U.S. patent application number 16/368330 was filed with the patent office on 2019-07-18 for methods and pharmaceuticals for treatment of viral infections of the eye.
The applicant listed for this patent is Tamir Biotechnology, Inc.. Invention is credited to Luis SQUIQUERA, Jamie SULLEY.
Application Number | 20190216904 16/368330 |
Document ID | / |
Family ID | 56194615 |
Filed Date | 2019-07-18 |
United States Patent
Application |
20190216904 |
Kind Code |
A1 |
SQUIQUERA; Luis ; et
al. |
July 18, 2019 |
METHODS AND PHARMACEUTICALS FOR TREATMENT OF VIRAL INFECTIONS OF
THE EYE
Abstract
Viral infections of the eye, and particularly viral infections
in the Herpesviridae and Adenoviridae families, can be treated by
administration of a pharmaceutical made up of an enzymatically
active ribonuclease and a vehicle. Advantageously, the
enzymatically active ribonuclease is ranpirnase, the '805 variant,
rAmphinase 2, and Amphinase 2, and the vehicle is an aqueous
solution.
Inventors: |
SQUIQUERA; Luis; (Buenos
Aires, AR) ; SULLEY; Jamie; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tamir Biotechnology, Inc. |
Short Hills |
NJ |
US |
|
|
Family ID: |
56194615 |
Appl. No.: |
16/368330 |
Filed: |
March 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15180270 |
Jun 13, 2016 |
10293032 |
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16368330 |
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62175961 |
Jun 15, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/08 20130101; A61P
27/02 20180101; A61P 31/20 20180101; A61P 31/22 20180101; A61K
38/465 20130101; A61K 47/02 20130101; C12Y 301/27 20130101; A61P
31/12 20180101; Y02A 50/393 20180101; Y02A 50/30 20180101; Y02A
50/385 20180101; Y02A 50/382 20180101; A61K 9/0048 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A61K 47/02 20060101 A61K047/02; A61K 9/08 20060101
A61K009/08; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of treating a herpesviral infection of the eye,
comprising the step of administering to the eye a therapeutically
effective dose of a ribonuclease that is a member of the
ribonuclease A superfamily.
2. The method of claim 1, wherein the eye is a human eye and the
ribonuclease is selected from a group consisting of a. ranpirnase;
b. the '805 variant; c. Amphinase 2; and d. rAmphinase 2.
3. The method of claim 1, wherein the ribonuclease is
non-irritating to the eye as determined using a. the Globally
Harmonized System of Classification Evaluation Criteria and b. the
European Economic Community Ocular Evaluation Criteria.
4. The method of claim 1, wherein the eye is a human eye and the
ribonuclease is functionally equivalent to a ribonuclease selected
from a group consisting of: a. ranpirnase; b. the '805 variant; c.
Amphinase 2; and d. rAmphinase 2.
5. A method for treating a herpesviral infection of the human eye,
the method comprising administering an ophthalmic composition to
the eye, the composition comprising: a. a therapeutically effective
dose of a ribonuclease that is a member of the ribonuclease A
superfamily and that is non-irritating to the eye as determined
using i. the Globally Harmonized System of Classification
Evaluation Criteria and ii. the European Economic Community Ocular
Evaluation Criteria; and b. a vehicle.
6. The method of claim 5, wherein the ribonuclease is selected from
a group consisting of: a. ranpirnase; b. the '805 variant; c.
Amphinase 2; and d. rAmphinase 2.
7. The method of claim 5, wherein the vehicle is an aqueous
solution.
8. The method of claim 5, wherein the ribonuclease is the
functional equivalent of a ribonuclease selected from a group
consisting of a. ranpirnase; b. the '805 variant; c. Amphinase 2;
and d. rAmphinase 2.
9. A method of treating a herpesviral infection of an eye, the
method comprising the step of topically administering to the eye a
therapeutically effective amount of a ranpirnase.
10. The method according to claim 9, wherein the ranpirnase is a
recombinant ranpirnase.
11. The method according to claim 9, wherein the ranpirnase is SEQ
ID NO: 1 or SEQ ID NO: 2.
12. The method according to claim 9, wherein the herpesviral
infection is the cause of chorioretinitis, viral keratitis, viral
conjunctivitis, or cytomegalovirus retinitis.
13. The method according to claim 9, wherein the herpesviral
infection is caused by an type I Herpes simplex virus, human
cytomegalovirus, or Herpes zoster virus.
14. The method according to claim 9, wherein the ranpirnase is
formulated as an ophthalmic pharmaceutical composition.
15. The method according to claim 14, wherein the ophthalmic
pharmaceutical composition is a solution, a suspension, a
nanosuspension, an emulsion, a microemulsion, a nanoemulsion, an
ointment, a gel, a hydrogel, liposomes, niosomes, nanoparticles, or
dendrimers.
16. The method according to claim 14, wherein the ophthalmic
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier and/or a pharmaceutically acceptable
excipient.
17. The method according to claim 9, wherein the ranpirnase is
administered as eye drops.
18. The method according to claim 9, wherein the eye is a human
eye.
19. A pharmaceutical composition comprising: a. a therapeutically
effective dose of a ribonuclease that is a member of the
ribonuclease A superfamily and that is non-irritating to an eye as
determined using i. the Globally Harmonized System of
Classification Evaluation Criteria and ii. the European Economic
Community Ocular Evaluation Criteria; and b. a vehicle.
20. The pharmaceutical composition of claim 19, wherein the
ribonuclease is selected from a group consisting of: a. ranpirnase;
b. the '805 variant; c. Amphinase 2; and d. rAmphinase 2.
21. The pharmaceutical composition of claim 19, wherein the
ribonuclease is the functional equivalent of a ribonuclease
selected from a group consisting of a. ranpirnase; b. the '805
variant; c. Amphinase 2; and d. rAmphinase 2.
22. The pharmaceutical composition of claim 19, wherein the
ribonuclease is ranpirnase present in an amount of 0.1%.
23. The pharmaceutical composition of claim 19, formulated for
topical administration to the eye.
24. The pharmaceutical composition of claim 23, wherein the
composition is formulated as a solution, a suspension, a
nanosuspension, an emulsion, a microemulsion, a nanoemulsion, an
ointment, a gel, a hydrogel, liposomes, niosomes, nanoparticles, or
dendrimers.
25. The pharmaceutical composition of claim 19, wherein the
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier and/or a pharmaceutically acceptable excipient.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates to viral infections of the eye, and
more particularly relates to methods and pharmaceuticals for
treatment of viral infections of the eye. In its most immediate
sense, the invention relates to treatment of human eye infections
caused by Herpesviridae viruses (including but not limited to Human
cytomegalovirus, herpes zoster virus, and varicella zoster virus)
and Adenoviridae viruses.
[0002] Viral diseases of the eye can have significant consequences.
Type 1 herpes simplex virus can cause conjunctivitis and keratitis,
Human cytomegalovirus can cause retinitis, adenovirus types 8, 19,
29, and 37 can cause epidemic keratoconjunctivitis, and adenovirus
types 3, 4, and 7 can cause pharyngoconjuctival fever. Herpes
zoster virus (HZV), a member of the Herpesviridae family, can cause
severe eye disease when affecting the trigeminal area. Herpes
zoster ophthalmicus, a severe form of acute herpes zoster, results
from the reactivation of varicella zoster virus (VZV) (another
member of the Herpesviridae family) in the trigeminal (fifth
cranial) nerve. Any branch of the nerve may be affected, though the
frontal branch within the first division of the trigeminal nerve is
most commonly involved. This frontal branch innervates nearly all
of the ocular and periocular structures. Herpes zoster ophthalmicus
at this particular location can lead to blindness and requires a
fast and effective therapeutic approach.
[0003] Human cytomegalovirus (CMV) is another member of the
Herpesviridae family. At least 60% of the US population has been
exposed to CMV, with a prevalence of more than 90% in high-risk
groups (e.g., unborn babies whose mothers become infected with CMV
during pregnancy, people with HIV, and transplant recipients).
[0004] CMV retinitis is one of the most common opportunistic
infections in persons with AIDS or pharmacologically induced
immunosuppression. Individuals with CMV retinitis typically exhibit
a progressive decrease in visual acuity, which may progress to
blindness. Long-term CMV treatment is necessary to prevent
retinitis relapse.
[0005] Immune reconstitution syndrome (IRIS) is reported in 16%-63%
of HIV-infected patients with CMV retinitis following the
initiation of HAART (Highly Active Antiretroviral Treatment). CMV
IRIS may manifest as painless floaters, blurred vision, photopia,
decreased visual acuity, or ocular pain. Some patients may develop
macular edema leading to vision loss or proliferative
vitreoretinopathy, spontaneous vitreal hemorrhage, and retinal
detachment.
[0006] It is known to treat viral eye infections with acyclovir but
such treatment is not entirely satisfactory. Topical acyclovir must
be applied frequently and causes irritation of the eye. Oral
acyclovir causes significant adverse side effects. Other antiviral
medications such as gancyclovir, valacyclovir and valgancyclovir
are used to treat viral eye infections, and such treatments are
also not entirely satisfactory. Gancyclovir is administered
intravenously, and therefore cannot be used outside e.g. a hospital
setting. Oral antiviral medications such as valacyclovir and
valgancyclovir have disadvantages; they are known to cause fever,
rash, diarrhea, and hematologic effects (e.g., neutropenia, anemia,
thrombocytopenia). In some cases neutropenia may respond to
lowering the dose or using drugs that stimulate the production of
neutrophils by the bone marrow as granulocyte colony-stimulating
factor [G-CSF], or granulocyte-macrophage colony-stimulating factor
[GM-CSF]. These toxic effects can be difficult to manage.
[0007] It would therefore be advantageous to provide a better
method and a better pharmaceutical for treating viral eye
infections in humans.
[0008] Various enzymatically active ribonucleases, including
ranpirnase and other proteins that are highly homologous to it, are
known to have antiviral activity, and to have activity against
viruses in the Herpesviridae family (specifically including but not
limited to Herpes simplex virus types 1 and 2 and Human
cytomegalovirus) and also against type 2 adenovirus. However,
proteins are known to be highly irritating to the eye due to an
intense inflammatory response mediated by T-cells. For this reason,
although ranpirnase and other related proteins have been
investigated for use against various viral infections, they have
not been investigated for use against viral infections of the
eye.
[0009] Despite the expectation that proteinaceous ranpirnase would
cause irritation in the eye, irritation of topically applied
ranpirnase in the eye was studied in a rabbit model. In this
experiment, ranpirnase was demonstrated to be non-irritating as
determined using the Globally Harmonized System of Classification
Evaluation Criteria and the European Economic Community Ocular
Evaluation Criteria. This was a remarkable result, because
administration of a foreign protein to the eye can produce corneal
irritation. As a result, ranpirnase and other proteins that are
highly homologous to it are expected to be useful in treating viral
infections of the human eye.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention will be better understood with reference to
the following exemplary and non-limiting drawings, in which:
[0011] FIG. 1 shows the scale used for scoring ocular lesions
observed in a rabbit that has undergone a Draize test;
[0012] FIG. 2 shows the results of a Draize test in which a
ranpirnase solution was applied to the right eye of three
rabbits;
[0013] FIG. 3 shows the results of the Draize test of FIG. 2 in
which the left eye of each of the rabbits was untreated; and
[0014] FIG. 4 shows the European Economic Community Ocular
Evaluation Criteria used to classify the ocular irritation caused
by a test article in a Draize test.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0015] Ranpirnase is a proteinaceous enzymatically active
ribonuclease (the word "ribonuclease" is frequently abbreviated as
"RNase") that is disclosed and claimed in U.S. Pat. No. 5,559,212.
U.S. Pat. Nos. 5,728,805, 6,239,257, 7,229,824 and 8,518,399
disclose three other proteinaceous enzymatically active
ribonucleases that are highly homologous to ranpirnase: [0016] a)
the ribonuclease of SEQ ID NO:2 in U.S. Pat. No. 5,728,805, herein
referred to as the "'805 variant"; [0017] b) the ribonuclease of
SEQ ID NO:2 in U.S. Pat. No. 6,239,257, herein referred to as
"Amphinase 2", and; [0018] c) the ribonuclease of SEQ ID NO:59 of
U.S. Pat. No. 7,229,824, herein referred to as "rAmphinase 2".
[0019] U.S. Pat. No. 8,518,399 discloses that ranpirnase, the '805
variant, and rAmphinase 2 have antiviral activity against
Herpesviridae viruses, specifically including but not limited to
Herpes simplex types 1 and 2 and Human cytomegalovirus. Based upon
its similarities with these three enzymatically active
ribonucleases, it is believed that Amphinase 2 will have these
activities as well.
[0020] Commonly-owned copending patent application Ser. No.
14/736,170 filed Jun. 10, 2015 and published as US 2015/0376584 A1
discloses that ranpirnase has antiviral activity against a number
of viruses, including type 2 adenovirus. As stated therein, the
viruses in the Adenoviridae family are very closely related and the
demonstrated antiviral activity of ranpirnase against any one virus
within the Adenoviridae family is strong evidence that ranpirnase
will have the same anti-replication activity against all viruses
within the Adenoviridae family. Furthermore, as stated in that
pending patent application, to a person of ordinary skill in this
art, the similarities of homology and activity of these three other
ribonucleases is strong evidence that these three other
ribonucleases will have the same activity as ranpirnase has. Hence,
although in vitro experiments have not yet been repeated using the
'805 variant, Amphinase 2, or rAmphinase 2, a person of ordinary
skill in this art would conclude that these three ribonucleases
will likewise be active against all the viruses in the Adenoviridae
family.
[0021] Thus, ranpirnase, the '805 variant, Amphinase 2, and
rAmphinase 2 have either been demonstrated to be active against
type 1 Herpes simplex virus and viruses in the Adenoviridae family
or would be expected to be so active. For this reason, a person of
ordinary skill in the art would expect that all four of these
proteinaceous enzymatically active ribonucleases will be useful
against viral infections of the human eye.
[0022] All four of these ribonucleases are members of the
ribonuclease A superfamily. Such ribonucleases are
pyrimidine-specific endonucleases found in high quantity in the
pancreas of certain mammals and of some reptiles. They are involved
in endonucleolytic cleavage of 3'-phosphomononucleotides and
3'-phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic
phosphate intermediates. Other members of this superfamily include
bovine seminal vesicle and brain ribonucleases, kidney
non-secretory ribonucleases, liver-type ribonucleases, angiogenin;
eosinophil cationic protein, pancreatic ribonucleases from
different species including human, and bovine pancreatic
ribonucleases.
[0023] Ranpirnase, which is disclosed in U.S. Pat. No. 5,559,212,
and was previously known by the ONCONASE trademark, is a
ribonuclease isolated from oocytes of the leopard frog Rana
pipiens. The amino acid sequence of ranpirnase is provided in SEQ
ID NO: 1. Ranpirnase has been tested and found to be cytotoxic to
cancer cells because of its enzymatic activity against RNA.
[0024] A variant of ranpirnase (hereinafter, the "'805 variant") is
disclosed in U.S. Pat. No. 5,728,805. The '805 variant is also a
ribonuclease, and has likewise been found to be cytotoxic to
certain cancer cells. The '805 variant is a close variant of
ranpirnase; its amino acid sequence is identical to that of
ranpirnase except that it has valine instead of isoleucine at
position 11, asparagine instead of aspartic acid at position 20,
and arginine instead of serine at position 103 of the ranpirnase
amino acid sequence. The '805 variant has been referred to as
"Val11, Asn20, Arg103-Ranpirnase". The amino acid sequence of the
'805 variant is provided in SEQ ID NO:2.
[0025] Amphinase 2 is also a ribonuclease. It is the protein
identified as 2325p4 in U.S. Pat. No. 6,239,257 and it too has been
found to be cytotoxic to cancer cells. The amino acid sequence of
Amphinase 2 is provided in SEQ ID NO: 3.
[0026] Recombinant Amphinase 2 ("rArphinase 2") is similar to
Amphinase 2, but has a Met residue at position -1 and lacks glycan
moieties that are located in Amphinase 2 at positions 27 and 91.
rAmphinase 2 is described in U.S. Pat. No. 7,229,824. The amino
acid sequence of rAmphinase 2 is provided in SEQ ID NO: 4.
[0027] The term "functional equivalent" is intended to mean any
protein that differs from any naturally occurring ribonuclease by
the deletion, addition or substitution of one or more amino acids,
but that retains ribonuclease activity. For example, the '805
variant is a functional equivalent of ranpirnase, because it
comprises three amino acid substitutions compared to the ranpirnase
amino acid sequence, but still has RNase activity.
[0028] The disclosures of U.S. Pat. Nos. 5,559,212, 5,728,805,
6,239,257, 7,229,824, 8,518,399, 8,663,964 and published patent
application US 2015/0376584 A1 are all incorporated by reference
herein in their entireties for all purposes.
[0029] As discussed above, it has been found that a ribonuclease of
the RNase A superfamily, in particular ranpirnase, can be used in
the treatment of a viral infection of the eye. A "viral infection
of the eye" is a disease which shows symptoms predominantly in the
eye of a subject and which is caused by viruses rather than
bacteria. Viral diseases of the eye include, but are not limited
to, conjunctivitis and keratitis, retinitis, keratoconjunctivitis,
chorioretinitis, pharyngoconjuctival fever and CMV retinitis.
[0030] Viral conjunctivitis, also known as pink eye, is
characterized by inflammation of the outermost layer of the white
part of the eye and the inner surface of the eyelid. Viral
conjunctivitis is typically caused by an adenovirus. Other viruses
that can be responsible for conjunctival infection include herpes
simplex virus (HSV), varicella-zoster virus (VZV), enterovirus 70,
Coxsackie virus A24, molluscum contagiosum and human
immunodeficiency virus (HIV).
[0031] Viral keratitis is an inflammation of the cornea which is
predominantly caused by herpes simplex virus.
[0032] Chorioretinitis is an inflammation of the choroid (thin
pigmented vascular coat of the eye) and retina of the eye. It is a
form of posterior uveitis. Chorioretinitis can be caused by
infection with cytomegalovirus (CMV), Varicella-Zoster (HZV),
dengue fever, West Nile virus or lymphocytic choriomeningitis virus
(LCMV).
[0033] In particular, viral infections of the eye can be caused by
a virus from the Herpesviridae family of viruses and by an
adenovirus selected from the group consisting of 3, 4, 7, 8, 19,
29, and 37.
[0034] Herpesviridae are viruses having a double-stranded, linear
DNA genome which are classified in Baltimore class I. Viruses from
the Herpesviridae family causing viral infections of the eye
include, but are not limited to, type I Herpes simplex virus, human
cytomegalovirus and Herpes zoster virus, in particular Herpes
zoster ophthalmicus.
[0035] Adenoviridae are double-stranded DNA viruses which are
classified in Baltimore class I. Adenovirus types 8, 19, 29, and 37
can cause epidemic keratoconjunctivitis, and adenovirus types 3, 4,
and 7 can cause pharyngoconjuctival fever types 8, 19, 29, and 37
can cause epidemic keratoconjunctivitis, and Adenovirus types 3, 4,
and 7 can cause pharyngoconjuctival fever.
[0036] The terms "treating" and "treatment," as used herein, refer
to administering to a subject having a viral infection of the eye a
therapeutically effective dose of a ribonuclease such as
ranpirnase, a ranpirnase variant such as the '805 variant,
Arnphinase 2, or rAmphinase 2. As used herein, the term "treating"
covers any treatment of a viral infection of the eye which results
in a desired pharmacologic and/or physiologic effect, including
arresting disease development, causing regression of the disease,
limiting spread of the virus from one cell to another within an
individual, limiting replication of a virus in an individual,
limiting entry of a virus into the cell of an individual and
reducing the number of viruses in an individual or a tissue of this
individual.
[0037] The term "therapeutically effective dose" refers to an
amount of a pharmaceutical that results in an improvement or
remediation of the symptoms of a disease or condition to be
treated. A therapeutically effective dose of a ribonuclease such as
ranpirnase, '805 variant, Amphinase 2, or rAmphinase 2, delays or
minimizes the onset of, or hastens or increases recovery of a
subject from, a viral infection of the eye in a subject. The RNase
may reduce the viral titer in the eye of the infected subject, or
may prevent the viral titer in the eye of the infected subject from
increasing. A therapeutically effective dose of a ribonuclease may
provide a therapeutic benefit in the treatment or management of a
viral infection of the eye by reducing the spread of the virus from
one cell to another and may also prevent disease and/or reduce the
severity of symptoms.
[0038] A therapeutically effective dose can be determined by the
skilled person as a matter of routine experimentation. The
therapeutically effective dosage of the pharmaceutical composition
can be determined readily by the skilled artisan, for example, from
animal studies. In addition, human clinical studies can be
performed to determine the preferred effective dose for humans by a
skilled artisan. Such clinical studies are routine and well known
in the art. The precise dose to be employed will also depend on the
route of administration. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal test
systems.
[0039] The RNase may be administered to a subject in need thereof
in a single dose or in multiple doses. The RNase may be
administered to the subject until symptoms resolve and/or until the
subject is no longer at risk of a virus infection. The schedule on
which the administration of the RNase (advantageously, ranpirnase)
commences, and on which a single dose or multiple doses of the
RNase (advantageously, ranpirnase) is or are administered to the
subject, and the duration of dosing, can be determined by a person
of ordinary skill. These factors may depend on factors such as the
severity of symptoms, patient response, etc.
[0040] The administration of a therapeutically effective dose of
the RNase (advantageously, ranpirnase) may reduce the virus titer
in the eye compared to a control that is infected with the virus
but not treated with the RNase.
[0041] The administration of a therapeutically effective dose of
the RNase (advantageously, ranpirnase) may lead to a reduction of
the virus titer below the detection level. Determination of virus
titers is for example discussed in Reischl (1996) Front Biosci. 1:e
72-7, Application of molecular biology-based methods to the
diagnosis of infectious diseases.
[0042] The ribonuclease (advantageously, ranpirnase) is preferably
administered topically to the eye, i.e. is administered directly to
the eye and not elsewhere on the body. The topical administration
may be by e.g. eye drops, a suspension, an emulsion, an ointment, a
solution, a gel, liposomes, nanoparticles, microemulsions,
nanoemulsions, nanosuspensions, niosomes, dendrimers and hydrogels.
Alternatively, the ribonuclease (advantageously, ranpirnase) may be
administered by the intravitreal, intracameral, subconjunctival,
subtenon, retrobulbar or posterior juxtascleral route.
[0043] The ribonuclease will be a component of a pharmaceutical
that includes a vehicle. The term "vehicle" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. These agents are generally safe,
non-toxic and neither biologically nor otherwise undesirable.
[0044] The vehicle may contain ingredients such as an excipient, a
surfactant, an ingredient to adjust the pH of the composition and
to buffer within a certain pH range, and a tonicity agent to adjust
the tonicity of the composition. Such ingredients are known, and
persons of ordinary skill in the art can select such of them as
will produce a formulation having appropriate characteristics when
combined with the RNase (advantageously, ranpirnase).
[0045] It is possible to determine whether a substance (here, a
ribonuclease) is non-irritating to the eye in a Draize test using
the Globally Harmonized System of Classification Evaluation
Criteria and using the European Economic Community Ocular
Evaluation Criteria. In the Draize test the RNase is placed in the
conjunctival sac of a rabbit's eye and the eye is examined at 1,
24, 48 and 72 hours after instillation of ranpirnase. The criteria
evaluated are corneal opacity, iris lesion, conjunctival redness
and conjunctival edema. If the score for each of these criteria is
zero, the tested substance is considered non-irritating to the eye.
FIGS. 1 and 4 show, respectively, the scale used for scoring ocular
lesions observed in a rabbit that has undergone a Draize test and
the European Economic Community Ocular Evaluation Criteria used to
classify the ocular irritation caused by a test article in a Draize
test.
[0046] As stated above, before the present invention, no person of
ordinary skill in the art would administer to the eye ranpirnase or
any of the other three above-identified proteinaceous enzymatically
active ribonucleases. However, such administration of ranpirnase
has been modeled using the Draize test and the results of this
experiment demonstrate that ranpirnase is non-irritating as defined
by two accepted standards.
Example
[0047] A 0.1% mL solution made up of 0.1% ranpirnase in a
proprietary aqueous solution used as a vehicle was used as a test
article. Three rabbits were used; each was a male New Zealand White
rabbit that was approximately 16 weeks old at the time of the
experiment and that weighed 3.3 to 3.4 kg.
[0048] After administration of two drops of Tetracaine
pre-anesthetic to the corneal surface of both eyes of each rabbit,
the test article was placed in the conjunctival sac of the right
eye of each rabbit by gently pulling the lower lid away from the
eyeball; the lids were gently held together for approximately one
second to limit the loss of the test material. The left eye of each
rabbit remained untreated and served as the control. The eyes of
the animals were examined at 1 (+15 minutes), 24, 48, and 72 hours
(.+-.1 hour) after installation of the test article. The grades of
ocular reaction according to Draize (FIG. 1) were manually recorded
at each examination (FIGS. 2 and 3). As can be seen in FIG. 2,
there was an ocular reaction in each animal one hour
post-instillation of the test article but in every instance that
reaction was completely resolved by 24 hours and thereafter.
[0049] To determine the degree of irritation caused by the test
article using the European Economic Community Ocular Evaluation
Criteria (FIG. 4), the total ocular irritation scores for the
examinations at 24, 48, and 72 hours were individually added for
corneal opacity, iris lesion, conjunctival redness, and
conjunctival edema and the mean scores for these scoring parameters
were compared to the European Economic Community Ocular Evaluation
Criteria. Because all these scores (and therefore the calculated
mean scores) were zero, the test subject was considered to be
non-irritating as defined by the European Economic Community Ocular
Evaluation Criteria.
[0050] To determine the degree of irritation caused by the test
article using the Globally Harmonized System of Classification
Evaluation Criteria, the 24-, 48-, and 72-hour scores were added
separately for each animal and each total divided by 3 (three time
points) to yield the individual mean scores for each animal.
Because all these scores (and therefore the calculated quotients)
were zero, the test subject was considered to be non-irritating as
defined by the Globally Harmonized System of Classification
Evaluation Criteria.
[0051] Hence, these test data demonstrate a new and unexpected
result: ranpirnase delivered to the eye in an aqueous solution is
non-irritating as defined by the Globally Harmonized System of
Classification Evaluation Criteria and by the European Economic
Community Ocular Evaluation Criteria, even though ranpirnase is a
protein (which would be expected to be irritating to the eye).
[0052] Although this experiment was carried out using a solution of
ranpirnase in a proprietary aqueous vehicle, a person of ordinary
skill in the art would consider it likely that solutions of the
three above-identified proteinaceous enzymatically active
ribonucleases would behave in the same way because of their
similarities to ranpirnase in respect of activity and homology.
[0053] Although at least one preferred embodiment of the invention
has been described above, this description is not limiting and is
only exemplary. The scope of the invention is defined only by the
claims, which follow:
Sequence CWU 1
1
41104PRTRana pipiens 1Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile
Thr Asn Thr Arg Asp1 5 10 15Val Asp Cys Asp Asn Ile Met Ser Thr Asn
Leu Phe His Cys Lys Asp 20 25 30Lys Asn Thr Phe Ile Tyr Ser Arg Pro
Glu Pro Val Lys Ala Ile Cys 35 40 45Lys Gly Ile Ile Ala Ser Lys Asn
Val Leu Thr Thr Ser Glu Phe Tyr 50 55 60Leu Ser Asp Cys Asn Val Thr
Ser Arg Pro Cys Lys Tyr Lys Leu Lys65 70 75 80Lys Ser Thr Asn Lys
Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val 85 90 95His Phe Val Gly
Val Gly Ser Cys 1002104PRTartificialvariant '805 of ranpirnase 2Glu
Asp Trp Leu Thr Phe Gln Lys Lys His Val Thr Asn Thr Arg Asp1 5 10
15Val Asp Cys Asn Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp
20 25 30Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile
Cys 35 40 45Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu
Phe Tyr 50 55 60Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr
Lys Leu Lys65 70 75 80Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu
Asn Gln Ala Pro Val 85 90 95His Phe Val Gly Val Gly Arg Cys
1003114PRTRana pipiens 3Lys Pro Lys Glu Asp Arg Glu Trp Glu Lys Phe
Lys Thr Lys His Ile1 5 10 15Thr Ser Gln Ser Val Ala Asp Phe Asn Cys
Asn Arg Thr Met Asn Asp 20 25 30Pro Ala Tyr Thr Pro Asp Gly Gln Cys
Lys Pro Ile Asn Thr Phe Ile 35 40 45His Ser Thr Thr Gly Pro Val Lys
Glu Ile Cys Arg Arg Ala Thr Gly 50 55 60Arg Val Asn Lys Ser Ser Thr
Gln Gln Phe Thr Leu Thr Thr Cys Lys65 70 75 80Asn Pro Ile Arg Cys
Lys Tyr Ser Gln Ser Asn Thr Thr Asn Phe Ile 85 90 95Cys Ile Thr Cys
Arg Asp Asn Tyr Pro Val His Phe Val Lys Thr Gly 100 105 110Lys
Cys4115PRTartificialrAmphinase 2 4Met Lys Pro Lys Glu Asp Arg Glu
Trp Glu Lys Phe Lys Thr Lys His1 5 10 15Ile Thr Ser Gln Ser Val Ala
Asp Phe Asn Cys Asn Arg Thr Met Asn 20 25 30Asp Pro Ala Tyr Thr Pro
Asp Gly Gln Cys Lys Pro Ile Asn Thr Phe 35 40 45Ile His Ser Thr Thr
Gly Pro Val Lys Glu Ile Cys Arg Arg Ala Thr 50 55 60Gly Arg Val Asn
Lys Ser Ser Thr Gln Gln Phe Thr Leu Thr Thr Cys65 70 75 80Lys Asn
Pro Ile Arg Cys Lys Tyr Ser Gln Ser Asn Thr Thr Asn Phe 85 90 95Ile
Cys Ile Thr Cys Arg Asp Asn Tyr Pro Val His Phe Val Lys Thr 100 105
110Gly Lys Cys 115
* * * * *