CA2160704A1 - Ocular delivery of nucleic acid - Google Patents
Ocular delivery of nucleic acidInfo
- Publication number
- CA2160704A1 CA2160704A1 CA002160704A CA2160704A CA2160704A1 CA 2160704 A1 CA2160704 A1 CA 2160704A1 CA 002160704 A CA002160704 A CA 002160704A CA 2160704 A CA2160704 A CA 2160704A CA 2160704 A1 CA2160704 A1 CA 2160704A1
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- CA
- Canada
- Prior art keywords
- ribozyme
- rna
- nucleic acid
- carbopol
- pharmaceutical composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Ophthalmology & Optometry (AREA)
- Inorganic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
Pharmaceutical composition comprising polyacrylic acid and a therapeutically effective amount of a nucleic acid.
Description
2160704`-DESCRIPTION
Ocular Deliverv of Nucleic Acid Backcround of the Invention This invention relates to therapeutic drug delivery vehicles for ocular tissue.
Ac~erman and Jones, U.S. Patent 2,923,692, describe mucilaginous compositions formed from carboxylic polymers, such as polymers of acrylic acid. These polymers are said to be useful as thickening agents. The mucilaginous compositions are said to be useful as textile printing pastes or printing inks, for cosmetics, polishing and cleaning compositions.
Carbopol has been used in some ocular delivery systems. For example, Davies et al., 9 Pharm. Res. 1137, 1992, discusses use of carb~pol in phospholipid vessels for entrapment of tropicamide; and Weinreb and Tani, 46 lS J. Pasenter. Sci. Technol. 51, 1992, discuss use of a polyacrylic acid polymer with Betaxolol for ocular delivery.
Summar~ of the Invention Applicant describes nucleic acid formulations designed for ocular delivery formed from polymers of acrylic acid. In particular, applicant describes the use of enzymatic RNA in such a formulation, useful for delivery of that enzymatic RNA to ocular tissue. In one example, an enzymatic RNA containing five 2'-O-methylated riboses in stems I and III of a hammerhead-type ribozyme (see below) were formulated in carbopol 974, which is a polymer of acrylic acid. Upon hydration with water, the polymer formed a viscous solution at 1~ concentration when the pH was between 4 and 4.5. Neutralization with ammonium hydroxide resulted in the viscous solution forming a complete gel, in which the internal compartment had a high intrinsic viscosity. Addition of cation to W094~4g~ ~ ~ ~ PCT~S94/04609 this gel caused the gel to collapse, thereby allowing release of the associated enzymatic RNA.
Thus, in a first aspect, the invention features a therapeutic composition containing a polyacrylic acid at a concentration sufficient to form a gel (e.a., between 0.5 and 1.5~), and a therapeutically effective amount of a nucleic acid molecule. In particularly preferred embodiments, the nucleic acid is RNA, e.q., enzymatic RNA.
Polyacrylic acids useful in the invention are described by Ackerman and Jones, supra, and are well known to those of ordinary skill in the relevant art. In addition, nucleic acid molecules useful for delivery are well known in the art and include enzymatic RNA molecules, or ribozymes.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. It is said that such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc.
Natl. Acad. of Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA
target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology WOg4~4g~ 2 1..6 0 7 0~ ~ PCT~S94/04609 (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the riboæyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA
target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
- This class of chemicals exhibits a high degree of specificity for cleavage of the intended target mRNA.
Consequently, the ribozyme agent will only affect cells expressing that particular gene, and will not be toxic to normal tissues.
By "enzymatic RNA molecule" it is meant an RNA
molecule which has complementarity in a substrate binding region to a specified mRNA target, and also has an enzymatic activity which is active to specifically cleave that mRNA. That is, the enzymatic RNA molecule is able to intermolecularly cleave mRNA and thereby inactivate a target mRNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA
molecule to the target RNA to allow the cleavage to occur.
One hundred percent complementarity is preferred, but complementarity as low as 50-75~ may also be useful in W094/~g~ 21 6 0 7 ~ PCT~S94/0~09 this invention. For in vivo treatment, complementarity between 30 and 45 bases is preferred.
In preferred embodiments, the enzymatic RNA molecule is formed in a h~mmerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I
intron or RNAseP-like RNA (in association with an RNA
guide sequence). Examples of such hammerhead motifs are described by Rossi et al., 8 Aids Research and Human Retroviruses 183, 1992, of hairpin motifs by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences", filed September 20, 1989, which is a continuation-in-part of U.S. Serial No. 07/247,100, filed September 20, 1988;
Hampel and Tritz, 28 BiochemistrY 4g29, 1989; and Hampel et al., 18 Nucleic Acids Research 299, 1990, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 31 BiochemistrY 16, 1992, of the RNAseP
motif by Guerrier-Takada, et al., 35 Cell 849, 1983, and of the group I intron by Cech et al., U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic RNA molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
By "therapeutically effective amount" is meant that amount sufficient to have an effect on the disease phenotype or condition to be treated. For example, in treatment of viral infections, such as herpes simplex virus, in ocular tissue it is the amount sufficient to lower the amount of viral nucleic acid present within the ocular tissue, or to reduce the number of viral forming units within that tissue.
In particularly preferred embodiments, the invention features administration of enzymatic RNA or ribozymes effective to treat herpes simplex virus, for example, - 2I6070~ ~ -those molecules described by Draper, U.S. Serial No.
07/882,921, filed May 14, 1992, entitled "Method and Reagent for Inhibiting Herpes Simplex Virus Replicationl', Draper, U.S. Serial No. 07/948,359, filed September 18, 1992, and Draper, U.S. Serial No. 07/987,133, filed December 7, 1992, all hereby incorporated by reference herein and assigned to the same assignee as the present application.
In a related aspect, the invention features a method ld for treatment of ocular tissue by administering therapeutic nucleic acid to that tissue by use of a polyacryllc acid polymer, with or without an additional chelator.
The composition and methods of this invention are significantly advantageous over administration of free nucleic acid to an eye, increasing the dose delivered to an eye up to about six-fold, facilitating patient compliance by decreasing the number of required administrations, protecting the ribozyme from nuclease degradation.
The methods of this invention are also suitable for administration of ribozymes to other tissues, including lung, gut, esophagus, skin, nasal, vaginal and even veins.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments The drawings will first briefly be described.
Drawinas Fig. 1 is a graphical representation showing ocular delivery of ribozymes using unpolymerized carbopol 974.
Fig. 2 is a histogram showing ribozyme uptake by ccular tissue in relationship to percent carbopol 974.
216070g - -Fig. 3 is a copy of various autoradiograms showing retention of ribozymes by ocular tissue after delivery in water, or in polymerized or unpolymerized carbopol 974.
Fig. 4 is a copy of various autoradiograms showing degradation of delivered ribozymes by micrococcal nuclease.
Fig. 5 is a dose response curve showing total amount of ocular associated ribozyme prior to extraction (B), and the amount of ribozyme extracted from eye (C). (Ribozyme 10` extraction efficiency was 50~.) PolvacrYlic Acid Polyacrylic acids useful in the present invention are - generally described in the art cited above. Below are provided examples of use of various polyacrylic acids as ocular delivery vehicles for ribozymes and other nucleic acids. Specifically, five forms of polyacrylic acid were obtained from B.F. Goodrich, including carbnpol 974, Pemulens TR1 and TR2, and carbopol 940. Prior to testing in vivo in mice, in vitro studies were performed to determine the compatibility of these polymers with the nucleic acid. These examples are not limiting in the present invention, but are provided merely to illustrate to those of ordinary skill in the art the breadth of the invention. While ribozymes exemplify the use of nucleic acid for delivery, they are not limiting in the invention.
Applicant has found that these polyacrylic acids are useful for topical delivery, e.a., ocular, buccal or vaginal delivery, of nucleic acids. Unpolymerized polyacrylic acid including the nucleic acid is a fluid, but when placed in contact with cationic groups or neutral pH (as found in tears) it forms a gel and allows slow release of the nucleic acid. Thus, it forms a useful pharmaceutical delivery vehicle composition. The following examples illustrate these properties and the use of such polyacrylic acids.
. 216D704 Exam~le 1: RibozYme/PolYacr~lic Acid Mixtures Carbopol is a polymer of acrylic acid and has some useful properties as an ocular delivery vehicle. Upon hydration with water, a viscous solution forms at 1%
s concentration when the pH is between 4 and 4.5 (unpolymerized carbapol). Neutralization with ammonium hydroxide results in the viscous solution forming a complete gel, in which the internal compartment has a high intrinsic viscosity (polymerized carb~pol). Addition of 10- a cationic moiety causes the gel to collapse. This was demonstrated by adding a hammerhead ribozyme (any one can be used) to the acid solution of the polymer on a piece of parafilm. Addition of water washed the ribozyme off the parafilm, as did addition of ammonium hydroxide followed by a phosphate buffered saline wash (1~0 mM NaCl, PBS).
Addition of ammonium hydroxide followed by the water wash, however, did not wash the ribozyme off the parafilm. The same results were observed when a collagen shield was substituted for the parafilm.
Specifically, carb~pol dry powder was hydrated with an aqueous phase containing a ribozyme. The concentration of ribozyme was 10 mg/ml and the carbopol was hydrated to 0.5~. Upon hydration, the viscous solution had a- pH
between 3 and 4. The solution was polymerized by bringing the pH to 7 with 0.2 M NH40H. The degree of ribozyme association was determined by layering 1 ml 30% ficoll 400 in water over the polymerized gel, followed by 1 ml 10 ficoll with 1 ml water layered over the top. The tubes were centrifuged and the polymerized gel with the ribozyme floated to the top. 75~ of the ribozyme was at the 10~
ficoll/water interface. In the absence of carbopol, no ribozyme was located at the 10% ficoll/water interface.
In another method, which was essentially the same as above, the same amount of ribozyme was located at the 10~
ficoll/water interface when the ribozyme was added to a solution of carbopol, rather than hydrating the dry powder with a solution of ribozyme.
--- 216070 1 f The carbopol hydrated with ribozyme, or a 1/1 solution of 1~ carbopol and 1 to 2 mg/ml ribozyme, were added to a piece of parafilm. The gel was polymerized by the addition of ammonium hydroxide. The strip of parafilm s was washed in water 3X to remove any non-polymer associated ribozyme. The strips were washed with phosphate buffered saline (0.8% NaCl, 0.02% KCl and 10 mM
Na phosphate), pH 7.4, and the strips were counted. 100~
of the ribozyme was retained on the parafilm after 3x water wash. 0% remained after lx wash with PBS.
Example 2: Ocular Retention of Delivered Ribozvme The following procedure was used to measure ribozyme delivery to ocular tissue. Mice were anesthetized with sodium pentobarbital with a dose of 50 mg/kg. Ten ~g of a hammerhead-type ribozyme was enzymatically labeled with 32P~-ATP at the 5' end. The radiolabeled ribozyme was used to spike cold ribozyme yielding a specific activity between 7.5 X 104 and 1.5 X 105 DPM/~g. The ribozyme was mixed 1:1 with a 1% unpolymerized carbOpol solution in water. A dose of 0.5 ~g, 1 ~g, 5 ~g, 10 ~g and 20 ~g was applied to the mouse eye in 0.5% carbapol in a volume of 2 ~l. The ~n;m~ls were euthenized at 3, 10, 30 and 60 minutes after administration. The eyes were surgically removed, washed in 10 ml PBS 3X, added to 200 ~l lysis buffer [4M guanidinium isothiocynate 0.5~ sarcosyl 25 mM
sodium citrate] and immediately frozen in liquid nitrogen.
The eyes were thawed by sonication in a strong bath sonicator (Scientific Products, Hicksville, Long Island).
The freezing and bath sonicator thawing was repeated 2 more times. The total RNA from the eyes was extracted using a chloroform/phenol extraction procedure "Guanidinium Methods for Total RNA Preparation,ll Current Protocols in Molecular BiolooY, (Ed. F.M. Ausubell) Greene Publishing Assoc., and Wiley-Interscience, John Wiley &
Sons, New York, pp. 4.2.1-4.2.8, (1992). The ethanol precipitated RNA was re-suspended in 10 ~l gel loading 216070~
buffer [95~ formamide, 20 mM EPTA, 0.5% xylene cyanol, 0.05% bromophenol blue]. Two ~l was counted for radioactivity and 8 ~l applied to a 20~ acrylamide gel.
The following studies were performed with a hammerhead ribozyme that contains five 2'-O-methyl riboses in stem I and stem III of the ribozyme:
1. The optimal percent of carbopol 974 was determined using the above protocol, and applying 1 ~g of ribozyme to-each eye in 0.0~, 0.05~, 0.10~ and 0.5%
i0 carbopol. The results are summarized in Fig. 2. 0.5%
carbopol was selected to be the optimum. No higher concentration of carbOpol was tested because the solution was too viscous, and reproducability of dose delivery was compromised.
2. A time course for retention of intact ribozyme was performed using carbopol 974. One ~g of the radiolabeled ribozyme was applied to the eye with 0.5~
carbapol 974, and the eyes were removed 3, 10, 30 and 60 minutes after application. Two mice were used per time point. The results are shown in Fig. 1. The results showed that an optimum eye retention was obtained 30 minutes after administration, yielding 4 pmol retained by the eye from an 83 pmol dose.
Ocular Deliverv of Nucleic Acid Backcround of the Invention This invention relates to therapeutic drug delivery vehicles for ocular tissue.
Ac~erman and Jones, U.S. Patent 2,923,692, describe mucilaginous compositions formed from carboxylic polymers, such as polymers of acrylic acid. These polymers are said to be useful as thickening agents. The mucilaginous compositions are said to be useful as textile printing pastes or printing inks, for cosmetics, polishing and cleaning compositions.
Carbopol has been used in some ocular delivery systems. For example, Davies et al., 9 Pharm. Res. 1137, 1992, discusses use of carb~pol in phospholipid vessels for entrapment of tropicamide; and Weinreb and Tani, 46 lS J. Pasenter. Sci. Technol. 51, 1992, discuss use of a polyacrylic acid polymer with Betaxolol for ocular delivery.
Summar~ of the Invention Applicant describes nucleic acid formulations designed for ocular delivery formed from polymers of acrylic acid. In particular, applicant describes the use of enzymatic RNA in such a formulation, useful for delivery of that enzymatic RNA to ocular tissue. In one example, an enzymatic RNA containing five 2'-O-methylated riboses in stems I and III of a hammerhead-type ribozyme (see below) were formulated in carbopol 974, which is a polymer of acrylic acid. Upon hydration with water, the polymer formed a viscous solution at 1~ concentration when the pH was between 4 and 4.5. Neutralization with ammonium hydroxide resulted in the viscous solution forming a complete gel, in which the internal compartment had a high intrinsic viscosity. Addition of cation to W094~4g~ ~ ~ ~ PCT~S94/04609 this gel caused the gel to collapse, thereby allowing release of the associated enzymatic RNA.
Thus, in a first aspect, the invention features a therapeutic composition containing a polyacrylic acid at a concentration sufficient to form a gel (e.a., between 0.5 and 1.5~), and a therapeutically effective amount of a nucleic acid molecule. In particularly preferred embodiments, the nucleic acid is RNA, e.q., enzymatic RNA.
Polyacrylic acids useful in the invention are described by Ackerman and Jones, supra, and are well known to those of ordinary skill in the relevant art. In addition, nucleic acid molecules useful for delivery are well known in the art and include enzymatic RNA molecules, or ribozymes.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. It is said that such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc.
Natl. Acad. of Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA
target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology WOg4~4g~ 2 1..6 0 7 0~ ~ PCT~S94/04609 (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the riboæyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA
target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
- This class of chemicals exhibits a high degree of specificity for cleavage of the intended target mRNA.
Consequently, the ribozyme agent will only affect cells expressing that particular gene, and will not be toxic to normal tissues.
By "enzymatic RNA molecule" it is meant an RNA
molecule which has complementarity in a substrate binding region to a specified mRNA target, and also has an enzymatic activity which is active to specifically cleave that mRNA. That is, the enzymatic RNA molecule is able to intermolecularly cleave mRNA and thereby inactivate a target mRNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA
molecule to the target RNA to allow the cleavage to occur.
One hundred percent complementarity is preferred, but complementarity as low as 50-75~ may also be useful in W094/~g~ 21 6 0 7 ~ PCT~S94/0~09 this invention. For in vivo treatment, complementarity between 30 and 45 bases is preferred.
In preferred embodiments, the enzymatic RNA molecule is formed in a h~mmerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I
intron or RNAseP-like RNA (in association with an RNA
guide sequence). Examples of such hammerhead motifs are described by Rossi et al., 8 Aids Research and Human Retroviruses 183, 1992, of hairpin motifs by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences", filed September 20, 1989, which is a continuation-in-part of U.S. Serial No. 07/247,100, filed September 20, 1988;
Hampel and Tritz, 28 BiochemistrY 4g29, 1989; and Hampel et al., 18 Nucleic Acids Research 299, 1990, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 31 BiochemistrY 16, 1992, of the RNAseP
motif by Guerrier-Takada, et al., 35 Cell 849, 1983, and of the group I intron by Cech et al., U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic RNA molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
By "therapeutically effective amount" is meant that amount sufficient to have an effect on the disease phenotype or condition to be treated. For example, in treatment of viral infections, such as herpes simplex virus, in ocular tissue it is the amount sufficient to lower the amount of viral nucleic acid present within the ocular tissue, or to reduce the number of viral forming units within that tissue.
In particularly preferred embodiments, the invention features administration of enzymatic RNA or ribozymes effective to treat herpes simplex virus, for example, - 2I6070~ ~ -those molecules described by Draper, U.S. Serial No.
07/882,921, filed May 14, 1992, entitled "Method and Reagent for Inhibiting Herpes Simplex Virus Replicationl', Draper, U.S. Serial No. 07/948,359, filed September 18, 1992, and Draper, U.S. Serial No. 07/987,133, filed December 7, 1992, all hereby incorporated by reference herein and assigned to the same assignee as the present application.
In a related aspect, the invention features a method ld for treatment of ocular tissue by administering therapeutic nucleic acid to that tissue by use of a polyacryllc acid polymer, with or without an additional chelator.
The composition and methods of this invention are significantly advantageous over administration of free nucleic acid to an eye, increasing the dose delivered to an eye up to about six-fold, facilitating patient compliance by decreasing the number of required administrations, protecting the ribozyme from nuclease degradation.
The methods of this invention are also suitable for administration of ribozymes to other tissues, including lung, gut, esophagus, skin, nasal, vaginal and even veins.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments The drawings will first briefly be described.
Drawinas Fig. 1 is a graphical representation showing ocular delivery of ribozymes using unpolymerized carbopol 974.
Fig. 2 is a histogram showing ribozyme uptake by ccular tissue in relationship to percent carbopol 974.
216070g - -Fig. 3 is a copy of various autoradiograms showing retention of ribozymes by ocular tissue after delivery in water, or in polymerized or unpolymerized carbopol 974.
Fig. 4 is a copy of various autoradiograms showing degradation of delivered ribozymes by micrococcal nuclease.
Fig. 5 is a dose response curve showing total amount of ocular associated ribozyme prior to extraction (B), and the amount of ribozyme extracted from eye (C). (Ribozyme 10` extraction efficiency was 50~.) PolvacrYlic Acid Polyacrylic acids useful in the present invention are - generally described in the art cited above. Below are provided examples of use of various polyacrylic acids as ocular delivery vehicles for ribozymes and other nucleic acids. Specifically, five forms of polyacrylic acid were obtained from B.F. Goodrich, including carbnpol 974, Pemulens TR1 and TR2, and carbopol 940. Prior to testing in vivo in mice, in vitro studies were performed to determine the compatibility of these polymers with the nucleic acid. These examples are not limiting in the present invention, but are provided merely to illustrate to those of ordinary skill in the art the breadth of the invention. While ribozymes exemplify the use of nucleic acid for delivery, they are not limiting in the invention.
Applicant has found that these polyacrylic acids are useful for topical delivery, e.a., ocular, buccal or vaginal delivery, of nucleic acids. Unpolymerized polyacrylic acid including the nucleic acid is a fluid, but when placed in contact with cationic groups or neutral pH (as found in tears) it forms a gel and allows slow release of the nucleic acid. Thus, it forms a useful pharmaceutical delivery vehicle composition. The following examples illustrate these properties and the use of such polyacrylic acids.
. 216D704 Exam~le 1: RibozYme/PolYacr~lic Acid Mixtures Carbopol is a polymer of acrylic acid and has some useful properties as an ocular delivery vehicle. Upon hydration with water, a viscous solution forms at 1%
s concentration when the pH is between 4 and 4.5 (unpolymerized carbapol). Neutralization with ammonium hydroxide results in the viscous solution forming a complete gel, in which the internal compartment has a high intrinsic viscosity (polymerized carb~pol). Addition of 10- a cationic moiety causes the gel to collapse. This was demonstrated by adding a hammerhead ribozyme (any one can be used) to the acid solution of the polymer on a piece of parafilm. Addition of water washed the ribozyme off the parafilm, as did addition of ammonium hydroxide followed by a phosphate buffered saline wash (1~0 mM NaCl, PBS).
Addition of ammonium hydroxide followed by the water wash, however, did not wash the ribozyme off the parafilm. The same results were observed when a collagen shield was substituted for the parafilm.
Specifically, carb~pol dry powder was hydrated with an aqueous phase containing a ribozyme. The concentration of ribozyme was 10 mg/ml and the carbopol was hydrated to 0.5~. Upon hydration, the viscous solution had a- pH
between 3 and 4. The solution was polymerized by bringing the pH to 7 with 0.2 M NH40H. The degree of ribozyme association was determined by layering 1 ml 30% ficoll 400 in water over the polymerized gel, followed by 1 ml 10 ficoll with 1 ml water layered over the top. The tubes were centrifuged and the polymerized gel with the ribozyme floated to the top. 75~ of the ribozyme was at the 10~
ficoll/water interface. In the absence of carbopol, no ribozyme was located at the 10% ficoll/water interface.
In another method, which was essentially the same as above, the same amount of ribozyme was located at the 10~
ficoll/water interface when the ribozyme was added to a solution of carbopol, rather than hydrating the dry powder with a solution of ribozyme.
--- 216070 1 f The carbopol hydrated with ribozyme, or a 1/1 solution of 1~ carbopol and 1 to 2 mg/ml ribozyme, were added to a piece of parafilm. The gel was polymerized by the addition of ammonium hydroxide. The strip of parafilm s was washed in water 3X to remove any non-polymer associated ribozyme. The strips were washed with phosphate buffered saline (0.8% NaCl, 0.02% KCl and 10 mM
Na phosphate), pH 7.4, and the strips were counted. 100~
of the ribozyme was retained on the parafilm after 3x water wash. 0% remained after lx wash with PBS.
Example 2: Ocular Retention of Delivered Ribozvme The following procedure was used to measure ribozyme delivery to ocular tissue. Mice were anesthetized with sodium pentobarbital with a dose of 50 mg/kg. Ten ~g of a hammerhead-type ribozyme was enzymatically labeled with 32P~-ATP at the 5' end. The radiolabeled ribozyme was used to spike cold ribozyme yielding a specific activity between 7.5 X 104 and 1.5 X 105 DPM/~g. The ribozyme was mixed 1:1 with a 1% unpolymerized carbOpol solution in water. A dose of 0.5 ~g, 1 ~g, 5 ~g, 10 ~g and 20 ~g was applied to the mouse eye in 0.5% carbapol in a volume of 2 ~l. The ~n;m~ls were euthenized at 3, 10, 30 and 60 minutes after administration. The eyes were surgically removed, washed in 10 ml PBS 3X, added to 200 ~l lysis buffer [4M guanidinium isothiocynate 0.5~ sarcosyl 25 mM
sodium citrate] and immediately frozen in liquid nitrogen.
The eyes were thawed by sonication in a strong bath sonicator (Scientific Products, Hicksville, Long Island).
The freezing and bath sonicator thawing was repeated 2 more times. The total RNA from the eyes was extracted using a chloroform/phenol extraction procedure "Guanidinium Methods for Total RNA Preparation,ll Current Protocols in Molecular BiolooY, (Ed. F.M. Ausubell) Greene Publishing Assoc., and Wiley-Interscience, John Wiley &
Sons, New York, pp. 4.2.1-4.2.8, (1992). The ethanol precipitated RNA was re-suspended in 10 ~l gel loading 216070~
buffer [95~ formamide, 20 mM EPTA, 0.5% xylene cyanol, 0.05% bromophenol blue]. Two ~l was counted for radioactivity and 8 ~l applied to a 20~ acrylamide gel.
The following studies were performed with a hammerhead ribozyme that contains five 2'-O-methyl riboses in stem I and stem III of the ribozyme:
1. The optimal percent of carbopol 974 was determined using the above protocol, and applying 1 ~g of ribozyme to-each eye in 0.0~, 0.05~, 0.10~ and 0.5%
i0 carbopol. The results are summarized in Fig. 2. 0.5%
carbopol was selected to be the optimum. No higher concentration of carbOpol was tested because the solution was too viscous, and reproducability of dose delivery was compromised.
2. A time course for retention of intact ribozyme was performed using carbopol 974. One ~g of the radiolabeled ribozyme was applied to the eye with 0.5~
carbapol 974, and the eyes were removed 3, 10, 30 and 60 minutes after application. Two mice were used per time point. The results are shown in Fig. 1. The results showed that an optimum eye retention was obtained 30 minutes after administration, yielding 4 pmol retained by the eye from an 83 pmol dose.
3. A dose response curve was tested from 0.5 ~g to 20 ~g (42 pmol to 1660 pmols) ribozyme. The ribozyme was administered with 0.5% carkopol in a volume of 2 ~l. The results show the uptake was linear over the dose range tested.
The above experiments show that 5~ of the administered dose is retained by the eye after 3 minutes and 10 minutes topical administration to the mouse eye.
Free ribozyme yields 1~ retention. We also found that unpolymerized carbopol-containing ribozyme yields a higher percentage of uptake, compared to administration of polymerized carbopol-containing ribozyme.
`- ~ 21 6070~-Example 3: Ocular Retention To determine if the ribozyme was in the eye as opposed to being bound to the outside, a comparison of washes was made. The mouse eyes were washed with water, PBS or PBS + micrococcal nuclease. The PBS wash removed 80% of the ribozyme from the polymerized car~ pol, but only 20~ from the unpolymerized carbQpol. The amount of ribozyme administered without car~ pol was unchanged. The micrococcal nuclease treatment showed a 40~ reduction in the amount of ribozyme retained by the eye. However, it was observed that the carbopol inhibited the micrococcal nuclease activity. This may have been due to a variety of reasons, but one control that was run ,was to inhibit the enzyme by the addition of EDTA. Carbo.pol is composed of acrylic acid monomers, which havea terminal carboxy group capable of binding mono-, di- and tri- valent cations.
Thus, the inhibition may be the result of the carkopol chelating the required divalent co-factor. An advantage of this observation is that the ribozyme complexed with car~opol is resistant to nuclease degradation.
Specifically, the following experiment was designed to determine if the retained ribozyme was intracellular or just bound to the surface of the eye. The study used 1 ~g of a hammerhead-type ribozyme with 0.5~ carbopol 974.
Time points were taken 3 and 10 minutes after administration and two mice were used per time point.
Free ribozyme was compared to carbopol 974 in the polymerized and unpolymerized form. To polymerize the gel, 1 ~l of ribozyme and 1 ~l of 1~ carbopol were mixed on a piece of parafilm. Addition of 1 ~l of 0.2 M NH40H
caused the mixture to polymerized gel. This was then applied to the eye as a smear. The eyes were washed in water, PBS and PBS followed by treatment with micrococcal nuclease treatment for 1 minute. Addition of PBS to carbopol caused the collapse of the gel and release of the ribozyme as shown in in vitro experiments. Other results ,. .. ..
showed that within this time frame, the micrococcal nuclease degraded 50% of 1 ~g ribozyme.
The results are shown in Fig. 4. The top panel (A) is free ribozyme, the middle panel (B) is unpolymerized carbopol, and the bottom panel (C) is polymerized carbopol. The results show that the carbopol yielded higher retention of the ribozyme compared to free ribozyme. Furthermore, the unpolymerized carb~pol gave better retention of the ribozyme than the polymerized a carbopol. The fact that less was removed by the PBS wash indicates that the ribozyme may be intracellular.
Ccular Tissue Localization of CarboDol/Ribozyme Ocular fixation followed by tissue sectioning was used to determine t,he tissue distribution of the ribozyme lS within the eye. The ribozyme was labeled at the 5' end with 33P. This isotope is a low energy b-emitter which produces discrete silver grains when exposed to photographic film. 1 ~g of ribozyme was applied to the eye in 0.5~ carbopol at acidic pH. At 10 minutes and 30 minutes after administration, the mice were euthanized, the eyes were removed and washed. One eye was fixed in glutaraldehyde or frozen, and the ribozyme was extracted from the other eye and analyzed by gel electrophoresis.
The fixed eyes were cut into 8 ~m sections and exposed to a photographic emulsion. Silver grains were over the epithelium and stromal layers of the cornea. The density of the grains decreased from the outermost layer to the innermost layer. These results showed that the ribozyme was not just sticking to the outer surface of the eye but was penetrating into the cells of the tissue.
Pharmaceutical Compositions The above formulations can be administered as noted above, or co-formulated with standard pharmaceutically acceptable carriers or diluents described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co.
WOg4~4g~ - s ? 6~ ~0 7 ~ 4 PCT~S94/04609 :. J
(A-G, Gennaro ed., 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers and dyes may be provided. Id at 1449. In addition, antioxidants and suspending agents may be used. Id.
Other embodiments are within the following claims.
The above experiments show that 5~ of the administered dose is retained by the eye after 3 minutes and 10 minutes topical administration to the mouse eye.
Free ribozyme yields 1~ retention. We also found that unpolymerized carbopol-containing ribozyme yields a higher percentage of uptake, compared to administration of polymerized carbopol-containing ribozyme.
`- ~ 21 6070~-Example 3: Ocular Retention To determine if the ribozyme was in the eye as opposed to being bound to the outside, a comparison of washes was made. The mouse eyes were washed with water, PBS or PBS + micrococcal nuclease. The PBS wash removed 80% of the ribozyme from the polymerized car~ pol, but only 20~ from the unpolymerized carbQpol. The amount of ribozyme administered without car~ pol was unchanged. The micrococcal nuclease treatment showed a 40~ reduction in the amount of ribozyme retained by the eye. However, it was observed that the carbopol inhibited the micrococcal nuclease activity. This may have been due to a variety of reasons, but one control that was run ,was to inhibit the enzyme by the addition of EDTA. Carbo.pol is composed of acrylic acid monomers, which havea terminal carboxy group capable of binding mono-, di- and tri- valent cations.
Thus, the inhibition may be the result of the carkopol chelating the required divalent co-factor. An advantage of this observation is that the ribozyme complexed with car~opol is resistant to nuclease degradation.
Specifically, the following experiment was designed to determine if the retained ribozyme was intracellular or just bound to the surface of the eye. The study used 1 ~g of a hammerhead-type ribozyme with 0.5~ carbopol 974.
Time points were taken 3 and 10 minutes after administration and two mice were used per time point.
Free ribozyme was compared to carbopol 974 in the polymerized and unpolymerized form. To polymerize the gel, 1 ~l of ribozyme and 1 ~l of 1~ carbopol were mixed on a piece of parafilm. Addition of 1 ~l of 0.2 M NH40H
caused the mixture to polymerized gel. This was then applied to the eye as a smear. The eyes were washed in water, PBS and PBS followed by treatment with micrococcal nuclease treatment for 1 minute. Addition of PBS to carbopol caused the collapse of the gel and release of the ribozyme as shown in in vitro experiments. Other results ,. .. ..
showed that within this time frame, the micrococcal nuclease degraded 50% of 1 ~g ribozyme.
The results are shown in Fig. 4. The top panel (A) is free ribozyme, the middle panel (B) is unpolymerized carbopol, and the bottom panel (C) is polymerized carbopol. The results show that the carbopol yielded higher retention of the ribozyme compared to free ribozyme. Furthermore, the unpolymerized carb~pol gave better retention of the ribozyme than the polymerized a carbopol. The fact that less was removed by the PBS wash indicates that the ribozyme may be intracellular.
Ccular Tissue Localization of CarboDol/Ribozyme Ocular fixation followed by tissue sectioning was used to determine t,he tissue distribution of the ribozyme lS within the eye. The ribozyme was labeled at the 5' end with 33P. This isotope is a low energy b-emitter which produces discrete silver grains when exposed to photographic film. 1 ~g of ribozyme was applied to the eye in 0.5~ carbopol at acidic pH. At 10 minutes and 30 minutes after administration, the mice were euthanized, the eyes were removed and washed. One eye was fixed in glutaraldehyde or frozen, and the ribozyme was extracted from the other eye and analyzed by gel electrophoresis.
The fixed eyes were cut into 8 ~m sections and exposed to a photographic emulsion. Silver grains were over the epithelium and stromal layers of the cornea. The density of the grains decreased from the outermost layer to the innermost layer. These results showed that the ribozyme was not just sticking to the outer surface of the eye but was penetrating into the cells of the tissue.
Pharmaceutical Compositions The above formulations can be administered as noted above, or co-formulated with standard pharmaceutically acceptable carriers or diluents described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co.
WOg4~4g~ - s ? 6~ ~0 7 ~ 4 PCT~S94/04609 :. J
(A-G, Gennaro ed., 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers and dyes may be provided. Id at 1449. In addition, antioxidants and suspending agents may be used. Id.
Other embodiments are within the following claims.
Claims (7)
1. Pharmaceutical composition comprising polyacrylic acid at a concentration sufficient to form a gel and a therapeutically effective amount of a nucleic acid.
2. The pharmaceutical composition of claim 1, wherein said nucleic acid is an enzymatic RNA molecule.
3. The pharmaceutical composition of claim 2, wherein said RNA molecule is in a hammerhead motif.
4. The pharmaceutical composition of claim 2, wherein said RNA molecule is in a hairpin, hepatitis Delta virus, group I intron, or RNAseP RNA mQtif.
5. The pharmaceutical composition of claim 3, wherein said ribozyme comprises between 30 and 45 bases complementary to a mRNA.
6. The pharmaceutical composition of claim 1, wherein said polyacrylic acid is not polymerized.
7. Method for delivery of a nucleic acid comprising administering the pharmaceutical composition of claim 1.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US5472293A | 1993-04-28 | 1993-04-28 | |
| US08/054,722 | 1993-04-28 | ||
| PCT/US1994/004609 WO1994024983A2 (en) | 1993-04-28 | 1994-04-27 | Ocular delivery of nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2160704A1 true CA2160704A1 (en) | 1994-11-10 |
Family
ID=21993084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002160704A Abandoned CA2160704A1 (en) | 1993-04-28 | 1994-04-27 | Ocular delivery of nucleic acid |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0695186A1 (en) |
| AU (1) | AU6714094A (en) |
| CA (1) | CA2160704A1 (en) |
| WO (1) | WO1994024983A2 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6146886A (en) * | 1994-08-19 | 2000-11-14 | Ribozyme Pharmaceuticals, Inc. | RNA polymerase III-based expression of therapeutic RNAs |
| US6040295A (en) | 1995-01-13 | 2000-03-21 | Genemedicine, Inc. | Formulated nucleic acid compositions and methods of administering the same for gene therapy |
| EP1038016A2 (en) * | 1997-12-16 | 2000-09-27 | Valentis Inc. | Needle-free injection of formulated nucleic acid molecules |
| CA2401327C (en) | 2000-03-03 | 2014-05-06 | Valentis, Inc. | Nucleic acid formulations comprising poly-amino acids for gene delivery and methods of use |
| WO2007149493A2 (en) | 2006-06-22 | 2007-12-27 | Immugen Inc | Restoration of hearing loss |
| AU2008213999B2 (en) | 2007-02-06 | 2014-04-17 | Tai June Yoo | Treatment and prevention of neurodegenerative diseases using gene therapy |
| EP2383286A1 (en) | 2010-04-30 | 2011-11-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treatment of retinal degenerative diseases |
| DE102010049036B4 (en) | 2010-10-21 | 2021-11-18 | Gramm medical healthcare GmbH | Aqueous solution for rinsing tissues or organs |
| US20150038557A1 (en) | 2012-02-24 | 2015-02-05 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and compositions for treatment of retinal degenerative diseases |
| AU2017221441A1 (en) | 2016-02-17 | 2018-10-04 | Children's Medical Center Corporation | FFA1 (GPR40) as a therapeutic target for neural angiogenesis diseases or disorders |
| US20220265695A1 (en) * | 2019-07-26 | 2022-08-25 | Proqr Therapeutics Ii B.V. | Opthalmic compositions comprising viscosifying polymers and nucleic acids |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4283393A (en) * | 1979-03-13 | 1981-08-11 | Merck & Co., Inc. | Topical application of interferon inducers |
| IE66830B1 (en) * | 1987-08-12 | 1996-02-07 | Hem Res Inc | Topically active compositions of double-stranded RNAs |
| IT1232318B (en) * | 1989-09-07 | 1992-01-28 | Crinos Industria Farmaco | TOPICAL COMPOSITIONS CONTAINING DEPOLYMERATED NUCLEIC ACIDS FOR SKIN AND BODY COSMETICS. |
-
1994
- 1994-04-27 EP EP94914922A patent/EP0695186A1/en not_active Withdrawn
- 1994-04-27 WO PCT/US1994/004609 patent/WO1994024983A2/en not_active Application Discontinuation
- 1994-04-27 AU AU67140/94A patent/AU6714094A/en not_active Abandoned
- 1994-04-27 CA CA002160704A patent/CA2160704A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994024983A3 (en) | 1995-01-19 |
| AU6714094A (en) | 1994-11-21 |
| WO1994024983A2 (en) | 1994-11-10 |
| EP0695186A1 (en) | 1996-02-07 |
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