Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
The present application claims priority to the applications, GB 2112199.1 and PCT/GB2022/052189, with the effective filing dates of 26 Aug 2021 and 25 Aug 2022.
Claim Status
This Office Action is in response to Applicant’s Response to Restriction Requirement filed,
14 July 2026.
Applicant’s election with traverse of Group I (claims 1-19) and species (
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) in the reply filed on 14 July 2026 is acknowledged. The traversal is on the ground(s) that the contribution made by each claimed invention was not assessed or considered as a whole. Applicant argues that even if compounds 4 and 14 fall into Formula (I), disclosure of a compound within Formula (I) does not, by itself, establish that the complete method common to Groups I-III lack novelty or inventive step. Applicant asserts that Groups I-III make a contribution over the prior art and that claims 20-23 expressly require all limitations of the staining method of claim 1 while adding further analysis, imaging, condition-detection, or sample limitations and thus form a single general inventive concept.
Applicant’s remarks are acknowledged and are persuasive. The restriction requirement is accordingly withdrawn.
Claims 1-23 are pending and are under consideration in the instant office action.
Claim Interpretation
The Examiner notes that the specification defines a mitochondrial condition as a mitochondrial disease or condition involving or that may lead to mitochondrial dysfunction where mitochondria fail to produce enough energy for the body or parts of the body to function properly (page 13, lines 6-8).
Information Disclosure Statement
The Information Disclosure Statement filed 3 December 2025 and the references cited therein have been considered, unless indicated otherwise.
Drawings
1. The drawings are objected to under 37 CFR 1.83(a) because they fail to show discernible features in the images of mitochondria in Figures 2-6 as described in the specification (pages 14-16). Any structural detail that is essential for a proper understanding of the disclosed invention should be shown in the drawing. MPEP § 608.02(d). Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
2. Claim(s) 1-23 are rejected under 35 U.S.C. 103 as being unpatentable over Shen (RSC Adv. 2017, 7, 10922-10927, see IDS filed 3 Dec 2025) in view of Berge (J. Pharm. Sci.¸1977, 66(1), 1-20) and Haugland (U.S. Patent No. 5,459,268, issued 17 Oct 1995, see IDS filed 3 Dec 2025) as evidenced by Wikipedia 1 (“Eukaryote,” Wikipedia, 2026, <en.wikipedia.org/wiki/Eukaryote>, accessed 12 Aug 2026) and Wikipedia 2 (“Yeast,” Wikipedia¸2026, <en.wikipedia.org/wiki/Yeast>, accessed 12 Aug 2026).
Shen teaches syntheses and biological application of three Si-rhodamine probes (abstract). Shen teaches that rhodamines are well-known fluorescent dyes that display water solubility, high fluorescence quantum yields, and high molar extinction coefficients and are widely used in various fluorescent probes for bioimaging (page 10922, column 1, paragraph 1). Shen teaches that near-infrared fluorescence dyes are attractive for biological applications because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence in the living systems (page 10922, column 1, paragraph 1). Shen teaches the previous improvements in rhodamine dyes created Janelia Fluor dyes that increased brightness and photostability (page 10922, column 1, paragraph 2). Shen teaches that the Janelia Fluor dyes, however, were prone to nucleophilic attack on the ninth carbon atom of the xanthene ring, which induced drastic changes in optical properties, and thus, probes with high stability are highly desired for practical images in vivo (page 10922, column 1, paragraph 1; page 10922, column 2, paragraph 1). Shen teaches AZSiR probes:
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, and AZSiR-1 and AZSiR-2 fall into Formula (I). Mapping shown below:
18/686,693 Formula (I) components
18/686,693 Formula (I)
AZSiR-1 and AZSir-2 probes (Shen)
Y
Unsubstituted azetidine ring
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34
32
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Z
Unsubstituted azetidine ring
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29
42
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X
SiR13R14; R13 = C1-8 alkyl; R14 = C1-8 alkyl
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23
42
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R1
AZSiR-1: R1 = H; AZSiR-2: R1 = C1-8 alkyl
AZSiR-1:
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35
40
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; AZSiR-2:
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36
43
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R2 – R5
H
AZSiR-1:
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35
40
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; AZSiR-2:
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36
43
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Rv, Rx, Rx, Ry, R6, R7
H
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40
75
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Shen teaches that the AZSiR-1 and AZSiR-3 kept 94% absorption intensity while AZSiR-2 kept 80% absorption intensity after irradiation for 30 minutes (Chart 1, page 10922; page 10924, column 1, paragraph 1). Shen teaches that the AZSiR probes have low cytotoxicity and that the bulky methyl groups on the 2-position of the pendant phenyl ring are beneficial to improve fluorescence quantum yield and prevent nucleophilic attack for good chemical stability (page 10294, column 1, paragraph 2; page 10924, column 2, paragraph 2; page 10925, column 1, paragraph 1). Shen teaches that the AZSiR probes have high fluorescence quantum yields, good photo-chemistry stability, live cell permeability, and low cytotoxicity, which make them excellent probes for live-cell imaging (page 10925, column 1, paragraph 1). Shen additionally teaches incubating HeLa cells with AZSiR probes and that they have good live cell permeability (page 10925, column 1, paragraph 1).
Regarding claim 1, Shen fails to teach a method of staining mitochondria via providing a sample containing mitochondria and incubating the sample with a composition of Formula (I), wherein the composition has counterion (thus making it a salt).
Berge teaches that chemical, biological, physical, and economic characteristics of medicinal agents can be manipulated and optimized by conversion to a salt form (page 1, column 1, paragraph 1). Berge teaches that salt-forming agents are often chosen empirically and the preferred form is selected via cost of raw materials, ease of crystallization, and percent yield (page 1, column 2, paragraph 1). Berge teaches that monoprotic hydrochlorides have been by far the most frequent choice of available anionic salt-forming radicals, outnumbering sulfates six to one and account for 42.98% of FDA-approved commercially marketed salts (page 2, column 2, paragraph 1; Table 1, page 2). However, Berge teaches a list of several, finite anions to form a salt of a compound and their popularity in previously FDA-approved commercially marketed salts (Table 1, page 2).
Haugland teaches dyes of fluorescent substituted 3’,6’-diaminoxanthenes, which selectively localize within mitochondria (abstract). Haugland teaches staining mitochondria in living cells and that fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable (column 1, lines 7-9; column 1, lines 15-17). Haugland teaches that specific cellular structures can be monitored with respect to their spatial and temporal distribution in diverse environments and that dyes can be used to determine ionic, electrical, or metabolic properties of cellular organelles (column 1, lines 20-24). Haugland teaches that mitochondria are the intracellular organelles responsible for aerobic metabolism in eukaryotic cells and that there is a need in biology to detect and observe mitochondria particularly in cells, as a specific application or in conjunction with additional labeling or other components (column 1, lines 25-30). Haugland teaches that due to the strong proton gradient across the mitochondrial membrane, cationic substances have been found to selectively localize with functioning mitochondria, which is useful for imaging (column 1, lines 31-38). Haugland teaches that the dyes therein provide bright mitochondrial staining at much lower concentrations (column 1, lines 60-62). Haugland teaches that cationic dye is sequestered in mitochondria in an equilibrium process and that mitochondrial staining can only be maintained by functioning mitochondria (column 1, lines 65-66; column 2, lines 1-3). Haugland teaches that attempts to fix stained cells generally results in cell death, and thus loss of mitochondrial potential and mitochondrial stain and that cells that are killed prior to staining do not stain well due to lack of potential across the membrane (column 2, lines 3-7). Haugland teaches that the compounds of the present invention indicate cells that possessed functioning mitochondria at the time of staining even after fixation and is a more meaningful indicator of metabolic activity than membrane permeability (column 2, lines 18-22). Haugland specifically teaches preparing a labeling solution of the dye, introducing the dye to the sample containing mitochondria, incubating the sample for a time sufficient to produce a detectable fluorescent response, and observing or analyzing the staining pattern in the sample (column 8, lines 60-65).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the instantly claimed invention to isolate the AZSiR probes of Shen as the salt for good solubility as taught by Berge and then utilize the probes for staining mitochondria akin to the methods as taught by Haugland to image mitochondria, which is attractive for biological applications due to minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence in the living systems, because:
-Shen teaches syntheses and biological application of three Si-rhodamine probes,
-Shen teaches that rhodamines are well-known fluorescent dyes that display water solubility, high fluorescence quantum yields, and high molar extinction coefficients and are widely used in various fluorescent probes for bioimaging,
-Shen teaches that near-infrared fluorescence dyes are attractive for biological applications because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence in the living systems,
-Shen teaches the previous improvements in rhodamine dyes created Janelia Fluor dyes that increased brightness and photostability,
-Shen teaches that the Janelia Fluor dyes, however, were prone to nucleophilic attack on the ninth carbon atom of the xanthene ring, which induced drastic changes in optical properties, and thus, probes with high stability are highly desired for practical images in vivo,
-Shen teaches AZSiR probes:
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349
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, which AZSiR-1 and -3 kept 94% absorption intensity while AZSiR-2 kept 80% absorption intensity after irradiation for 30 minutes,
-Shen teaches that the AZSiR probes have low cytotoxicity and that the bulky methyl groups on the 2-position of the pendant phenyl ring are beneficial to improve fluorescence quantum yield and prevent nucleophilic attack for good chemical stability,
-Shen teaches that the AZSiR probes have high fluorescence quantum yields, good photo-chemistry stability, live cell permeability, and low cytotoxicity, which make them excellent probes for live-cell imaging,
-Shen additionally teaches incubating HeLa cells with AZSiR probes and that they have good live cell permeability,
-Berge teaches that chemical, biological, physical, and economic characteristics of medicinal agents can be manipulated and optimized by conversion to a salt form,
-Berge teaches that salt-forming agents are often chosen empirically and the preferred form is selected via cost of raw materials, ease of crystallization, and percent yield,
-Berge teaches that monoprotic hydrochlorides have been by far the most frequent choice of available anionic salt-forming radicals, outnumbering sulfates six to one and account for 42.98% of FDA-approved commercially marketed salts,
-Berge teaches a list of several, finite anions to form a salt of a compound and their popularity in previously FDA-approved commercially marketed salts,
-Haugland teaches dyes of fluorescent substituted 3’,6’-diaminoxanthenes, which selectively localize within mitochondria,
-Haugland teaches staining mitochondria in living cells and that fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable,
-Haugland teaches that specific cellular structures can be monitored with respect to their spatial and temporal distribution in diverse environments and that dyes can be used to determine ionic, electrical, or metabolic properties of cellular organelles,
-Haugland teaches that due to the strong proton gradient across the mitochondrial membrane, cationic substances have been found to selectively localize with functioning mitochondria, which is useful for imaging,
-Haugland teaches that mitochondria are the intracellular organelles responsible for aerobic metabolism in eukaryotic cells and that there is a need in biology to detect and observe mitochondria particularly in cells, as a specific application or in conjunction with additional labeling or other components,
-Haugland teaches that the dyes therein provide bright mitochondrial staining at much lower concentrations,
-Haugland teaches that cationic dye is sequestered in mitochondria in an equilibrium process and that mitochondrial staining can only be maintained by functioning mitochondria,
-Haugland teaches that attempts to fix stained cells generally results in cell death, and thus loss of mitochondrial potential and mitochondrial stain and that cells that are killed prior to staining do not stain well due to lack of potential across the membrane,
-Haugland teaches that the compounds of the present invention indicate cells that possessed functioning mitochondria at the time of staining even after fixation and is a more meaningful indicator of metabolic activity than membrane permeability, and
-Haugland specifically teaches preparing a labeling solution of the dye, introducing the dye to the sample containing mitochondria, incubating the sample for a time sufficient to produce a detectable fluorescent response, and observing or analyzing the staining pattern in the sample.
Accordingly, the combination of Shen, Berge, and Haugland teaches a method for staining mitochondria via providing a sample containing mitochondria and incubating the sample in a composition of a compound of Formula (I):
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248
255
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.
Regarding claim 2, Shen teaches AZSiR-1:
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(Chart 1, page 10922), wherein Y and Z are unsubstituted azetidines and Ra and Rb are H.
Regarding claim 3, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (II) and R8 and R9 are H.
Regarding claim 4, Berge teaches that the counterion is chloride (Table 1, page 2), which is a biologically compatible counterion as it is present in 42.98% of FDA-approved commercially marketed salts
Regarding claim 5, Berge teaches that the counterion is chloride (Table 1, page 2), which is a halide.
Regarding claim 6, Haugland teaches that cells are transferred to labeling medium containing the dye and incubated at 37 °C for 15-30 minutes (Example 14, column 19, lines 31-33).
Regarding claim 7, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (III) and R10 and R11 are H.
Regarding claim 8, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (IV) and R8, R9, R10, and R11 are H.
Regarding claim 9, Shen teaches AZSiR-1:
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(Chart 1, page 10922), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are H.
Regarding claim 10, Shen teaches AZSiR-2:
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112
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(Chart 1, page 10922), wherein R1 is a C1 alkyl.
Regarding claim 11, Shen teaches AZSiR-2:
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112
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(Chart 1, page 10922), wherein R1 is a methyl.
Regarding claim 12, Haugland teaches that the composition further comprises DMSO (Example 13, column 19, lines 21-22; column 9, lines 8-13), wherein DMSO is an organic agent.
Regarding claim 13, Haugland teaches that preferred organic solvent for preparing the stock solution of dye are DMSO, DMF (dimethylformaide), acetone, MeCN (acetonitrile), dioxane, and THF (tetrahydrofuran; column 9, lines 8-13).
Regarding claim 14, Haugland teaches that the sample containing mitochondria is a tissue sample (column 9, lines 34-36).
Regarding claim 15, Haugland teaches that the sample containing mitochondria is eukaryotic (column 9, line 29; Example 18, column 20, lines 54-55), which is plant, animal, or fungal as evidenced by Wikipedia 1 (page 1, column 1). Additionally, Haugland teaches that the sample containing mitochondria is from yeast (Example 17, column 20, line 40), which is a fungus as evidenced by Wikipedia 2 (page 1, paragraph 1). Also, Haugland teaches that the sample containing mitochondria is from goat’s blood and thus an animal (Example 16, column 20, lines 18-21).
Regarding claim 16, Haugland teaches that the sample containing mitochondria contains live mitochondria or live cells (column 20, lines 29-31; column 19, line 30).
Regarding claim 17, Haugland teaches that the sample containing mitochondria does not contain fixed cells (column 11, lines 6-7; Example 17, column 20, lines 40-51).
Regarding claim 18, Haugland teaches that the cationic species is in the range of 50-200 nM (Example 13, lines 20-27).
Regarding claim 19, Shen teaches AZSiR-1:
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(Chart 1, page 10922).
Regarding claim 20, Haugland teaches that after the sample has been stained, the fluorescently labeled mitochondria are observed and observation of the sample includes illuminating the stained sample with a wavelength of light appropriate to general a fluorescent response and visually examining the sample by use of a microscope or confocal microscope (column 13, lines 47-57).
Regarding claim 21, Haugland teaches that the xanthylium ring system absorbs light maximally at about 490 – 520 nm (column 4, lines 50-58). Additionally, Haugland teaches that the sample cells were illuminated at 546 nM and observed at 590 nm (Example 17, column 20, lines 43-45). Also, Shen teaches continuous irradiation of the AZSiR probes with a 660 nM LED laser (page 10923, column 2, paragraph 3; page 10924, column 1, paragraph 1).
Regarding claim 22, Haugland teaches a method of detecting a mitochondrial condition, in particular, the assessment of mitochondrial function as it relates to the viability of eukaryotic cells (column 3, lines 28-34).
Regarding claim 23, Haugland teaches that the sample containing mitochondria is eukaryotic (column 9, line 29; Example 18, column 20, lines 54-55), which is plant, animal, or fungal as evidenced by Wikipedia 1 (page 1, column 1). Additionally, Haugland teaches that the sample containing mitochondria is from yeast (Example 17, column 20, line 40), which is a fungus as evidenced by Wikipedia 2 (page 1, paragraph 1). Also, Haugland teaches that the sample containing mitochondria is from goat’s blood and thus an animal (Example 16, column 20, lines 18-21).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
3. Claims 1-11, 14-15, and 19-23 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 and 19-22 of copending Application No. 18/686,700 in view of Shen (RSC Adv. 2017, 7, 10922-10927, see IDS filed 3 Dec 2025) and Berge (J. Pharm. Sci.¸1977, 66(1), 1-20).
This is a provisional nonstatutory double patenting rejection. Although the claims at issue are not identical, they are not patentably distinct from each other.
18/686,700 claims a method for staining mitochondria via providing a sample containing mitochondria and incubating the sample in a composition of Formula (I):
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, wherein Y is a substituted or unsubstituted azetidine ring; Z is a substituted or unsubstituted azetidine ring or OR17; X is selected from O, S, O2, Se, NR12, P(O)R12, CR13R14, SiR13R14, Te, and GeR13R14; R1, R2, R3, R4, and R4 are each independently selected from H, C1-8 alkyl, OR15, C(O)OR16, NHCOR15, CONHR15, and halo; Rv, Rw, Rx, Ry, R6, R7 are each independently selected from H, C1-8 alkyl, and halo; R12, R13, R14, and R15 are each independently selected from H, C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, R16 is selected from C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and R17 is selected from H, C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; wherein the composition includes a counterion (claim 1 and 19).
Regarding claim 1, ‘700 fails to claim a specific compound falling into Formula (I) as ‘700 requires one of R1 – R5 is Q.
Shen teaches syntheses and biological application of three Si-rhoadamine probes (abstract). Shen teaches that rhodamines are well-known fluorescent dyes that display water solubility, high fluorescence quantum yields, and high molar extinction coefficients and are widely used in various fluorescent probes for bioimaging (page 10922, column 1, paragraph 1). Shen teaches that near-infrared fluorescence dyes are attractive for biological applications because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence in the living systems (page 10922, column 1, paragraph 1). Shen teaches the previous improvements in rhodamine dyes created Janelia Fluor dyes that increased brightness and photostability (page 10922, column 1, paragraph 2). Shen teaches that the Janelia Fluor dyes, however, were prone to nucleophilic attack on the ninth carbon atom of the xanthene ring, which induced drastic changes in optical properties, and thus, probes with high stability are highly desired for practical images in vivo (page 10922, column 1, paragraph 1; page 10922, column 2, paragraph 1). Shen teaches AZSiR probes:
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, and AZSiR-1 and AZSiR-2 fall into Formula (I). Mapping shown below:
18/686,693 Formula (I) components
18/686,693 Formula (I)
AZSiR-1 and AZSir-2 probes (Shen)
Y
Unsubstituted azetidine ring
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34
32
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Z
Unsubstituted azetidine ring
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29
42
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X
SiR13R14; R13 = C1-8 alkyl; R14 = C1-8 alkyl
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23
42
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R1
AZSiR-1: R1 = H; AZSiR-2: R1 = C1-8 alkyl
AZSiR-1:
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35
40
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; AZSiR-2:
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36
43
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R2 – R5
H
AZSiR-1:
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35
40
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; AZSiR-2:
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43
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Rv, Rx, Rx, Ry, R6, R7
H
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40
75
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Greyscale
Shen teaches AZSiR-1 and AZSiR-3 kept 94% absorption intensity while AZSiR-2 kept 80% absorption intensity after irradiation for 30 minutes (Chart 1, page 10922; page 10924, column 1, paragraph 1). Shen teaches that the AZSiR probes have low cytotoxicity and that the bulky methyl groups on the 2-position of the pendant phenyl ring are beneficial to improve fluorescence quantum yield and prevent nucleophilic attack for good chemical stability (page 10294, column 1, paragraph 2; page 10924, column 2, paragraph 2; page 10925, column 1, paragraph 1). Shen teaches that the AZSiR probes have high fluorescence quantum yields, good photo-chemistry stability, live cell permeability, and low cytotoxicity, which make them excellent probes for live-cell imaging (page 10925, column 1, paragraph 1). Shen additionally teaches incubating HeLa cells with AZSiR probes and that they have good live cell permeability (page 10925, column 1, paragraph 1).
Berge teaches that chemical, biological, physical, and economic characteristics of medicinal agents can be manipulated and optimized by conversion to a salt form (page 1, column 1, paragraph 1). Berge teaches that salt-forming agents are often chosen empirically and the preferred form is selected via cost of raw materials, ease of crystallization, and percent yield (page 1, column 2, paragraph 1). Berge teaches that monoprotic hydrochlorides have been by far the most frequent choice of available anionic salt-forming radicals, outnumbering sulfates six to one and account for 42.98% of FDA-approved commercially marketed salts (page 2, column 2, paragraph 1; Table 1, page 2). However, Berge teaches a list of several, finite anions to form a salt of a compound and their popularity in previously FDA-approved commercially marketed salts (Table 1, page 2).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the instantly claimed invention to isolate the AZSiR probes of Shen as the salt for good solubility as taught in Berge and then utilize the probes for staining mitochondria as claimed in ‘700 to image mitochondria, which are the intracellular organelles responsible for aerobic metabolism in eukaryotic cells and that there is a need in biology to detect and observe mitochondria particularly in cells, because:
-‘700 claims a method for staining mitochondria via providing a sample containing mitochondria and incubating the sample in a composition of Formula (I):
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273
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, wherein Y is a substituted or unsubstituted azetidine ring; Z is a substituted or unsubstituted azetidine ring or OR17; X is selected from O, S, O2, Se, NR12, P(O)R12, CR13R14, SiR13R14, Te, and GeR13R14; R1, R2, R3, R4, and R4 are each independently selected from H, C1-8 alkyl, OR15, C(O)OR16, NHCOR15, CONHR15, and halo; Rv, Rw, Rx, Ry, R6, R7 are each independently selected from H, C1-8 alkyl, and halo; R12, R13, R14, and R15 are each independently selected from H, C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, R16 is selected from C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and R17 is selected from H, C1-8 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; wherein the composition includes a counterion,
-Shen teaches syntheses and biological application of three Si-rhoadamine probes,
-Shen teaches that rhodamines are well-known fluorescent dyes that display water solubility, high fluorescence quantum yields, and high molar extinction coefficients and are widely used in various fluorescent probes for bioimaging,
-Shen teaches that near-infrared fluorescence dyes are attractive for biological applications because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence in the living systems,
-Shen teaches the previous improvements in rhodamine dyes created Janelia Fluor dyes that increased brightness and photostability,
-Shen teaches that the Janelia Fluor dyes, however, were prone to nucleophilic attack on the ninth carbon atom of the xanthene ring, which induced drastic changes in optical properties, and thus, probes with high stability are highly desired for practical images in vivo,
-Shen teaches AZSiR probes:
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, which AZSiR-1 and -3 kept 94% absorption intensity while AZSiR-2 kept 80% absorption intensity after irradiation for 30 minutes,
-Shen teaches that the AZSiR probes have low cytotoxicity and that the bulky methyl groups on the 2-position of the pendant phenyl ring are beneficial to improve fluorescence quantum yield and prevent nucleophilic attack for good chemical stability,
-Shen teaches that the AZSiR probes have high fluorescence quantum yields, good photo-chemistry stability, live cell permeability, and low cytotoxicity, which make them excellent probes for live-cell imaging,
-Shen additionally teaches incubating HeLa cells with AZSiR probes and that they have good live cell permeability,
-Berge teaches that chemical, biological, physical, and economic characteristics of medicinal agents can be manipulated and optimized by conversion to a salt form,
-Berge teaches that salt-forming agents are often chosen empirically and the preferred form is selected via cost of raw materials, ease of crystallization, and percent yield,
-Berge teaches that monoprotic hydrochlorides have been by far the most frequent choice of available anionic salt-forming radicals, outnumbering sulfates six to one and account for 42.98% of FDA-approved commercially marketed salts, and
-Berge teaches a list of several, finite anions to form a salt of a compound and their popularity in previously FDA-approved commercially marketed salts.
Accordingly, the combination of ‘700, Shen, and Berge teaches a method for staining mitochondria via providing a sample containing mitochondria and incubating the sample in a composition of a compound of Formula (I):
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.
Regarding claim 2, Shen teaches AZSiR-1:
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(Chart 1, page 10922), wherein Y and Z are unsubstituted azetidines and Ra and Rb are H.
Regarding claim 3, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (II) and R8 and R9 are H.
Regarding claim 4, Berge teaches that the counterion is chloride (Table 1, page 2), which is a biologically compatible counterion as it is present in 42.98% of FDA-approved commercially marketed salts
Regarding claim 5, Berge teaches that the counterion is chloride (Table 1, page 2), which is a halide.
Regarding claim 6, ‘700 claims incubating the sample for a predetermined time in the range of 10 min to 2 hours and at a predetermined temperature in the range of 20-39 °C (claim 20).
Regarding claim 7, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (III) and R10 and R11 are H.
Regarding claim 8, Shen teaches AZSiR-1:
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(Chart 1, page 10922), which is a compound of Formula (IV) and R8, R9, R10, and R11 are H.
Regarding claim 9, Shen teaches AZSiR-1:
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(Chart 1, page 10922), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are H.
Regarding claim 10, Shen teaches AZSiR-2:
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(Chart 1, page 10922), wherein R1 is a C1 alkyl.
Regarding claim 11, Shen teaches AZSiR-2:
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(Chart 1, page 10922), wherein R1 is a methyl.
Regarding claim 14, ‘700 claims that the sample containing mitochondria is a tissue sample (claim 21).
Regarding claim 15, ‘700 claims that the sample containing mitochondria is a plant, animal, or fungal tissue sample, a sample of plants, animal, or fungal cells, or isolated plant, animal, or fungal mitochondria (claim 22).
Regarding claim 19, Shen teaches AZSiR-1:
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(Chart 1, page 10922).
Regarding claim 20, ‘700 claims a method of analyzing mitochondria via staining a sample of mitochondria via a compound, illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and observing or imaging a magnified image of the sample (claim 25).
Regarding claim 21, ‘700 claims the appropriate wavelength is in the range of 400 – 800 nm (claim 26).
Regarding claim 22, ‘700 claims a method of detecting a mitochondrial condition via staining a sample of mitochondria (claim 27).
Regarding claim 23, ‘700 claims that the sample of mitochondria is a plant, animal, or fungal tissue sample, a sample of plant, animal, or fungal cells, or isolated plant, animal, or fungal mitochondria (claim 28).
Conclusion
No claim is allowed.
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/MADELINE M. DEKARSKE/Examiner, Art Unit 1622
/JAMES H ALSTRUM-ACEVEDO/Supervisory Patent Examiner, Art Unit 1622
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