braula spp., Musca spp.,
Hydrotaea spp., Stomoxys spp., Haematobia spp., Morellia spp.,
Fannia spp., Glossina spp., Calliphora spp., Lucilia spp.,
Chrysomyia spp., Wohlfahrtia spp., Sarcophaga spp., Oestrus spp.,
Hypoderma spp., Gasterophilus spp., Hippobosca spp., Lipoptena spp.
and Melophagus spp. [0206] From the order of the Siphonapterida,
for example Pulex spp., Ctenocephalides spp., Xenopsylla spp. and
Ceratophyllus spp. [0207] From the order of the Heteropterida, for
example, Cimex spp., Triatoma spp., Rhodnius spp. and Panstrongylus
spp. [0208] From the order of the Blattarida, for example Blatta
orientalis, Periplaneta americana, Blattela germanica and Supella
spp. [0209] From the subclass of the Acaria (Acarina) and the
orders of the Meta- and Mesostigmata, for example, Argas spp.,
Ornithodorus spp., Otobius spp., Ixodes spp., Amblyomma spp.,
Boophilus spp., Dermacentor spp., Haemophysalis spp., Hyalomma
spp., Rhipicephalus spp., Dermanyssus spp., Raillietia spp.,
Pneumonyssus spp., Sternostoma spp. and Varroa spp. [0210] From the
order of the Actinedida (Prostigmata) and Acaridida (Astigmata),
for example, Acarapis spp., Cheyletiella spp., Ornithocheyletia
spp., Myobia spp., Psorergates spp., Demodex spp., Trombicula spp.,
Listrophorus spp., Acarus spp., Tyrophagus spp., Caloglyphus spp.,
Hypodectes spp., Pterolichus spp., Psoroptes spp., Chorioptes spp.,
Otodectes spp., Sarcoptes spp., Notoedres spp., Knemidocoptes spp.,
Cytodites spp. and Laminosioptes spp.
[0211] The compositions of the invention are also suitable for
controlling arthropods which infest agricultural productive
livestock, such as, for example, cattle, sheep, goats, horses,
pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks,
geese and bees, other pets, such as, for example, dogs, cats, caged
birds and aquarium fish, and also so-called test animals, such as,
for example, hamsters, guinea pigs, rats and mice. By controlling
these arthropods, cases of death and reduction in productivity (for
meat, milk, wool, hides, eggs, honey etc.) should be diminished, so
that more economic and easier animal husbandry is possible by use
of the active compounds according to the invention.
[0212] iv. Industrial Pests
[0213] The pesticidal compositions of the invention are also useful
against insects which destroy industrial materials.
[0214] These pests include, but are not limited to: [0215] Beetles,
such as Hylotrupes bajulus, Chlorophorus pilosis, Anobium
punctatum, Xestobium rufovillosum, Ptilinus pecticornis, Dendrobium
pertinex, Ernobius mollis, Priobium carpini, Lyctus brunneus,
Lyctus africanus, Lyctus planicollis, Lyctus linearis, Lyctus
pubescens, Trogoxylon aequale, Minthes rugicollis, Xyleborus spec.
Tryptodendron spec. Apate monachus, Bostrychus capucins,
Heterobostrychus brunneus, Sinoxylon spec. Dinoderus minutus.
[0216] Hymenopterons, such as Sirex juvencus, Urocerus gigas,
Urocerus gigas taignus, Urocerus augur. [0217] Termites, such as
Kalotermes flavicollis, Cryptotermes brevis, Heterotermes indicola,
Reticulitermes flavipes, Reticulitermes santonensis, Reticulitermes
lucifugus, Mastotermes darwiniensis, Zootermopsis nevadensis,
Coptotermes formosanus. Bristletails, such as Lepisma
saccharina.
[0218] Industrial materials in the present connection are to be
understood as meaning non-living materials, such as, preferably,
plastics, adhesives, sizes, paper and card, leather, wood and
processed wood products and coating compositions.
[0219] Wood and processed wood products are materials to be
protected, especially preferably, from insect infestation.
[0220] Wood and processed wood products which can be protected by
the agents according to the invention or mixtures comprising these
are to be understood as meaning, for example: building timber,
wooden beams, railway sleepers, bridge components, boat jetties,
wooden vehicles, boxes, pallets, containers, telegraph poles, wood
paneling, wooden windows and doors, plywood, chipboard, joinery or
wooden products which are used quite generally in house-building or
in building joinery.
[0221] v. Enclosed Spaces/Household Pests
[0222] The pesticidal compositions of the invention are also
suitable for controlling animal pests, in particular insects,
arachnids and mites, which are found in enclosed spaces such as,
for example, dwellings, factory halls, offices, vehicle cabins and
the like. They can be employed alone or in combination with other
active compounds and auxiliaries in domestic pesiticide products
for controlling these pests. They are active against sensitive and
resistant species and against all developmental stages. These pests
include, but are not limited to: [0223] From the order of the
Scorpionidea, for example, Buthus occitanus. [0224] From the order
of the Acarina, for example, Argas persicus, Argas reflexus,
Bryobia ssp., Dermanyssus gallinae, Glyciphagus domesticus,
Ornithodorus moubat, Rhipicephalus sanguineus, Trombicula
alfreddugesi, Neutrombicula autumnalis, Dermatophagoides
pteronissimus, Dermatophagoides forinae. [0225] From the order of
the Araneae, for example, Aviculariidae, Araneidae. [0226] From the
order of the Opiliones, for example, Pseudoscorpiones chelifer,
Pseudoscorpiones cheiridium, Opiliones phalangium. [0227] From the
order of the Isopoda, for example, Oniscus asellus, Porcellio
scaber. [0228] From the order of the Diplopoda, for example,
Blaniulus guttulatus, Polydesmus spp. [0229] From the order of the
Chilopoda, for example, Geophilus spp. [0230] From the order of the
Zygentoma, for example, Ctenolepisma spp., Lepisma saccharina,
Lepismodes inquilinus. [0231] From the order of the Blattaria, for
example, Blatta orientalies, Blattella germanica, Blattella
asahinai, Leucophaea maderae, Panchlora spp., Parcoblatta spp.,
Periplaneta australasiae, Periplaneta americana, Periplaneta
brunnea, Periplaneta fuliginosa, Supella longipalpa. [0232] From
the order of the Saltatoria, for example, Acheta domesticus. [0233]
From the order of the Dermaptera, for example, Forficula
auricularia. [0234] From the order of the Isoptera, for example,
Kalotermes spp., Reticulitermes spp. [0235] From the order of the
Psocoptera, for example, Lepinatus spp., Liposcelis spp. [0236]
From the order of the Coleptera, for example, Anthrenus spp.,
Attagenus spp., Dermestes spp., Latheticus oryzae, Necrobia spp.,
Ptinus spp., Rhizopertha dominica, Sitophilus granarius, Sitophilus
oryzae, Sitophilus zeamais, Stegobium paniceum. [0237] From the
order of the Diptera, for example, Aedes aegypti, Aedes albopictus,
Aedes taeniorhynchus, Anopheles spp., Calliphora erythrocephala,
Chrysozona pluvialis, Culex quinquefasciatus, Culex pipiens, Culex
tarsalis, Drosophila spp., Fannia canicularis, Musca domestica,
Phlebotomus spp., Sarcophaga carnaria, Simulium spp., Stomoxys
calcitrans, Tipula paludosa. [0238] From the order of the
Lepidoptera, for example, Achroia grisella, Galleria mellonella,
Plodia interpunctella, Tinea cloacella, Tinea pellionella, Tineola
bisselliella. [0239] From the order of the Siphonaptera, for
example, Ctenocephalides canis, Ctenocephalides felis, Pulex
irritans, Tunga penetrans, Xenopsylla cheopis. [0240] From the
order of the Hymenoptera, for example, Camponotus herculeanus,
Lasius fuliginosus, Lasius niger, Lasius umbratus, Monomorium
pharaonis, Paravespula spp., Tetramorium caespitum. [0241] From the
order of the Anoplura, for example, Pediculus humanus capitis,
Pediculus humanus corporis, Phthirus pubis. [0242] From the order
of the Heteroptera, for example, Cimex hemipterus, Cimex
lectularius, Rhodinus prolixus, Triatoma infestans.
[0243] vi. Parasites of Animal and Human.
[0244] The pesticidal compositions of the invention are also
suitable for controlling parasites which can infest animals and
humans. They can be employed alone or in combination with other
active compounds for controlling these pests and symptoms related
to the infestation of such pests. They are active against sensitive
and resistant species and against all developmental stages. These
pests include, but are not limited to ticks, lice, mites,
strongyles, nematode parasites, such as ascarids (Ascaris)
(including Ascaris lumbricoides (Large Roundworm of Man)),
filarias, hookworms, pinworms (including Enterobius
vermicularis--(The Human Pinworm)) whipworms (Trichuris trichiura),
Trichinella spiralis, Dirofilaria immitis (heartworms), Haemonchus
contortus, and Myrmeconema neotropicum.
[0245] vii. Fouling Pests
[0246] The pesticidal compositions of the invention are also
suitable for controlling pests which cause fouling of ships and
other objects which come into contact with saltwater or brackish
water, such as hulls, screens, nets, buildings, moorings and
signalling systems, against fouling.
[0247] Such fouling includes, but is not limited to, fouling by
sessile Oligochaeta, such as Serpulidae, and by shells and species
from the Ledamorpha group (goose barnacles), such as various Lepas
and Scalpellum species, or by species from the Balanomorpha group
(acorn barnacles), such as Balanus or Pollicipes species, increases
the frictional drag of ships and, as a consequence, leads to a
marked increase in operation costs owing to higher energy
consumption and additionally frequent residence in the dry
dock.
[0248] viii. Pests Expressing .beta.-Lacatamase
[0249] The invention is based, at least in part, on the discovery
that when a pest, e.g., an arthopod, a nematode, an insect, or a
parasite, ingests an enzyme-cleavable .beta.-lactamase specific
construct, the construct is cleaved by .beta.-lactamases that are
produced by the pest, resulting in the release of free
photosensitizer within the insect. The pest then dies when exposed
to light. In accordance with the invention, the pest is an animal
that expresses .beta.-lactamase. In particular embodiments, In
accordance with the invention, the pest is an animal that expresses
a .beta.-lactamase comprising the protein domain sequence:
TABLE-US-00003 (SEQ ID NO: 1)
ILTEKRKILVDCGDPWNGTQIIQALSKYSLNCDDITDLIITHGHSDHCG
NLSLFQQAKIYMGDDMAKDGIYEGIWTLDDFVKIRPTPGHTDRSIIVLD
TEYGTVAIVGDIFEEENDDDSWKENSKYPEEQQKSRKIILKEADWIIPG H (GenBANK
protein sequence XP_001891895) or a fragment thereof.
[0250] In certain embodiments, the pest expresses a
.beta.-lactamase comprising a protein domain of at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, identity
(e.g., when compared to the overall length of the protein sequence)
to SEQ ID NO:1 or a fragment thereof.
[0251] In still other embodiments, the pest is an animal that
expresses a .beta.-lactamase comprising the protein domain
sequence:
TABLE-US-00004 (SEQ ID NO: 2)
TNTYIIGTGKRRILLDAGDENVPEYIGHLKKVISDERILINDIIVSHWH
HDHIGGVDEVLDIIENKDSCKVWKFPRADAPDGTIRNANINHLKHGQKF
NIEGATLEVLHTPGHTTDHVVLVLHEDNSLFSADCILGEGSTVEEDLYE
YTKSLQAIQDAKPSVIYPG (GenBANK protein sequence XP_001656361) or a
fragment thereof.
[0252] In certain embodiments, the pest expresses a
.beta.-lactamase comprising a protein domain of at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, identity
(e.g., when compared to the overall length of the protein sequence)
to SEQ ID NO:2 or a fragment thereof.
[0253] To determine the percent identity or similarity of two
protein domain sequences, the sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in the sequence
of a first protein sequence for optimal alignment with a second
protein sequence). As used herein, the terms "percent identity" and
"percent similarity" are used interchangeably.
[0254] When a position in the first sequence is occupied by the
same amino acid residue as the corresponding position in the second
sequence, then the molecules are similar at that position. The
percent similarity between the two sequences is a function of the
number of similar positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positions.times.100),
advantageously taking into account the number of gaps and size of
said gaps necessary to produce an optimal alignment.
[0255] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. A particular, non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc.
Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated
into the NBLAST and XBLAST programs (version 2.0) of Altschul et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to nucleic acid molecules of
the invention. BLAST polypeptide searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to polypeptide molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (1997) Nucleic Acids
Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another particular, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an
algorithm is incorporated into the ALIGN program available, for
example, at the GENESTREAM network server, IGH Montpellier, FRANCE
or at the ISREC server. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
[0256] Alternatively, the percent identity between two protein
sequences can be determined using the GAP program in the GCG
software package, using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight
of 2, 3, or 4.
[0257] In other embodiments, percent identity is determined at the
polynucleotide level. A .beta.-lactamase polynucleotide is one that
encodes a .beta.-lactamase polypeptide. In particular embodiments,
a polynucleotide encoding a .beta.-lactamase polypeptide are
identified by hybridizing the polynucleotide sequence with a
.beta.-lactamase probe. Hybridization conditions are known to those
skilled in the art and can be found in Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons,
Inc. (1995), sections 2, 4 and 6. Additional stringent conditions
can be found in Molecular Cloning: A Laboratory Manual, Sambrook et
al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
chapters 7, 9 and 11. A particular, non-limiting example of
stringent hybridization conditions includes hybridization in
4.times. sodium chloride/sodium citrate (SSC), at about
65-70.degree. C. (or hybridization in 4.times.SSC plus 50%
formamide at about 42-50.degree. C.) followed by one or more washes
in 1.times.SSC, at about 65-70.degree. C. A particular,
non-limiting example of highly stringent hybridization conditions
includes hybridization in 1.times.SSC, at about 65-70.degree. C.
(or hybridization in 1.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 0.3.times.SSC,
at about 65-70.degree. C. A particular, non-limiting example of
reduced stringency hybridization conditions includes hybridization
in 4.times.SSC, at about 50-60.degree. C. (or alternatively
hybridization in 6.times.SSC plus 50% formamide at about
40-45.degree. C.) followed by one or more washes in 2.times.SSC, at
about 50-60.degree. C. Ranges intermediate to the above-recited
values, e.g., at 65-70.degree. C. or at 42-50.degree. C. are also
intended to be encompassed by the present invention. SSPE
(1.times.SSPE is 0.15 M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25 mM
EDTA, pH 7.4) can be substituted for SSC (1.times.SSC is 0.15 M
NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes each after
hybridization is complete. The hybridization temperature for
hybrids anticipated to be less than 50 base pairs in length should
be 5-10.degree. C. less than the melting temperature (T.sub.m) of
the hybrid, where T.sub.m is determined according to the following
equations. For hybrids less than 18 base pairs in length,
T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For
hybrids between 18 and 49 base pairs in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M).
[0258] It will also be recognized by the skilled practitioner that
additional reagents can be added to hybridization and/or wash
buffers to decrease non-specific hybridization to membranes, for
example, nitrocellulose or nylon membranes, including but not
limited to blocking agents (e.g., BSA or salmon or herring sperm
carrier DNA), detergents (e.g., SDS), chelating agents (e.g.,
EDTA), Ficoll, PVP and the like. When using nylon membranes, in
particular, an additional, non-limiting example of stringent
hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or, alternatively, 0.2.times.SSC, 1% SDS).
[0259] vii. Combinations
[0260] The pesticidal compositions of the invention can be used as
a mixture with other known active compounds, such as additional
pesticide materials, fungicides, insecticides, acaricides,
nematicides, bird repellents, plant nutrients and agents which
improve soil structure, is also possible. The pesticidal
composition of the present invention may include attractants such
as cockroach pheromones (e.g., sex attractants, aggregation
pheromones) or food-based attractants (e.g.,
methylcyclopentenalone, maltol, fenugreek and other
flavorings).
[0261] For example, and without limitation, the pesticidal
compositions of the invention can be used as a mixture with known
acaricides, nematicides or insecticides.
[0262] Suitable Insecticides/Acaricides/Nematicides include, but
are not limited to, the following compounds: [0263] abamectin,
acephate, acetamiprid, acrinathrin, alanycarb, aldicarb,
aldoxycarb, alpha-cypermethrin, alphamethrin, amitraz, avermectin,
AZ 60541, azadirachtin, azamethiphos, azinphos A, azinphos M,
azocyclotin, Bacillus popilliae, Bacillus sphaericus, Bacillus
subtilis, Bacillus thuringiensis, baculoviruses, Beauveria
bassiana, Beauveria tenella, bendiocarb, benfuracarb, bensultap,
benzoximate, betacyfluthrin, bifenazate, bifenthrin,
bioethanomethrin, biopermethrin, bistrifluoron, BPMC, bromophos A,
bufencarb, buprofezin, butathiofos, butocarboxim, butylpyridaben,
cadusafos, carbaryl, carbofuran, carbophenothion, carbosulfan,
cartap, chloethocarb, chlorethoxyfos, chlorfenapyr,
chlorfenvinphos, chlorfluazuron, chlormephos, chlorpyrifos,
chlorpyrifos M, chlovaporthrin, chromafenozide, cis-resmethrin,
cispermethrin, clocythrin, cloethocarb, clofentezine,
clothianidine, cyanophos, cycloprene, cycloprothrin, cyfluthrin,
cyhalothrin, cyhexatin, cypermethrin, cyromazine, deltamethrin,
demeton M, demeton S, demeton-S-methyl, diafenthiuron, diazinon,
dichlorvos, dicofol, diflubenzuron, dimethoate, dimethylvinphos,
dinetofuran, diofenolan, disulfoton, docusat-sodium, dofenapyn,
eflusilanate, emamectin, empenthrin, endosulfan, Entomopfthora
spp., esfenvalerate, ethiofencarb, ethion, ethiprole, ethoprophos,
etofenprox, etoxazole, etrimfos, fenamiphos, fenazaquin, fenbutatin
oxide, fenitrothion, fenothiocarb, fenoxacrim, fenoxycarb,
fenpropathrin, fenpyrad, fenpyrithrin, fenpyroximate, fenthion,
fenvalerate, fipronil, fluazinam, fluazuron, flubrocythrinate,
flucycloxuron, flucythrinate, flufenoxuron, flumethrin,
flupyrazofos, flutenzine, fluvalinate, fonophos, fosmethilan,
fosthiazate, fubfenprox, furathiocarb, granulosis viruses,
halofenozide, HCH, heptenophos, hexaflumuron, hexythiazox,
hydroprene, imidacloprid, indoxacarb, isazofos, isofenphos,
isoxathion, ivermectin, nuclear polyhedrosis viruses,
lambda-cyhalothrin, lufenuron, malathion, mecarbam, metaldehyde,
methamidophos, Metharhizium anisopliae, metharhizium flavoviride,
methidathion, methiocarb, methoprene, methomyl, methoxyfenozide,
metolcarb, metoxadiazone, mevinphos, milbemectin, milbemycin,
monocrotophos, naled, nitenpyram, nithiazine, novaluron, omethoate,
oxamyl, oxydemethon M, Paecilomyces fumosoroseus, parathion A,
parathion M, permethrin, phenthoate, phorate, phosalone, phosmet,
phosphamidon, phoxim, pirimicarb, pirimiphos A, pirimiphos M,
profenofos, promecarb, propargite, propoxur, prothiofos, prothoate,
pymetrozine, pyraclofos, pyresmethrin, pyrethrum, pyridaben,
pyridathion, pyrimidifen, pyriproxyfen, quinalphos, ribavirin,
salithion, sebufos, silafluofen, spinosad, spirodiclofen, sulfotep,
sulprofos, tau-fluvalinate, tebufenozide, tebufenpyrad,
tebupirimiphos, teflubenzuron, tefluthrin, temephos, temivinphos,
terbufos, tetrachlorvinphos, tetradifon, theta-cypermethrin,
thiacloprid, thiamethoxam, thiapronil, thiatriphos, thiocyclam
hydrogen oxalate, thiodicarb, thiofanox, thuringiensin,
tralocythrin, tralomethrin, triarathene, triazamate, triazophos,
triazuron, trichlophenidine, trichlorfon, triflumuron,
trimethacarb, vamidothion, vaniliprole, Verticillium lecanii, YI
5302, zeta-cypermethrin, zolaprofos,
(1R-cis)-[5-(phenylmethyl)-3-furanyl]-methyl-3-Rdihydro-2-oxo-3(2H)-furan-
-ylidene)-methyl]-2,2-dimethylcyclopropanecarboxylate,
(3-phenoxyphenyl)-methyl-2,2,3,3-tetramethylcyclopropanecarboxylate,
1-[(2-chloro-5-thiazolyl)methyl]tetrahydro-3,5-dimethyl-N-nitro-1,-3,5-tr-
iazine-2(1H)-imine,
2-(2-chloro-6-fluorophenyl)-4-[4-(1,1-dimethylethyl)phenyl]-4,5-dihydro-o-
-xazole, 2-(acetyloxy)-3-dodecyl-1,4-naphthalenedione,
2-chloro-N-[[[4-(1-phenylethoxy)-phenyl]-aminol-carbonyl]-benzamide,
2-chloro-N-[[[4-(2,2-dichloro-1,1-difluoroethoxy)-phenyl]-aminol-carbonyl-
]-benzamide, 3-methylphenyl propylcarbamate,
4-[4-(4-ethoxyphenyl)-4-methylpentyl]-1-fluoro-2-phenoxybenzene,
4-chloro-2-(1,1-dimethylethyl)-5-[[2-(2,6-dimethyl-4-phenoxyphenoxy)ethyl-
-]thio]-3(2H)-pyridazinone,
4-chloro-2-(2-chloro-2-methylpropyl)-5-[(6-iodo-3-pyridinyl)methoxy]-3(2H-
--)-pyridazinone,
4-chloro-5-[(6-chloro-3-pyridinyl)methoxy]-2-(3,4-dichlorophenyl)-3(2H)-p-
-yridazinone, Bacillus thuringiensis strain EG-2348,
[2-benzoyl-1-(1,1-dimethylethyl)-hydrazinobenzoic acid,
2,2-dimethyl-3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl
butanoate,
[3-[(6-chloro-3-pyridinyl)methyl]-2-thiazolidinylidene]-cyanamide,
dihydro-2-(nitromethylene)-2H-1,3-thiazine-3(4H)-carboxaldehyde,
ethyl[2-[[1,6-dihydro-6-oxo-1-(phenylmethyl)-4-pyridazinyl]oxy]ethyl]-car-
-bamate, N-(3,4,4-trifluoro-1-oxo-3-butenyl)-glycine,
N-(4-chlorophenyl)-3-[4-(difluoromethoxy)phenyl]-4,5-dihydro-4-phenyl-1H--
pyrazole-1-carboxamide,
N-[(2-chloro-5-thiazolyl)methyl]-N'-methyl-N''-nitro-guanidine,
N-methyl-N'-(1-methyl-2-propenyl)-1,2-hydrazinedicarbothioamide,
[0295] N-methyl-N'-2-propenyl-1,2-hydrazinedicarbothioamide,
O,O-diethyl-[2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate,
N-cyanomethyl-4-trifluoromethyl-nicotinamide,
3,5-dichloro-1-(3,3-dichloro-2-propenyloxy)-4-[3-(5-trifluoromethylpyridi-
n-2-yloxy)-propoxy]-benzene.
[0264] It is also possible to admix the pesticidal compositions of
the invention with other known active compounds, such as
herbicides, fertilizers and growth regulators, or safeners or
semiochemicals.
IV. PHOTOACTIVATION
[0265] Typically, administration of a pesticidal composition
according to the invention is followed by a sufficient period of
time to allow accumulation thereof in the pest. The
.beta.-lactamase cleavage site of the linker is cleaved by
.beta.-lactamase produced by the pest. As a result, the
photosensitizers are no longer quenched. The photosensitizers can,
subsequently, be activated by irradiation. This is accomplished by
applying light of a suitable wavelength and intensity, for an
effective length of time, so as to kill the pest. As used herein,
"irradiation" refers to the use of light to induced a chemical
reaction of a photosensitizer.
[0266] Photoactivating dosages depend on various factors, including
the amount of the pesticide administered, the wavelength of the
photoactivating light, the intensity of the photoactivating light,
and the duration of illumination by the photoactivating light.
Thus, the dose can be adjusted to a therapeutically effective dose
by adjusting one or more of these factors. Such adjustments are
within the level of ordinary skill in the art.
[0267] Irradiation of the appropriate wavelength for a given
compound may be administered by a variety of methods. Methods for
irradiation include, but are not limited to, the administration of
laser, nonlaser, or broad band light. Irradiation can be produced
by extracorporeal or intraarticular generation of light of the
appropriate wavelength. Light used in the invention may be
administered using any device capable of delivering the requisite
power of light including, but not limited to, fiber optic
instruments, arthroscopic instruments, or instruments that provide
transillumination. Delivery of the light to a recessed, or
otherwise inaccessible physiological location can be facilitated by
flexible fiber optics (implicit in this statement is the idea that
one can irradiate either a broad field, such as the lung or a lobe
of the lung, or a narrow field where bacterial cells may have
localized). The source of the light needed to inactivate the pest
can be an inexpensive diode laser or a non-coherent light
source.
[0268] The photosensitizer compositions of the invention should be
stable during the course of at least a single round of treatment by
continued or pulsed irradiation, during which the photosensitizer
within the composition would, preferably, be repeatedly excited to
the energized state, undergoing multiple rounds of generation of
singlet oxygen.
[0269] The suitable wavelength, or range of wavelengths, will
depend on the particular photosensitizer(s) used, and can range
from about 350 nm to about 550 nm, from about 550 nm to about 650
nm, from about 650 nm to about 750 nm, from about 750 nm to about
850 nm and from about 850 nm to about 950 nm.
[0270] In specific embodiments, pests are illuminated with red
light. Given that red and/or near infrared light best penetrates
animal tissues, photosensitizers with strong absorbances in the
range of about 600 nm to about 900 nm are optimal for PDT. For
photoactivation, the wavelength of light is matched to the
electronic absorption spectrum of the photosensitizer so that the
photosensitizer absorbs photons and the desired photochemistry can
occur. Wavelength specificity for photoactivation generally depends
on the molecular structure of the photosensitizer. Photoactivation
can also occur with sub-ablative light doses. Determination of
suitable wavelength, light intensity, and duration of illumination
is within ordinary skill in the art.
[0271] In a particular embodiment, the pest are illuminated with
sunlight (i.e. solar radiation). Such exposure can be direct,
indirect, focussed or diffused.
[0272] The effective penetration depth, .delta..sub.eff, of a given
wavelength of light is a function of the optical properties of the
tissue, such as absorption and scatter. The fluence (light dose) in
a tissue is related to the depth, d, as: e.sup.-d/.delta..sub.eff.
Typically, the effective penetration depth is about 2 to 3 mm at
630 nm and increases to about 5 to 6 nm at longer wavelengths
(about 700 to about 800 nm) (Svaasand and Ellingsen, (1983)
Photochem Photobiol. 38:293-299). Altering the biologic
interactions and physical characteristics of the photosensitizer
can alter these values. In general, photosensitizers with longer
absorbing wavelengths and higher molar absorption coefficients at
these wavelengths are more effective photodynamic agents.
[0273] The light for photoactivation can be produced and delivered
to the site of infestation by any suitable means known in the art.
Photoactivating light can be delivered to the site of infestation
from a light source, such as a laser or optical fiber. Preferably,
optical fiber devices that directly illuminate the site of
infestation deliver the photoactivating light. For example, the
light can be delivered by optical fibers threaded through small
gauge hypodermic needles. Light can be delivered by an appropriate
intravascular catheter, such as those described in U.S. Pat. Nos.
6,246,901 and 6,096,289, which can contain an optical fiber.
Optical fibers can also be passed through arthroscopes. In
addition, light can be transmitted by percutaneous instrumentation
using optical fibers or cannulated waveguides. For open surgical
sites, suitable light sources include broadband conventional light
sources, broad arrays of light-emitting diodes (LEDs), and
defocused laser beams.
[0274] Delivery can be by all methods known in the art, including
transillumination. Some photosensitizers can be activated by near
infrared light, which penetrates more deeply into biological tissue
than other wavelengths. Thus, near infrared light is advantageous
for transillumination. Transillumination can be performed using a
variety of devices. The devices can utilize laser or non-laser
sources, (e.g., lightboxes or convergent light beams).
[0275] Where pesticidal activity is desired, the dosage of
pesticidal composition, and light activating the photosensitizer
composition, is administered in an amount sufficient to produce a
phototoxic species effective to kill the pest
[0276] Irradiation of the appropriate wavelength for a given
compound may be administered by a variety of wavelengths. Methods
for irradiation include, but are not limited to, the administration
of laser, nonlaser, or broad band light. Irradiation can be
produced by extracorporeal or intraarticular generation of light of
the appropriate wavelength. Light used in the invention may be
administered using any device capable of delivering the requisite
power of light including, but not limited to, fiber optic
instruments, arthroscopic instruments, or instruments that provide
transillumination.
[0277] The wavelength and power of light can be adjusted according
to standard methods known in the art to control the production of
phototoxic species. Thus, under certain conditions (e.g., low
power, low fluence rate, shorter wavelength of light or some
combination thereof), a fluorescent species is primarily produced
from the photosensitizer and any reactive species produced has a
negligible effect. These conditions are easily adapted to bring
about the production of a phototoxic species. For example, where
the photosensitizer is chlorin.sub.e6, the light dose administered
to produce a fluorescent species and an insubstantial reactive
species is less than about 10 J/cm, preferably less than about 5
J/cm and more preferably less than about 1 J/cm. Determination of
suitable wavelength, light intensity, and duration of illumination
for any photosensitizer is within the level of ordinary skill in
the art.
V. DETECTION OF PESTS
[0278] In certain embodiments, the administration of the
compositions of the invention, followed by photoactivation, does
not kill the pest but instead results in the pest fluorescing. As
such, in certain embodiments, the invention provides a method for
detecting the presence of pests, the method comprising the steps
of: contacting the pest with a photosensitizer composition of the
invention; cleaving one or more moieties cleavable by
a.beta.-lactamase expressed by the pest to dequench the
photosensitizer composition, light-activating the composition to
produce a fluorescent species, thereby causing the pest to
fluoresce and observing the fluorescence thereby detecting the
presence of pests.
[0279] In still other embodiments, the administration of the
pesticidal compositions of the invention, followed by
photoactivation, results in both the termination of the pest and
the pest fluorescing. As such, in certain embodiments, the
invention provides a method of eliminating and detecting a pest,
the method comprising the steps of: contacting the pest with a
photosensitizer composition of the invention; cleaving one or more
moieties cleavable by the .beta.-lactamase to dequench the
photosensitizer composition and light-activating the composition to
produce a fluorescent, phototoxic species, thereby eliminating the
pest and causing the pest to fluoresce and observing the
fluorescence thereby also detecting the presence of the pest.
[0280] The invention is additionally described by way of the
following illustrative, non-limiting Examples that provide a better
understanding of the present invention and of its many
advantages.
Examples
Example 1
Preparation of Conjugates Comprising Polymer, .beta.-Lactam Moiety
and Photosensitizer
[0281] In one approach, the synthesis of the conjugates is based on
cephalosporin, the most often used .beta.-lactam. It is conceivable
to develop penem or carbapenem derivatives subsequently.
[0282] In the following, the photosensitizer (a porphyrin molecule
with at least one propionic side chain) is represented by
PS--CH.sub.2--CH.sub.2--COOH. The polymer used in the synthetic
routes shown below is a linear or branched poly(ethylene glycol)
with propionic acid groups (PEG-CH.sub.2--CH.sub.2--COOH) (Senter,
P. D., et al. (1995) Bioconjug. Chem. 6:389-394). However, the
chemistry is applicable to similar polymeric materials containing
available carboxylic side chains. In order to be released upon
enzymatic hydrolysis, the porphyrin molecule is preferably linked
at the 3'-position of the cephalosporin. The
cephalosporin-porphyrin moiety obtained can then be conjugated to
the polymer using the amino group on the .beta.-lactam ring.
[0283] The preparation of three different conjugates is proposed,
where the porphyrin and cephalosporin are linked via an ester:
##STR00001##
or via a carbamate group:
##STR00002##
##STR00003##
The preparation of a cephalosporin-prophyrin ester comprises the
following steps:
[0284] A. Protection of the amino-group in the .beta.-lactam
ring
[0285] There are several ways to protect the amino group. One is
represented below (Hanessian, S., et al. (1993) Can. J. Chem.
71:896-906):
##STR00004##
[0286] Protected cephalosporin derivatives are commercially
available. Other protecting groups include (Albrecht, H. A., et
al., (1990) J. Med. Chem. 33:77-86; Albrecht, H. A., et al. (1991)
J. Med. Chem. 34:2857-2864; Alexander, R. P., et al. (1991)
Tetrahedron Lett. 32:3269-3272):
##STR00005##
[0287] For example, the following molecule (which comes with a
protected amino group) is called cephalothin.
##STR00006##
[0288] B. Binding of the porphyrin at the 3'-position of the
cephalosporin via an ester function [0289] i. Through a diazomethyl
intermediate (Mobashery, S., et al. (1986) J. Biol. Chem.
261:7879-7887)
##STR00007## ##STR00008##
[0290] In this scheme, pNBz=para-nitro-benzyl. [0291] ii. Through a
halogenated intermediate (Mobashery, S., et al. (1986) J. Biol.
Chem. 261:7879-7887)
[0291] ##STR00009## [0292] iii. Through a hydroxymethyl
intermediate (Hanessian, S., et al. (1993) Can. J. Chem.
71:896-906)
##STR00010##
[0293] C. Deprotection of the amino-group in the B-lactam ring
(Albrecht, H. A., et al. (1991) J. Med. Chem. 34:669-675)
##STR00011##
Deprotection of the amino group is also very often carried out
using Penicillin-G amidase (PGA) (Vrudhula, V. M., et al. (1995) J.
Med. Chem. 38:1380-1385).
[0294] D. Conjugation of the cephalosporin-porphyrin moiety to a
polymer (Senter, P. D., et al. (1995) Bioconjug. Chem.
6:389-394)
##STR00012##
[0295] The preparation of a cephalosporin-porphyrin carbamate
comprises the following steps:
[0296] A. Protection of the amino-group in the .beta.-lactam ring
(see above)
[0297] B. Binding of the porphyrin at the 3'-position of the
cephalosporin via a carbamate [0298] i. Direct coupling between the
porphyrin and cephalosporin (Alexander, R. P., et al. (1991)
Tetrahedron Lett. 32:3269-3272; Rodrigues, M. L., et al. (1995)
Chem. & Biol. 2:223-227; Smith, K. M., et al. (1987)
Heterocycles 26:1947-1963)
[0298] ##STR00013## [0299] ii. Coupling through a linker
(Alexander, R. P., et al. (1991) Tetrahedron Lett. 32:3269-3272;
Rodrigues, M. L., et al. (1995) Chem. & Biol. 2:223-227;
Boutorine, A. S., et al. (1996) J. Am. Chem. Soc.
118:9469-9476)
##STR00014##
[0300] C. Deprotection of the amino-group in the .beta.-lactam ring
(see above)
[0301] D. Conjugation of the cephalosporin-porphyrin moiety to a
polymer (Senter, P.D., et al. (1995) Bioconjug. Chem. 6:389-394)
(see above)
[0302] Of additional note, if, after these chemical modifications,
the cephalosporin derivatives described above retain their
properties as substrates for .beta.-lactamases, one can expect to
observe the enzyme-dependent release of three different porphyrin
moieties:
PS-Ch.sub.2-CH.sub.3:
##STR00015##
[0303] PS--CH.sub.2--CH.sub.2--NH.sub.2
##STR00016##
[0304] and
PS--CH.sub.2--CH.sub.2--CO--NH--(CH.sub.2).sub.4--NH.sub.2:
##STR00017##
[0305] Example 2
Development of Carbamate-Linked Photosensitizer, Inactive (with or
without Light) while Linked and Light-Activatable Only When
Released by the .beta.-lactamase Enzyme-mediated Cleavage
[0306] The lactam ring opening of the prodrugs releases the
photosensitizer and make it light-activatable for photokilling
(FIG. 1).
Synthesis
##STR00018##
[0308] Commercially available 7-aminochephalosporanic acid was
reacted with phenylacetyl chloride under Shotten-Baumann reaction
conditions to achieve an amino protected chephalosporin molecule.
This was further de-esterified using tetrabutylammonium hydroxide
as a base to yield easily functionalizable hydroxy end group on
cephalosporin. The last step of the synthesis was achieved in a
one-pot reaction sequence. Toluidine Blue O (TBO) was converted
into its isocynate derivative in the presence of diphosgene. The
carbamate-linked prodrug was obtained by adding Cephalosporin
derivative to the same reaction mixture.
Synthesis of 7-[(2-phenylacetyl)amino] cephalosporanic acid
[0309] To a stirred mixture of sodium bicarbonate (2.1 g, 25 mmol)
in water (40 ml) and acetone (30 ml), added 7-(phenylacetyl)amino
cephalosporanic acid. Stirred this solution for nearly 15 min in
ice bath and slowly added phenylacetyl chloride (2.5 ml, 20 mmol)
over the period of 30 min. This reaction mixture was stirred
overnight and acidified to pH 2.0 with 1N hydrochloric acid.
Precipitates obtained were extracted with dichloromethane and
washed with water. Dried over magnesium sulphate and solvent
evaporated to give off-white solid. The solid sample was stirred
overnight in diethyl ether and filters to obtain crude product in
80% yield.
Synthesis of 7-[(2-phenylacetyl)amino]3-hydrodxymethy
cephalosporanic acid
[0310] To a suspension of 7-[(2-phenylacetyl)aminol cephalosporanic
acid (0.5 g, 1.28 mmol) in a a mixture of methane (4 ml) and water
(2.5 ml), triethylamine (0.21 ml, 1.54 mmol) was added in 15 min at
0-5.degree. C. To this solution, tetrabutylammonium hydroxide (30%
solution in water, 1.53 g, 1.92 mmol) was added at -18.degree. C.
in 30 minutes. The reaction mixture was maintained at -18.degree.
C. for nearly 7.0 h and acidified to pH 5.0 using glacial acetic
acid. Purification was done using C-18 reverse phase column and
pure product was obtained as white solid in 67% yield.
Synthesis of Cephalosporanic Acid-Toluidine BlueO Prodrug
[0311] To a magnetically stirred suspension of toludine blue 0 (0.1
g, 0.33 mmol) in anhydrous THF (3 ml) under nitrogen was added a
solution of tricholoromethyl chloroformate (19.7 .mu.l, 0.164 mmol)
over activated charcoal as a catalyst. The reaction mixture was
stirred at 55.degree. C. for 30 min. Progress of reaction was
monitored using mass spectroscopy for formation of isocynate
derivative of toludine blue 0. Cooled the flask to room temperature
and added a solution of 74(2-phenylacetyl)aminol 3-hydrodxymethy
cephalosporanic acid (0.15 g, 0.33 mmol) in anhydrous
dichloromethane (1 ml). The reaction flask was cooled to 0.degree.
C. and slowly added diisopropylethylamine (57.0 .mu.l, 0.33 mmol).
Stirred for 3.0 h and purified using C18 column with acetonirile
and water as eluting solvents. Pure product obtained as a blue
solid in 25% yield.
[0312] .sup.1H NMR spectra were obtained for
7-[(2-phenylacetyl)aminol cephalosporanic acid in CDCl.sub.3 as a
solvent, as well as for 7-[(2-phenylacetyl)amino]3-hydrodxymethy
cephalosporanic acid in DMSO-d.sub.6 as a solvent (FIG. 2). MS
spectra were obtained for 7-[(2-phenylacetyl)amino]3-hydrodxymethy
cephalosporanic acid and cephalosporanic acid-toluidine blue O
prodrug (FIG. 3).
[0313] UV-visible spectra revealed blue shift in the absorption
spectra of the prodrug, indicating extended conjugation, as well as
quenching, of carbamate linked TBO photosensitizer (FIG. 4).
Fluorescence spectra revealed nearly an 8-fold reduction in
fluorescence emission maxima at 635 nm excitation, indicating
quantitative quenching of the photosensitizer upon conjugation with
the cephalosporin moiety (FIG. 5).
Enzyme-Mediated Cleavage of the Prodrug
[0314] The prodrug obtained was further studied for release of
photosensitizer in presence of .beta.-lactamase from Enterobacter
cloacae. For the fluorescence emission study of the prodrug, the
solvent employed was water, and the excitation wavelength 635 nm in
the presence of beta-lactamase enzyme (from Enterobacter cloacae).
Time-dependent fluorescence emission was also measured for
photosensitizer release from the prodrug in the presence of enzyme.
The results indicate an easy release and nearly 5-fold increase in
excited stated properties within minutes of incubation of prodrug
with enzyme (FIG. 6). Thus, the prodrug was ynthesized and
characterized. Furthermore, the prodrug showed quantitative
quenching of the photosensitizer in the conjugated form.
Additionally, the product demonstrated lactamase-specific
activity.
Example 3
Cleavage of Carbamate-Linked Photosensitizer in Microfilarial B.
malayi
[0315] Brugia malayi Adults and 1st Stage Larvae (Microfilariae)
Activate the .beta.-LEAP Photosensitizer-Containing Construct by
Cleavage of the Cephalosporin Moiety. (FIG. 8)
[0316] Groups of 3 adult males, 3 adult females and 10,000
microfilariae were incubated with 10 .mu.M .beta.-LEAP in
triplicate in the wells of 96 well microtiter plates. Positive
control was commercially available Bacillus cereus .beta.-lactamase
(0.25 U/ml). Negative controls included reactions lacking worms and
reactions lacking .beta.-LEAP. Data was collected by a SpectraMax
fluorimeter every minute over a 3 hour period with excitation set
at 650 nm and emission at 700 nm. Increasing relative fluorescence
resulting from activation of .beta.-LEAP by enzymatic cleavage over
the first hour of data collection is shown.
Confocal Laser Scanning Microscopy of Brugia malayi Adults and
1.sup.st Stage Larvae Following Exposure to .beta.-LEAP and 650 nm
Light. (FIG. 9)
[0317] Worms recovered from the assay described above were fixed,
incubated with RNase and then their nucleic acids stained with
propidium iodide. Samples were mounted on glass slides in mounting
medium containing DAPI (also stains DNA). Images were acquired
using an Olympus FV1000 confocal laser scanning microscope. Images
of adult worms are at the region of the reproductive tissue. All
images are at 100.times. magnification and a 50 .mu.m scale bar is
provided for reference. The widespread red coloration indicates
fluorescence released from .beta.-LEAP, Blue is fluorescence from
DAPI and green is fluorescence from propidium iodide.
Confocal Laser Scanning Microscopy of an Adult Brugia malayi Female
Following Exposure to .beta.-LEAP and 650 nm Light. (FIG. 10)
[0318] A female worm recovered from the assay described above was
fixed, incubated with RNase and then it's nucleic acids stained
with propidium iodide. The worm was mounted on a glass slide in
mounting medium containing DAPI (also stains DNA). The images were
acquired using an Olympus FV1000 confocal laser scanning
microscope. Images are at the region of the reproductive tissue and
reveal the intestine. Panel A 20.times. magnification; Panel B
100.times. magnification. The widespread red coloration indicates
fluorescence released from .beta.-LEAP, Blue is fluorescence from
DAPI and green is fluorescence from propidium iodide.
Photodynamic Therapy with the Photosensitizer EtNBS Kills Adult
Brugia malayi. (FIG. 11)
[0319] Adult female worms were incubated in 10-fold serial
dilutions of EtNBS (the photosensitizer that is released from
.beta.-LEAP upon enzymatic cleavage) for 16 hr at 37.degree. C.
Worms were pooled into groups of 4 and irradiated with light (600
nm at 33 mW/cm.sup.2) for different time periods so as to deliver
different light doses. Irradiated worms were then returned to
standard culture conditions for 24 hrs and the viability of the 4
individual worms from each condition was assessed by use of the MTT
assay. The viability of worms from each group is expressed as
absorbance units (510 nm) and standard errors are presented. A dose
dependent decrease in viability of worms exposed to 1 .mu.M EtNBS
is apparent as light dose increases.
Example 4
Cleavage of Carbamate-Linked Photosensitizer in Aedes
albopictus
[0320] Aedes albopictus Cells Activate the .beta.-LEAP
Photosensitizer-Containing Construct by Cleavage of the
Cephalosporin Moiety. (FIG. 11)
[0321] The Aedes albopictus cell line C6/36 was grown to high
density in tissue culture flasks and then 1,000,000 cells were
transferred to each of 3 wells of a 96 well microtiter plate and
incubated with 10 .mu.M .beta.-LEAP. Positive control was
commercially available Bacillus cereus .beta.-lactamase (0.25
U/ml). Negative controls included reactions lacking cells and
reactions lacking .beta.-LEAP. Data was collected by a SpectraMax
fluorimeter every minute over a 3 hour period with excitation set
at 650 nm and emission at 700 nm Increasing relative fluorescence
units (RFU) resulting from activation of 3-LEAP by enzymatic
cleavage over the first hour of data collection is shown.
Example 5
Use of .beta.-LEAP in Industrial Crops Enzymatic Cleavage
Exemplary Formulations:
Pellet Formulation
[0322] 75 parts by mass of sawdust is impregnated with 20 parts by
mass of .beta.-LEAP and 5 parts by mass of cornstarch is added
thereto and the mixture is molded into bars to obtain a pellet
formulation (cylindrical form 5 to 10 mm long having a diameter of
about 2 mm).
Flowable (Emulsion) Formulation
[0323] 5 parts by mass of polyvinyl alcohol, 3 parts by mass of
DEMOL N (trade name; manufactured by Kao Corporation), 0.5 parts by
mass of ANTIFOAM E-20 (trade name; manufactured by Kao Corporation)
and 41.5 parts by mass of water are stirred and mixed. 50 parts by
mass of .beta.-LEAP is added thereto by dropwise to obtain a
flowable (emulsion) formulation.
Field Trials:
[0324] Field trials are run in accordance with pertinent protocols
and in conformance with USDA notification requirements. Field soil
is packed in 350 cm.sup.2 plastic pots and seedlings of corn,
wheat, sorghum, soybean, and tobacco are sown and covered with soil
of about 1 cm thickness. After water absorption and at 11 days
after seeding, .beta.-LEAP pellets are spread or a .beta.-LEAP
flowable formulation is diluted with a stock solution or water were
sprayed uniformly in a spray volume of 200 ml per m.sup.2. The test
is performed in a glass greenhouse box at a temperature of from 18
to 30.degree. C., and the soil is appropriately moistened from the
bottom side. Pests common to each type of plant are introduced into
the respective greenhouse box and allowed to ingest the
.beta.-LEAP. After 1 hour, the greenhouse boxes are exposed to
sunlight or red LED lighting. The elimination of the pests is
observed.
INCORPORATION BY REFERENCE
[0325] The contents of all references (including literature
references, issued patents, published patent applications, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated herein in their entireties by
reference.
EQUIVALENTS
[0326] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
21148PRTBrugia malayi 1Ile Leu Thr Glu Lys Arg Lys Ile Leu Val Asp
Cys Gly Asp Pro Trp 1 5 10 15 Asn Gly Thr Gln Ile Ile Gln Ala Leu
Ser Lys Tyr Ser Leu Asn Cys 20 25 30 Asp Asp Ile Thr Asp Leu Ile
Ile Thr His Gly His Ser Asp His Cys 35 40 45 Gly Asn Leu Ser Leu
Phe Gln Gln Ala Lys Ile Tyr Met Gly Asp Asp 50 55 60 Met Ala Lys
Asp Gly Ile Tyr Glu Gly Ile Trp Thr Leu Asp Asp Phe 65 70 75 80 Val
Lys Ile Arg Pro Thr Pro Gly His Thr Asp Arg Ser Ile Ile Val 85 90
95 Leu Asp Thr Glu Tyr Gly Thr Val Ala Ile Val Gly Asp Ile Phe Glu
100 105 110 Glu Glu Asn Asp Asp Asp Ser Trp Lys Glu Asn Ser Lys Tyr
Pro Glu 115 120 125 Glu Gln Gln Lys Ser Arg Lys Ile Ile Leu Lys Glu
Ala Asp Trp Ile 130 135 140 Ile Pro Gly His 145 2166PRTAedes
aegypti 2Thr Asn Thr Tyr Ile Ile Gly Thr Gly Lys Arg Arg Ile Leu
Leu Asp 1 5 10 15 Ala Gly Asp Glu Asn Val Pro Glu Tyr Ile Gly His
Leu Lys Lys Val 20 25 30 Ile Ser Asp Glu Arg Ile Leu Ile Asn Asp
Ile Ile Val Ser His Trp 35 40 45 His His Asp His Ile Gly Gly Val
Asp Glu Val Leu Asp Ile Ile Glu 50 55 60 Asn Lys Asp Ser Cys Lys
Val Trp Lys Phe Pro Arg Ala Asp Ala Pro 65 70 75 80 Asp Gly Thr Ile
Arg Asn Ala Asn Ile Asn His Leu Lys His Gly Gln 85 90 95 Lys Phe
Asn Ile Glu Gly Ala Thr Leu Glu Val Leu His Thr Pro Gly 100 105 110
His Thr Thr Asp His Val Val Leu Val Leu His Glu Asp Asn Ser Leu 115
120 125 Phe Ser Ala Asp Cys Ile Leu Gly Glu Gly Ser Thr Val Phe Glu
Asp 130 135 140 Leu Tyr Glu Tyr Thr Lys Ser Leu Gln Ala Ile Gln Asp
Ala Lys Pro 145 150 155 160 Ser Val Ile Tyr Pro Gly 165
* * * * *
References