DETAILED ACTION
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, filed September 1, 2023, is a national stage entry under 35 USC § 371 of International Patent Application No. PCT/US2022/071222, filed March 18, 2022, which claims the benefit of U.S. Provisional Patent Application Nos. 63/249,865 and 63/164,489, filed September 29, 2021 and March 22, 2021, respectively.
Status of the Claims
In the amendment filed September 1, 2023, claims 6-12, 16, 19-20, 22, 27-39, 44, and 46-52 are canceled. Claims 3-4, 13-15, 17-18, 21, 23, 26, 40, 42, and 45 are amended. Claims 1-5, 13-15, 17-18, 21, 23-26, 40-43, and 45 are currently pending.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on September 1, 2023 March 4, 2024 are acknowledged.
Claim Objections
An objection is raised to claim 40:
Claim 40 is indefinite due to the following informalities: the claim includes two instances of a typographical error in which it appears “form” is misspelled as “from.” Specifically, at line 4 the claim recites “to from a control culture,” and at line 6 recites “to from a plurality of test cultures.” Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. § 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. § 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 2 is indefinite:
Claim 2 is rejected under 35 U.S.C. § 112(b) or 35 U.S.C. § 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 is indefinite for reciting “wherein the one or more amino acids comprises a low concentration of asparagine, aspartic acid, glutamine, and/or glutamic acid,” because a person of ordinary skill in the art could not reasonably determine the metes and bounds of this limitation. In particular, one of skill in the art could not reasonably determine what constitutes a “low concentration” within the scope of claim 2. The instant specification describes different real examples having a concentration of one of the recited amino acids at a concentration within a range of about 0.1 to 2.0 g/L (see, for example, Table 2), and describes that similar results were observed up to 4 g/L (paragraph [0118]), but this is not sufficient to allow a person of skill in the art to determine any upper limit for “low concentration.”
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-5 and 13 are anticipated by Agapova:
Claims 1-5 and 13 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by the non-patent publication, Flexible nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis, eLife, 8, art. e41129, pgs. 1-22 (2019) by Agapova et al. (hereinafter, “Agapova”).
Claim 1 recites a composition for facilitating growth of M. tuberculosis, comprising:
one or more nitrogen sources comprising one or more amino acids or salts thereof, alone or in combination with one or more ammonium salts;
one or more carbon sources comprising glycerol, butyric acid, lactate, cholesterol, pyruvic acid, dextrose, citric acid, or a combination thereof;
one or more salts comprising MgSO4, KH2PO4, K2HPO4, NaH2PO4, or a combination thereof; and
one or more supplementary nutrients comprising ZnSO4, pyridoxine hydrochloride, biotin, Tween 80, oleic acid, albumin, ferric ammonium citrate, catalase, or a combination thereof.
Claim 1 further recites that the composition is formulated to support antibiotic susceptibility testing of M. tuberculosis at a beginning pH between 6.2 to 8.0 at 35-37 °C. The phrase, “is formulated to support antibiotic susceptibility testing of M. tuberculosis” recites an intended use that does not limit the physical makeup of the composition in any way, and thus the final clause, “wherein the composition is formulated to support antibiotic susceptibility testing of M. tuberculosis at a beginning pH between 6.2 to 8.0 at 35-37 °C” is construed simply to require that the composition have, prior to any use/inoculation, a pH between 6.2 and 8.0 at 35-37 °C.
Agapova discloses a study of nitrogen utilization by M. tuberculosis (Abstract), in which, inter alia, M. tuberculosis-laden filters were transferred to agar plates having growth medium (7H10), each supplemented with one of the twenty proteinogenic amino acids (pg. 3, Results, first paragraph) and also grown in 7H9 broth (liquid, i.e. non-agar containing) modified to have a single nitrogen source, such as a single amino acid (pg. 6, Amino acids are superior nitrogen sources, compared to NH4+, first paragraph; and pg. 19, Strains and growth media). Agapova teaches that this modified 7H9 growth medium (7H9x) includes, inter alia, a (i) a single amino acid (“nitrogen source of interest”), (ii) glycerol, (iii) KH2PO4 (monobasic potassium phosphate), (iv) ZnSO4 (zinc sulphate), and further teaches the media is set to pH 6.6 – pg. 19, Strains and growth media. While Agapova is silent as to the temperature at which the growth medium is set to 6.6, even if it was at room temperature, such a buffered aqueous solution would inherently have a pH within the recited range at a temperature of 35 or 37 °C. Agapova thus anticipates the composition of instant claim 1.
With respect to claim 2, Agapova teaches that different batches of the modified 7H9x growth medium has asparagine, aspartic acid, glutamine, or glutamic acid at 0.25, 0.5, 1 or 2 g/L (Figure 3a).
With respect to claim 3, Agapova teaches the preparation, incubation, and testing of a series of petri dishes having 7H10x medium, identical to standard 7H10 agar medium but with the glutamic acid replaced with each, one at a time, of the twenty proteinogenic amino acids, such as alanine (see, e.g. Figure 2a-f). This 7H10x medium is a composition of claim 1, having, inter alia, the amino acid/nitrogen source; glycerol; monopotassium phosphate; and zinc sulphate. Agapova doesn’t expressly state a pH of the 7H10x medium, but it is 6.6, as shown by the non-patent publication, Bacteriology of tuberculosis: laboratory methods, Am. J. Public Health Nations Health, 48, pgs. 844-853 (1958) by Middlebrook et al. (hereinafter, “Middlebrook”), particularly beginning at the bottom of the right column of pg. 845, Base Solids, through the continued materials listing at the top of the left column of pg. 846. Middlebrook shows the composition and pH of 7H10 medium, inherently present in the 7H10x agar medium of Agapova. Each of the sixteen batches of 7H10x medium Agapova that contains an amino acid other than Glu, Gln, Asp, or Asn is thus a composition of instant claim 3.
With respect to claim 4, Agapova teaches the 7H9x and growth medium has ferric ammonium citrate, which includes ammonium cations and citrate anions, i.e. ammonium citrate (pg. 19, Strains and growth media). Stated alternatively, when ferric ammonium citrate is dissolved in aqueous solution, as in the 7H10 medium of Agapova and/or a composition of the present claims, ammonium citrate is present in the solution. With respect to claim 5, Agapova teaches the 7H9x growth medium includes glycerol (pg. 19, Strains and growth media). With respect to claim 13, Agapova teaches the 7H9x growth medium is supplemented with ADC i.e. albumin-dextrose-catalase (pg. 19, Strains and growth media).
With respect to claim 15, the 7H9, 7H9x, 7H10, and 7H10x media of Agapova contain malachite green (see, for example, pg. 19, Strains and growth media). Malachite green is used in some situations as a pH indicator (see, for example, the non-patent publication, Malachite Green, a PubChem entry obtained at the url pubchem.ncbi.nlm.nih.gov/compound/Malachite-Green, created in 2005 and last modified in 2025, which states that that malachite green is used as a fungicide, dye, biological stain, reagent, and pH indicator – pg. 23). While the pH-sensitive properties of malachite green might not be relevant to methods of the present application, it nevertheless is capable of functioning as a pH indicator and thereby satisfies the requirement of instant claim 15.
Claims 1, 2, 4-5, 14-15, 17, 21, 23-26, 40-41, and 45 are anticipated by Heifets:
Claims 1, 2, 4-5, 14-15, 17, 21, and 23-26 are rejected under 35 U.S.C. 102(a)(1) as being an by U.S. Patent No. 6,579,694 to Heifets et al. (hereinafter, “Heifets”).
Claim 1 recites a composition for facilitating growth of M. tuberculosis, comprising:
one or more nitrogen sources comprising one or more amino acids or salts thereof, alone or in combination with one or more ammonium salts;
one or more carbon sources comprising glycerol, butyric acid, lactate, cholesterol, pyruvic acid, dextrose, citric acid, or a combination thereof;
one or more salts comprising MgSO4, KH2PO4, K2HPO4, NaH2PO4, or a combination thereof; and
one or more supplementary nutrients comprising ZnSO4, pyridoxine hydrochloride, biotin, Tween 80, oleic acid, albumin, ferric ammonium citrate, catalase, or a combination thereof.
Claim 1 further recites that the composition is formulated to support antibiotic susceptibility testing of M. tuberculosis at a beginning pH between 6.2 to 8.0 at 35-37 °C. The phrase, “is formulated to support antibiotic susceptibility testing of M. tuberculosis” does not appear to limit the composition in any way, and thus the final clause, “wherein the composition is formulated to support antibiotic susceptibility testing of M. tuberculosis at a beginning pH between 6.2 to 8.0 at 35-37 °C” is construed simply to require that the composition have, prior to any use/inoculation, a pH between 6.2 and 8.0 at 35-37 °C.
Heifets teaches an agar medium for drug-susceptibility testing of M. tuberculosis (Abstract), which includes an agar base suitable for growth of M. tuberculosis and animal serum (col. 6, lines 49-52). Heifets teaches that the agar base can be Middlebrook 7H10, and that the agar medium of Heifets can be at a pH between about 6.0 to about 6.25. As noted above (see the reference to Middlebrook), 7H10 is a medium of instant claim 1, and thus the modified 7H10 medium of Heifets is also a medium of instant claim 1.
With respect to claim 2, the 7H10-based medium of Heifets includes glutamic acid (see Middlebrook; see also Heifets col. 7, lines 24-27). With respect to claim 4, the 7H10 base of Heifets includes ferric ammonium citrate (see Middlebrook), which includes ammonium cations and citrate anions, i.e. ammonium citrate (pg. 19, Strains and growth media). Stated alternatively, when ferric ammonium citrate is dissolved in aqueous solution, as in the 7H10 medium of Heifets and/or a composition of the present claims, ammonium citrate is present in the solution. Similarly, with respect to claim 5, the 7H10 base of Heifets contains glycerol (see Middlebrook).
With respect to claim 14, Heifets teaches that various antimicrobial agents and tuberculosis drugs can be incorporated into the medium for the purpose of isolating M. tuberculosis cultures and for drug susceptibility testing (col. 7, lines 9-12).
With respect to claim 15, the modified 7H10 medium of Heifets contains malachite green, which is a pH indicator as discussed above in reference to Agapova.
Claim 17 recites a method for determining drug susceptibility of an antibiotic for treating a M. tuberculosis infection, comprising:
(a) inoculating a first medium comprising a composition accord of claim 1 with a clinical test sample of M. tuberculosis
(b) incubating the inoculated first medium in the presence or absence of the antibiotic under neutral pH conditions for a period of time and temperature sufficient to observe growth of the test sample in the absence of the antibiotic;
(c) comparing the growth of the test sample in the presence or absence of the antibiotic; and
(d) determining whether the test sample is sensitive or resistant to the antibiotic based on the comparison in step (c).
Heifets is applied as to claim 1, above, and further teaches a method for testing the drug susceptibility of a culture of M. tuberculosis (i.e. a method for determining drug susceptibility of an antibiotic for treating a M. tuberculosis infection – see Heifets col. 9, lines 52-54). This method includes the steps of: (a) inoculating an agar medium as set forth above, with a sample containing M. tuberculosis, wherein the agar medium further comprises an amount of at least one drug effective for selection against M. tuberculosis organisms that are susceptible to the drug; (b) incubating the inoculated agar medium for a time sufficient to detect growth of the M. tuberculosis in the absence of a growth-inhibiting drug; and, (c) measuring growth of the M. tuberculosis on the agar medium as compared to growth of the M. tuberculosis on the agar medium in the absence of the at least one drug. (col. 9, lines 54-66).
As noted, the agar medium of Heifets step (a), above, can have a Middlebrook 7H10 base (col. 7, lines 12-15) and is a composition of instant claim 1. The cited steps (a)-(c) of Heifets thus correspond to steps (a)-(c) of instant claim 17.
Heifets further teaches that a growth rate of the M. tuberculosis on the agar medium containing the at least one drug that is less than a pre-established drug-resistance level for the at least one drug, when compared to the growth rate of the M. tuberculosis on the agar medium in the absence of the at least one drug, indicates that the M. tuberculosis is susceptible to the at least one drug (col. 11, line 66 through col. 12, line 5). This corresponds to step (d) of instant claim 17. Heifets thus anticipates claim 17 in its entirety.
With respect to claim 18, Heifets teaches a working example (Example 3) in which four-segment plates having plain 7H11 agar medium are inoculated with different strains of M. tuberculosis, including both drug-resistant strains (resistant to isoniazid and/or rifampin) and pansusceptible strains (col. 24, lines 26-35). Each plate has a segment with no drug, and one or more segments with drug (e.g. two segments with different isoniazid concentration and one segment with rifampin – see col. 24, lines 34-39). The plates are then incubated and monitored for bacterial growth. This represents an example of a method of base claim 17, as the plate segments, having different drugs, are necessarily inoculated and incubated in parallel, and growth or lack thereof necessarily indicates whether the inoculum is sensitive or resistant to the antibiotic. This is further an example of a method of claim 18, in which a pansusceptible strain constitutes the control sample, and the other, resistant strain constitutes the test strain. More generally, Heifets teaches use of a quality control strain of M. tuberculosis which has previously been characterized against the drugs to be tested on a duplicate set of plates to confirm plate quality (col. 13, lines 15-19). Such a quality control strain constitutes a control sample of instant claim 18, for comparison to any test sample such that growth is expected in the quality control plate, to confirm that the result in the test plate is not artifactual.
With respect to claim 21, Heifets teaches determination of MICs (minimum inhibitory concentrations) of isoniazid (INH) and rifampin (RMP) for pansusceptible strains and for INH and RMP resistant strains (Example 3).
With respect to claims 23-25, Heifets teaches the antibiotic incorporated into the agar medium can be, for example, pyrazinamide, rifampin, isoniazid, or ethambutol, among others (col. 9, lines 19-34). With respect to claim 26, as noted, the 7H10 base of Heifets is ordinarily at a pH of about 6.6 (Middlebrook), although Heifets discloses that the agar medium may be set to a pH of 6.0 to 6.25 (col. 7, lines 51-53).
Claim 40 recites a method for screening a plurality of test compounds for their ability to inhibit the growth of Mycobacterium tuberculosis, comprising the steps of:
(a) inoculating a culture media comprising a composition of claim 1 with a M. tuberculosis isolate to from a control culture;
(b) inoculating in parallel a plurality of secondary cultures comprising the M. tuberculosis in step (a) to form a plurality of test cultures, wherein each of the plurality of test: cultures comprises a different test compound in the medium comprising the composition in step (a);
(c) incubating in parallel the first culture and the test cultures under neutral pH conditions for a period of time and temperature sufficient to observe growth of the control sample in the absence of the different test compounds in step (b);
(d) comparing the growth of the M. tuberculosis isolate in the presence or absence of the test compound; and
(e) determining whether one or more of the test compounds inhibit growth of the M. tuberculosis isolate, based on the comparison in step (d).
The limitation that the incubating of the first culture and the test culture be done “in parallel” is understood to require that they be incubated simultaneously.
Claim 40 is essentially the same as claim 17, except that it recites inoculating a plurality of test cultures, each comprising a different test compound (claim 40) rather than inoculating a single test sample with a single drug, and requires that the plurality of secondary cultures be inoculated at roughly the same time and that the resulting test cultures be incubated simultaneously.
Heifets is applied to claim 40 as to claim 17, above, and further teaches the methods can be applied against multiple drugs (different test compounds – see e.g. col. 9, lines 9-18), and more specifically teaches that the agar medium can contain a few different drugs (a plurality of test different test compounds) wherein each of the drugs is added to a portion of the agar medium and then plated into a separate plate, separate well, or separate segment of a segmented plate so that the drugs can be tested in one experiment against the same inoculum (col. 10, line 1-10). This embodiment clearly mandates that the various portions of agar medium containing different drugs be inoculated at roughly the same time (in parallel) and that the resulting inoculates having different drugs be incubated in parallel, particularly when occupying different well of the same plate. Claim 40 is therefore anticipated.
With respect to claims 41 and 45, Example 3 of Heifets is a study in which INH isoniazid and rifampicin are studied in parallel (col. 24, lines 26-32). As noted above, this Example describes a four segment plate having no drug, two concentrations of isoniazid, and one concentration of rifampin, again, inoculated and incubated in parallel (col. 24, lines 25-39). Heifets further teaches that this was performed using pansusceptible strains (col. 24, lines 26-28), such that growth was inhibited by both drugs (claim 41), and both antibiotics were known to inhibit growth of the culture (claim 45).
Claim Rejections - 35 USC § 103
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.
Claims 42-43 are obvious over Heifets and Martin:
Claims 42-43 are rejected under 35 U.S.C. § 103 as being unpatentable over Heifets, in view of the non-patent publication, Resazurin Microtiter Assay Plate Testing of Mycobacterium tuberculosis Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method, Antimicrob. Agents Chemother., 47, pgs. 3616-3619 (2003) by Martin et al. (hereinafter, “Martin”).
Claim 42 recited the method of claim 40 wherein each culture medium comprises a pH indicator, and wherein the comparison in step (d) comprises detection of a change in the color of the culture medium following growth of a test culture.
Heifets is applied to claim 42 as to claim 40, above, but does not explicitly disclose use of a pH indicator in the medium that will change color in response to mycobacterial growth, and consequent detection of this color change to monitor mycobacterial growth or lack thereof. It would have been obvious to use such a system to monitor mycobacterial growth in the method of Heifets, however, because such systems for monitoring bacterial growth in mycobacterial antibiotic susceptibility testing were well-known in the art. See, for example, Martin.
Martin teaches a colorimetric, microplate-based AST method based on resazurin pH sensitivity, testing 150 different M. tuberculosis isolates against several second-line drugs and getting results in 8 days (Abstract). Martin teaches inoculation of 7H9-S broth and distribution in 96-well plates, with varying concentration of different drugs and resazurin pH indicator (paragraph spanning the bottom of pg. 3616, right column, to the top of pg. 3617, left column, ). Martin further teaches that a change in color from blue to pink as growth (pg. 3617, left column, first paragraph), and that this system gave accurate results in comparison to alternative methods, but was rapid and relatively inexpensive (pg. 3618,left column, last paragraph). It would have been obvious to modify the method of Heifets to the non-agar, microtiter plate-based system of Martin, using resazurin for rapid detection to take advantage of rapid results with an inexpensive system.
With respect to claim 43, Martin states that the results can be determined by spectrophotometry or fluorometry (i.e. by plate reader).
Conclusion
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/ALEXANDER K. SHOWALTER/Examiner, Art Unit 1629
/JEFFREY S LUNDGREN/Supervisory Patent Examiner, Art Unit 1629