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 .
DETAILED ACTION
This application is a US national phase of PCT/IB2021/062381, filed December 28, 2021. Applicant’s Preliminary amendment filed June 24, 2024 is acknowledged. Claims 1-19 are canceled, and claims 20-37 are newly added.
Currently claims 20-37 are pending and under examination.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claims 20, 23, 27, and 29-30 are objected to because of the following informalities:
Claim 20, lines 8-9, needs to be changed to “separating the protective metabolites in liquid form from the strain of microorganism by centrifugation.” to reduce redundancy in claim language.
Claim 23, needs to be changed to “The method of claim 22, wherein each strain is grown separately to a concentration of at least 1-109 CFU/ml.” to remove superfluous claim language.
Claim 27, needs to be changed to “The method of claim 20, wherein the separating the protective metabolites includes centrifugations, wherein centrifugal acceleration at a temperature from +2 to +25 ⁰C.” to remove superfluous claim language.
Claim 29, need to be changed to “The method of claim 20, further comprising mixing the protective metabolites with an additive matrix after the centrifugation.” to maintain claim language consistency.
Claim 30, needs to add “…after the separating step.” for claim language consistency.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 20-37 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
Newly added claim 20 recites “a strain of microorganism that is compatible with human microbiome or normal human microflora”, which appears to depart from the claims as originally filed. The specification does not describe a ‘a strain of microorganism that is compatible with human microbiome or normal human microflora’. Therefore, these limitations constitute new matter. The claimed method as described in the specification is drawn to using a nutrient medium to grow strains of microorganisms approved for use included in the human microbiome or friendly to normal human microflora [0017]. If Applicant believes that such support is present in the specification and claimed priority documents, Applicant should point, with particularity, to where such support is to be found.
Claims 20-37 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of manufacturing protective metabolites derived from Bifidobacterium bifidum, Lactobacillus delbrueckii subsp. Bulgaricus, and Lactobacillus acidophilus by applying low-frequency and high-frequency vibrations in the range of 8-20 kHz and 70-250 kHz, respectively, for 1-20 minutes, then incubated for 60 minutes at 25 ⁰C to produce metabolites, does not reasonably provide enablement for a method of manufacturing protective metabolites derived from any strain of microorganism that is compatible with human microbiome applying life-inhibitory physical stress for any amount of time characterized as temperature shock, high-G force, a shock or shock-cavitation wave, then incubating the strain for any amount of time at any temperature to produce the metabolites. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
The breadth of the claims:
Claims 20-37 are drawn to a method for manufacturing an anti-infective agent (i.e. protective metabolites) by growing a strain of microorganism compatible with human microbiome, applying a life-inhibitory physical stress to the strain, and incubating the strain to produce the metabolites, then finally separating the metabolites by centrifugation.
The nature of the invention:
The nature of the invention relies upon using any microorganism that is compatible with human microbiome and applying life-inhibitory physical stress for any amount of time, characterized as temperature shock, high-G force, a shock or shock-cavitation wave, on the strain, then incubating for any amount of time at any temperature for producing protective metabolites.
Whether the specification would have been enabling as of the filing date involves consideration of the nature of the invention, the state of the prior art, and the level of skill in the art. The state of the prior art is what one skilled in the art would have known, at the time the application was filed, about the subject matter to which the claimed invention pertains. The relative skill of those in the art refers to the skill of those in the art in relation to the subject matter to which the claimed invention pertains at the time the application was filed. See MPEP § 2164.05(b). The state of the prior art provides evidence for the degree of predictability in the art and is related to the amount of direction or guidance needed in the specification as filed to meet the enablement requirement. The state of the prior art is also related to the need for working examples in the specification.
The state of the prior art:
A thorough review of the patent and non-patent literature indicates that the state of the art demonstrating applying life-inhibitory physical stress for any amount of time, characterized as temperature shock, high-G force, a shock or shock-cavitation wave, to any type of microorganism to produce protective metabolites is variable and lacking. As evidenced by Rose et al. (Infection and Immunity, 2025, vol. 93, issue 6, pages 1-19), anaerobic microbes under pressure of oxidative stress (caused by shock-cavitation wave) will generate detrimental secondary metabolites, but some commensal biota have evolved specialized mechanisms to address the threat of O2 resulting reactive oxygen species, however, these responses and the capacity to detoxify and neutralize oxygen and ROS vary significantly among species, resulting in an array of oxygen tolerance and susceptibility amongst commensal organisms, which reflects the oxygen tension present in their specific intestinal niches (pg. 10, para 1), thus underlining the extreme variability of among strains in response to stress.
Yoon et al. (J Ind Microbiol Biotechnol (2014) 41:415–424), discloses the production of jadomycin B, by Streptomyces venezuelae is a classic example of eliciting a secondary metabolite in response to stress, Jadomycin B belongs to the family of benzoxazolophenanthridine antibiotics that have potent antibiotic activity against Gram-positive pathogens such as Staphylococcus aureus and Staphylococcus epidermidis, and during normal culture of S. venezuelae (after heat-shock), relatively little jadomycin is produced, however shifting the temperature from 27 to 42 °C, causes greatly elevated yields of jadomycin B, which peaks at 12 h following heat shock with titers increasing to as high as 25 μg/ml (pg. 416, col. 2, para 3), thus underlining the variability in temperature incubation and time for producing said metabolites.
A review of the prior art does not find consistent or specific support of manufacturing protective metabolites using any microorganism by applying a life-inhibitory physical stress for any time period characterized as temperature shock, high-G force, a shock or shock-cavitation wave, then incubating at any temperature for any time period to produce said metabolites.
The level of one of ordinary skill:
While the level of one of ordinary skill practicing said invention would be high, the level of predictability is considered variable as evident in the prior art discussed above and is not considered to provide sufficient enablement to practice the claimed invention. Because the state of the prior art does not provide evidence of the degree of predictability that methods for producing protective metabolites using any type of microorganism by applying life-inhibitory physical stress for any time period characterized as temperature shock, high-G force, a shock or shock-cavitation wave, then incubating at any temperature for any time period to produce said metabolites, one of ordinary skill in the art would look for guidance or direction in the instant specification.
The level of predictability in the art:
“The “predictability or lack thereof” in the art refers to the ability of one skilled in the art to extrapolate the disclosed or known results to the claimed invention. If one skilled in the art can readily anticipate the effect of a change within the subject matter to which the claimed invention pertains, then there is predictability in the art. On the other hand, if one skilled in the art cannot readily anticipate the effect of a change within the subject matter to which that claimed invention pertains, then there is lack of predictability in the art. Accordingly, what is known in the art provides evidence as to the question of predictability.” (MPEP 2164.03).
The amount of direction provided by the inventor:
The amount of guidance or direction needed to enable the invention is inversely related to the amount of knowledge in the state of the art as well as the predictability in the art. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970). The “amount of guidance or direction” refers to that information in the application, as originally filed, that teaches exactly how to make or use the invention. The more that is known in the prior art about the nature of the invention, how to make, and how to use the invention, and the more predictable the art is, the less information needs to be explicitly stated in the specification. In contrast, if little is known in the prior art about the nature of the invention and the art is unpredictable, the specification would need more detail as to how to make and use the invention in order to be enabling. >See, e.g., Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1326 (Fed. Cir. 2004).
The existence of working examples:
The working embodiment in the instant application describes using Bifidobacterium bifidum, Lactobacillus delbrueckii subsp. Bulgaricus, and Lactobacillus acidophilus by applying low-frequency and high-frequency vibrations in the range of 8-20 kHz and 70-250 kHz, respectively, for 1-20 minutes, then incubated for 60 minutes at 25 ⁰C to produce protective metabolites, separated the metabolites by centrifugation, then tested for antimicrobial activity against Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans , and assessed antiviral activity against a variety of viral pathogens (pg. 6-9). The working embodiments do not describe any other type of microorganism that produces protective metabolites, nor any other life-inhibitory physical stress applied for any amount of time, other than the low- and high-frequency vibrations applied for 1-3 minutes, and does not describe any other time periods to incubate the stressed bacteria to produce the metabolites. While the MPEP 2164.02 states the specification need not contain an example if the invention is otherwise disclosed in such manner that one skilled in the art will be able to practice it without an undue amount of experimentation. In re Borkowski, 422 F.2d 904, 908, 164 USPQ 642, 645 (CCPA 1970), the lack of a working example, however, is a factor to be considered, especially in a case involving an unpredictable and undeveloped art.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure:
The prior art is undeveloped for the role that applying a life-inhibitory physical stress for any amount of time characterized as temperature shock, high-G force, a shock or shock-cavitation wave, to any type of microorganism, then incubating for any time period at any temperature, would predictably produce protective metabolites as broadly claimed. The specification does not provide sufficient guidance on using any type of microorganism to produce protective metabolites except for the described examples outlined on pages 6-9.
Without further guidance, one of skill in the art would have to practice a substantial amount of trial-and-error experimentation, an amount considered undue and not routine, to practice the instantly claimed invention.
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.
Claims 20-37 are 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.
The term “compatible with human microbiome or with normal human microflora” in claim 20 is a relative term which renders the claim indefinite. The term ‘compatible’ is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification merely discloses ‘friendly to normal human microflora’, but does not define an operational threshold (such as survival rate threshold, co-culture stability), thus the metes and bounds of the claim are not clearly defined.
Claim 20 recites “life-inhibitory physical stress to the strain, wherein the stress is any of temperature shock, high-G force, a shock or shock cavitation wave”. It is unclear what the term ‘life-inhibitory physical stress’ encompasses, as the specification discloses implemented stress (life-threatening) effect on the organisms, and that the stress produces protective stress proteins. Does the life-inhibitory stress kill the bacteria or inhibit replication or only induce the protective compounds? One of ordinary skill in the art would not be apprised of the metes and bounds of the claim, thus is indefinite. Furthermore, ‘shock and shock-cavitation wave’ is unclear, because as disclosed in the specification, the biomass mixture was subjected to a life-threatening impact using shock and cavitation equipment producing low-frequency and high-frequency vibrations [0042]. It is unclear if the first ‘shock’ is directed to only a shock induced by the shock and cavitation equipment, or another undefined shock (such as mechanical, pressure, electrical, etc.), thus is indefinite.
The term “protective metabolites” in claims 20 and 31 is a subjective term which renders the claim indefinite. The term “protective metabolites” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification does not specify what constitutes a protective metabolite, only disclosing content of toxic elements and antimicrobial activity of protective metabolites, and protective substances having the structure of peptidoglycans, glycoproteins and peptides, have a stimulating effect on the immune response of human cells by binding to the surface proteins of these cells, modulators of the growth of most representatives of normal human microflora as molecules providing quorum sensing, contains cytostatic and anti-inflammatory substances, and are commonly in mixture with amino acids, triglycerides, metalloproteins and proteins included into exo- and endo- metabolites causing suppressive action on pathogenic microorganisms and viruses [0016]. However, ‘protective metabolites’ could also be interpreted as those affording protection to the microorganism itself or to
Claim 20 also recites “…releasing the protective metabolites by separating, using a centrifuge, the protective metabolites in a liquid form from the strain of microorganism using a centrifuge.”. The limitation of ‘releasing the protective metabolites by separating with centrifugation’ is confusing as centrifugation separates components but does not necessarily release metabolites, especially in the case of intracellular metabolites, and if extracellular metabolites, are already released, thus creates uncertainty regarding what process is required.
Claim 21 recites “…the nutrient medium includes any of: Pancreatic casein hydrolysate (20.0-30.0 g/l); …etc.”. This appears to be an open-ended list of alternatives and is uncertain whether is the last member in this group of alternatives is Micronutrients. The parenthetical phrase (20.0-30.0 g/l) renders the claim indefinite because it is unclear whether the limitation within the parenthetical phrase is part of the claimed invention. To obviate this rejection, the Examiner suggests amending to “the nutrient medium includes in the amount of any of: Pancreatic casein hydrolysate at 20.0-30.0 g/l; ….etc.”. The claim also recites “Micronutrients (0.5-1.0)” but does not indicate a concentration is not preceded by the word “and”, thus is indefinite.
Claim 21 contains the trademark/trade name “Tween 80”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe polysorbate 80 and, accordingly, the identification/description is indefinite.
Claim 22 recites “…a biomass of strains from 2 to 100 human symbiont species are mixed, in equal proportions, or with a variation of up to 99.9% of a proportion.”. It is uncertain if a biomass of strains is mixed prior to using a nutrient medium to grow a strain of microorganism, or added during the step of incubating the strain to produce protective metabolites. It is unclear what a ‘variation of up to 99.9% of a proportion’ means. , as the specification indicates ‘from 2 to 100 human symbiont species are mixed, in equal parts, or with a variation of up to 99.9% of the part, i.e. 1:1 to 1:0.001 parts or use biomass of a single strain.’, which indicates a range of parts in a ratio, but the claim recites a proportion, which can be alternatively interpreted as 99.9% variation between genetic homology of the 2-100 species. Therefore, one of ordinary skill in the art would not be apprised of the metes and bounds of the claim, thus is indefinite.
Claim 24 recites “wherein the mixture of biomass of strains is sustained for at least 60 minutes at +20 to +28 ⁰C to produce the metabolites before starting the release of the metabolites.”. It is unclear which step in claim 20 this claim limitation is referring to as claim 20 does not recite any strain is sustained. Is the life-inhibitory physical stress performed for 60 minutes at 20-28 ⁰C, or is the stress performed, and then the microbes are incubated for 60 minutes in that temperature range? Furthermore the phrase ‘produce the metabolites before starting the release of the metabolites’ is unclear, as this could be interpreted as secretion of the metabolites from the cells, or the physical separation of the centrifugation, thus is indefinite.
Claim 28 recites “wherein, after the separating, liquids are combined in proportions according to a material balance formula for mixing liquids of different densities: Vk • pk = V1•p1 + V2•p2 + V3 • p3 + Vn • pn, where, V1 and Vn are volumes of supernatant liquid No. 1 and n, in ml; p1 and pn are density of supernatant liquid No.1 and n, g/ml; and Vk is the final volume, ml; pk is the final density, g/ml.”. It is unclear what liquids are combined in proportions and at which steps of the separation, and V2•p2 + V3 • p3 are also not defined in the claim, thus is indefinite. Furthermore, supernatant liquid No. 1 lacks antecedent basis in the claim. Also, ‘..and n, g/ml;…final volume, ml;…final density, g/ml.” needs to clearly define each variable as ‘in g/ml’ or ‘in ml’ for clarity in claim language.
Claim 32 recites “The method of claim 20, further comprising impregnation of the anti- infective agent into wipes or patches, followed by a subsequent drying stage.”, however it is unclear what constitutes ‘the anti-infective agent’, such as the protective metabolites or the microorganism, thus is indefinite.
Claims 23, 25-27, 29-30, and 33-37 are likewise rejected as being dependent on an indefinite claim.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 22 and 24 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 22 recites: the method of claim 20, wherein a single strain is used or a biomass of strains from 2 to 100 human symbiont species are mixed, in equal proportions, or with a variation of up to 99.9% of a proportion. Claim 24 recites: the method of claim 22, wherein the mixture of biomass of strains is sustained for at least 60 minutes…. Claim 20 recites “using a nutrient medium to grow a strain of microorganism…” and does not recite a biomass of strains, thus claims 22 and 24 are not further limiting the subject matter of the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 20-23, 29-30, 32, 34, and 36 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by O'Nejl et al. (RU 2656152 C2, hereinafter “O'Nejl”).
Regarding claims 20-22, and 30, O’Nejl teaches use of probiotic bacterium lysate for improvement and/or restoration of barrier function of skin (title). O’Nejl teaches probiotic bacteria, particularly, Lactobacillus reuteri, was grown according to standard procedures to a stable phase in Wilkins-Chalgren Broth (WCB) broth (Oxoid) at 37 ° C in a Mark 3 anaerobic workstation, which anticipates a nutrient medium containing yeast extract in claims 20-21, and a microorganism strain compatible with human microbiome and ‘single strain’ in claims 20 and 22 (pg. 19, last two lines). O’Nejl teaches the cultures were inactivated by placing in a water bath at 85 °C for 45 minutes, Gram stained to confirm cell lysis did not occur, and plated on growth media for experiments utilizing cell lysates (pg. 20, para 1), which anticipates the limitations of life-inhibitory physical stress by temperature shock, and incubating strain to produce protective metabolites. O’Nejl teaches the Lactobacillus reuteri were resuspended and centrifuged, washed, concentrated, and lysed, then sterilized filtered to remove whole bacteria, which anticipates the limitations of releasing the protective metabolites from the strain by centrifugation in claim 20 and sterilizing filtration in claim 30 (pg. 20, para 1). O’Nejl teaches the lysates from L. reuteri had a significant protective effect on the viability of normal human epidermal keratinocytes (NHKEK) cells exposed to Staphylococcus aureus, thus inherently anticipating the limitation of ‘protective metabolites’ (pg. 22, para 2).
Regarding claim 23, O’Nejl teaches each strain can be grown in the range of 104 to 1012 CFU, which encompasses ‘at least 109 CFU/mL’, thus anticipating the claim.
Regarding claim 29, O’Nejl teaches the lysates of L. reuteri was added to NHKEK cell cultures that were suspended in keratinocyte basal medium containing additives (pg. 20, para 2).
Regarding claim 32, O’Nejl teaches the probiotic lysates can be formulated to be suitable for transdermal administration as patches, thus anticipating the claim (pg. 18, para 1).
Regarding claim 34, O’Nejl teaches formulations of the lysate can be in the form of lozenges, which anticipates the claim (pg. 18, para 6).
Regarding claim 36, O’Nejl teaches the compounds of the invention can be formulated to treat conditions of the skin, thus anticipating the claim (pg. 13, para 5).
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.
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.
Claims 22, 24, 27-28, and 31 are rejected under 35 U.S.C. 103 as being unpatentable over O’Nejl.
As discussed above, O’Nejl anticipates claims 20-23, 29-30, 32, 34, and 36.
Regarding claim 22, O’Nejl does not explicitly teach a mixture of biomass strains from 2 – 100 human symbiont strains, mixed in equal proportions. However, O’Nejl teaches five different strains of lactic acid bacteria were tested for their protective effect on the NHKEK cells, and found that Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus plantarum, and L. reuteri all increased transepithelial electrical resistance (TEER), which is a measure of the functioning of Tight junctions (TJ), which are multi-protein complexes that fill the paracellular space between adjacent epithelial cells and restrict the transport through this path for small hydrophilic molecules and ions, thus improving barrier function (pg. 29, para 3; pg. 3, para 3-4).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to use a biomass of strains for producing protective metabolites comprising at least 2 human symbiont species, because O’Nejl teaches the four probiotic strain’ lysates released protective metabolites that conferred increased TJ functioning of the NHKEK cells. Thus it would have been obvious to one of ordinary skill in the art to combine these strains into a biomass mixture for manufacturing an anti-infective agent comprising protective metabolites.
Regarding claim 24, O’Nejl teaches that all probiotic strains (B. longum ATCC-51870, L. plantarum, ATCC-10241, L. reuteri, ATCC-55730, L. rhamnosus Goldwin and Gorbach, ATCC-53103, L. fermentum, ATCC-14932) were grown according to standard procedures to the stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37 ° C in Mark 3 anaerobic workstation (Don Whitley scientific, UK). Cultures were adjusted spectrophotometrically to contain 10 8 CFU / ml. 10 ml of these cultures were centrifuged, washed and then concentrated in 1 ml of keratinocyte basal medium. See Example 4, Materials and Methods, Preparation of probiotic lysates. For purposes of applying prior art, the examiner is interpreting ‘mixture of biomass of strains is sustained for at least 60 minutes’ to mean incubated.
O’Nejl does not teach the culture is grown for at least 60 minutes at 20-28 ⁰C to produce the protective metabolites, as recited in the claim. However, it is well-known and routine in the art to adjust incubation times and temperatures according to the needs of the microorganism’s growth and metabolic requirements, thus it would be obvious to one of ordinary skill in the art to modify these parameters accordingly. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claims 27-28, O’Nejl teaches the probiotic cells were pelleted in a microcentrifuge at 15,000 × g for 5 minutes and the cell free supernatant (CFS) was extracted for use, but does not indicate a temperature range or that the cell suspension was separated into liquid sediment through multiple steps of centrifugal separation, nor combined in the particular proportions according to the formula recited in claim 28. However, it is well-known and routine in the art to adjust concentration and temperatures according to the method, thus it would be obvious to one of ordinary skill in the art to modify these parameters accordingly. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Similarly, formulations of mixtures according to volume and density of cell cultures is routine and well-understood in microbiological practices, thus would be obvious to one of ordinary skill in the art to combine in proportions according to the recited material balance formula in claim 28.
Regarding claim 31, O’Nejl teaches adding a 0.85% NaCl solution to wash bacteria before centrifugation and resuspension in a keratinocyte medium, (See Example 1: Materials and Methods – Bacteria Cultivation). Therefore, it would have been prima facie obvious to combine the released protective metabolites with a salt solution as a diluent.
Claims 33, 35, and 37 are rejected under 35 U.S.C. 103 as being unpatentable over O’Nejl as applied to claims 20-23, 29-30, 32, 34, and 36 above, and further in view of Nader-Macias et al. (Current Women’s Health Reviews, 2008, 4, 240-257, hereinafter “Macias”).
O’Nejl teaches in some embodiments, the restoration or regeneration of the skin barrier includes the restoration or regeneration of the mucous membrane, including vagina, penis, urethra, bladder and anus (pg. 16, para 2).
O’Nejl does not teach the method comprises impregnating the metabolites into a tampon, adding to a suppository base, or injecting into internal genital organs.
However, Macias teaches clinical applications of lactic acid bacteria as probiotics in the urogenital tract (title). Macias teaches a study on patients with dysbiotic vaginal microbiota by inserting a tampon with a bacterial preparation (pg. 246, Table 1), which meets the limitation of claim 33. Macias teaches another study to evaluate the influence of 3-day antimicrobial therapy by administering lactobacillus vaginal suppositories (pg. 246, Table 1), which meets the limitation of claim 35. Macias also discloses a study wherein women were treated for 6 days with a douche containing lactobacilli (pg. 249, Table 1), which meets the limitation of ‘injectable into internal genital organs’ in claim 37.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the filing date of the claimed invention to utilize the method of producing protective metabolites that restore mucosal barrier function as taught by O’Nejl, and formulate the metabolites into tampons, suppositories, or injectable forms for vaginal treatment as taught by Macias with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to formulate the protective metabolites into the applicable carriers as taught by Macias to effectively treat vaginal dysbiosis.
Claims 25-26 are rejected under 35 U.S.C. 103 as being unpatentable over O’Nejl as applied to claims 20-23, 29-30, 32, 34, and 36 above, and further in view of Bayer et al. (INFECTION AND IMMUNITY, May 1990, p. 1344-1349, hereinafter “Bayer”), as evidenced by Rose et al. (Infection and Immunity, 2025 Volume 93 Issue 6, pgs. 1-19, hereinafter “Rose”).
As discussed above, O’Nejl anticipates claims 20-23, 29-30, 32, 34, and 36.
O’Nejl teaches the probiotic strains are stressed with heat inactivation. O’Nejl teaches a lysate may contain one or more soluble metabolites of the probiotic bacteria, and lysis can be carried out by chemical or physical disruption, for example, by adding an osmotic agent or enzyme to bacteria or by applying physical pressure, for example, by disruption by sound, for example, preparing a lysate by ultrasound (pg. 17, para 5).
O’Nejl does not teach the method includes shock-cavitation wave.
However, Bayer teaches oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa (title). Bayer teaches P. aeruginosa cultures were stressed with oxygen tension in the presence of pO2s of ~40 or 80 mm Hg by exposing the cells for 6 hours at 37 ⁰C by bubbling a gas mixture of 6% O2-94%N2 into the liquid culture for 5 minutes (pg. 1345, col. 1, last para), which meets the limitation of a ‘shock-cavitation wave’ in claim 25, and falls within the time range recited in claim 26. Bayer teaches the oxygen-enhanced exopolysaccharide seen in nonmucoid P. aeruginosa cells was, indeed, mucoid exopolysaccharide (MEP), which enhances attachment of mucoid strains to tracheal epithelium; resistance of mucoid strains to nonopsonic phagocytosis by monocyte-derived macrophages and neutrophils; mitigation of antibiotic (aminoglycoside) - diffusion in vitro, presumably through a barrier mechanism (pg. 1348, col. 1, para 1, 3).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to optimize the method of producing protective metabolites in a microorganism as taught by O’Nejl by applying an oxygen tension stress to bacterial culture for at least 5 minutes to induce production of protective metabolites as taught by Bayer. Although Bayer’s method utilizes P. aeruginosa to produce the protective metabolites, as evidenced by Rose, it is well-known in the art that due to their residence in the small intestine, Lactobacilli must tolerate higher oxygen levels than obligate anaerobes in the distal colon, as the small intestine has a significantly higher oxygen tension (pg. 11, para 2). Rose discloses Lactobacilli are the most frequently used probiotics, as they have the potential to enhance intestinal barrier integrity, confer protection from pathogens by producing antimicrobial peptides, occupy nutritional niches, and address inflammatory conditions, and in high-oxygen tension environments, many Lactobacilli express a manganese (Mn) containing SOD to neutralize superoxide radicals (pg. 11, para 2). Thus, one of ordinary skill in the art would be motivated to confer oxygen tension as taught by Bayer, to the lactobacilli strains in the method taught by O’Nejl, to effectively induce production of protective metabolites, as disclosed by Rose.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
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