DETAILED ACTION
Preliminary Amendment filed on 02/22/2024 is acknowledged. Claims 1-15 are pending in the application and are considered on merits.
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 .
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.
Claim 1-15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 15-28 of copending Application No. 17/798,720 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because both the instant claims and the currently patented claims expressly recite the same subject matter, it would have been obvious to one of ordinary skill in the art at the time the invention was made to employ both device and methods, as recited in both sets of claims.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
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.
Claim(s) 1-3, 5-7 and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Comas-Basté et al. (Journal of Pharmaceutical and Biomedical Analysis, 2017) (Baste), in view of Komericki et al. (Wien Klin Wochenschr, 2011) (Komericki), and Nelis et al. (Talanta, 2020) (Nelis).
Regarding claim 1, Baste teaches a method of diagnosis of histamine intolerance syndrome in a human subject suspected of suffering from insufficient histamine degradation activity (abstract). Specifically, Baste teaches that histamine intolerance is a disorder in histamine homeostasis due to reduced intestinal degradation of histamine, mainly caused by diamine oxidase (DAO) enzyme deficiency, and that a new approach for diagnosis of histamine intolerance could be the determination of histamine and its metabolites in urine (abstract). Baste further teaches that DAO performs oxidative deamination of histamine to imidazole acetaldehyde, which is later converted to imidazoleacetic acid, and that HNMT converts histamine to methylhistamine, which is later converted to N-methylimidazoleacetic acid. Thus, Baste teaches diagnosing histamine intolerance by evaluating histamine and histamine inactivation products in a bodily fluid sample, namely urine.
Baste further teaches comparing suspected histamine-intolerant individuals with healthy individuals because Baste teaches that individuals with insufficient DAO activity would have a distribution profile of histamine and metabolites significantly different from healthy individuals, and that individuals with symptoms associated with histamine intolerance would show higher urinary histamine and methylhistamine than the healthy population (page 379, par 2).
Baste does not expressly teach administering a preparation, solution, or suspension containing a known amount of stable-isotope-labeled histamine before obtaining the bodily fluid samples.
Komericki teaches administering a preparation or solution containing a known amount of histamine to human subjects suspected of histamine intolerance. Specifically, Komericki teaches that patients suspected to be histamine intolerant were recruited and that the first step consisted of open oral provocation of these patients with 75 mg of liquid histamine (page 17, par 2). Komericki further teaches that the open provocation used 75 mg of pure histamine in 100 mL of peppermint tea (page 17, par 2). Thus, Komericki teaches administering a preparation/solution containing a known amount of histamine to a human subject suspected of histamine intolerance.
Baste in view of Komericki does not expressly teach that the administered histamine is labeled by a stable isotope, determining isotopically labeled histamine and one or more other isotopically labeled inactivation products using HILIC LC-MS/MS, or using an aprotic solvent miscible with the aqueous sample to prepare the sample.
Nelis teaches an LC-MS/MS method for separating and quantifying histamine and its main metabolites in human urine samples, including imidazole acetaldehyde, imidazole acetic acid, methyl imidazole acetic acid, methylhistamine, and acetylhistamine (abstract). Nelis further teaches that histamine is metabolized by DAO to imidazole acetaldehyde, followed by oxidation to imidazole acetic acid, and that histamine is metabolized by HNMT to methylhistamine, followed by further metabolism to methyl imidazole acetic acid (page 1, par 1). Nelis also teaches that measuring histamine metabolites in body specimens provides a way to calculate histamine turnover (page 1, par 2).
Nelis teaches using hydrophilic interaction liquid chromatography with tandem mass spectrometry because the polar nature of histamine and its metabolites makes reversed phase chromatography insufficient, HILIC provides an alternative for polar compounds, and HILIC is well suited for MS detection (page 2, par 3-4). Nelis also teaches preparing urine samples by diluting urine with acetonitrile to a final volume comprising 95% acetonitrile and centrifuging the samples prior to injection (page 4, par 2). Acetonitrile is an aprotic solvent miscible with aqueous samples. Nelis further teaches use of a stable-isotope-labeled histamine compound, histamine-d4, in the LC-MS/MS method (page 4, par 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify Baste’s urine-based diagnostic method by including Komericki’s known oral histamine provocation before collecting the urine sample, because Baste seeks to diagnose histamine intolerance based on urinary histamine/metabolite profiles, and Komericki teaches that oral provocation with a standardized quantity of histamine was a known test procedure for subjects suspected of histamine intolerance. A person of ordinary skill would have been motivated to apply a standardized histamine load before urinary metabolite testing to reduce variability caused by uncontrolled dietary histamine exposure and to provide a defined histamine challenge for evaluating histamine degradation/inactivation.
It would further have been obvious to use a stable-isotope-labeled histamine as the known administered histamine in the modified method because Baste and Nelis both concern endogenous histamine and endogenous histamine metabolites in biological samples, and Nelis recognizes that the endogenous occurrence of histamine/metabolites complicates quantitative analysis (page 2, par 3). Nelis also teaches the use of deuterated histamine, histamine-d4, in LC-MS/MS analysis (page 4, par 3). A person of ordinary skill would have been motivated to label the administered histamine with a stable isotope so that the administered/test histamine and its corresponding inactivation products could be distinguished by mass spectrometry from endogenous histamine and endogenous metabolites while retaining the same relevant histamine degradation pathways. Such modification would have predictably improved the ability to calculate histamine inactivation activity attributable to the administered histamine load rather than background endogenous histamine.
It would further have been obvious to use the HILIC LC-MS/MS method and acetonitrile sample preparation taught by Nelis in the modified Baste/Komericki diagnostic method because Baste teaches that chromatographic methods coupled with sensitive detection systems, including mass spectrometry, are suitable for simultaneous separation and quantification of histamine and metabolites, and Nelis provides a HILIC LC-MS/MS method specifically developed to quantify histamine and its main metabolites in human urine. The motivation would have been to improve sensitivity, specificity, and analytical coverage of histamine degradation products and thereby provide a more complete histamine metabolism/turnover profile for diagnosing histamine intolerance.
Therefore, the combination of Baste, Komericki, and Nelis teaches or renders obvious a method of diagnosing histamine intolerance syndrome in a human subject suspected of insufficient histamine inactivation activity, comprising administering a known amount of stable-isotope-labeled histamine, obtaining a bodily fluid sample such as urine after a predetermined time, preparing the sample using an aprotic solvent such as acetonitrile, determining isotopically labeled histamine and isotopically labeled histamine inactivation products including imidazole acetic acid using HILIC LC-MS/MS, calculating histamine inactivation activity based on the amounts of histamine and imidazole acetic acid, and comparing the result with healthy subjects.
Regarding claim 2, Nelis teaches that the method of claim 1, further comprising a determination of methyl imidazole acetic acid in the sample (Fig. 3, page 1, par 1).
Furthermore, because the modified method of claim 1 administers stable-isotope-labeled histamine, a person of ordinary skill would have reasonably expected the methyl imidazole acetic acid generated from the administered stable-isotope-labeled histamine to also be isotopically labeled. It would have been obvious to determine the isotopically labeled methyl imidazole acetic acid by LC-MS/MS in order to distinguish the methyl imidazole acetic acid produced from the administered histamine load from endogenous methyl imidazole acetic acid.
Regarding claim 3, Baste teaches that the method of claim 1 further comprising a determination of methylhistamine in the sample (Abstract).
Nelis also teaches that the method of claim 1 further comprising a determination of methylhistamine in the sample (Fig. 3).
Furthermore, because the modified method of claim 1 administers stable-isotope-labeled histamine, a person of ordinary skill would have reasonably expected methylhistamine generated from the administered stable-isotope-labeled histamine to also be isotopically labeled. It would have been obvious to determine the isotopically labeled methylhistamine by LC-MS/MS in order to distinguish methylhistamine produced from the administered histamine load from endogenous methylhistamine.
Regarding claim 5, Nelis teaches that wherein the isotopically labelled histamine is selected from histamine 15N-labelled at positions at anyone or more nitrogen position or a histamine 13C-labelled at anyone or more carbon positions or a histamine deuterium labelled at anyone or more hydrogen position, or a histamine containing a combination of stable isotope labels (page 4, par 3).
Regarding claim 6, Nelis teaches that wherein the aprotic solvent is selected from acetonitrile, tetrahydrofuran, acrylonitrile, dioxane, ethanol, methanol preferably from acetonitrile (page 4, par 3).
Regarding claim 7, Baste teaches that an alternative diagnostic approach for histamine intolerance is the determination of histamine and its metabolites in urine, because individuals with insufficient DAO activity would have a distribution profile of histamine and metabolites significantly different from healthy individuals (abstract). Baste further teaches that individuals with symptoms associated with histamine intolerance would show higher urinary histamine and methylhistamine than the healthy population, and that the distribution profile of histamine and methylhistamine in urine could provide a complementary evaluation of DAO activity (page 380, par 1).
Nelis teaches determining histamine, methylhistamine, imidazole acetic acid, and methyl imidazole acetic acid in a urine sample by HILIC-MS/MS (Fig. 3). Nelis further teaches that histamine is degraded by DAO to imidazole acetaldehyde and then to imidazole acetic acid, and also degraded by HNMT to methylhistamine and then to methyl imidazole acetic acid (Fig. 1). Nelis further teaches that measurement of histamine metabolites in body specimens provides a way to calculate histamine turnover, and that measuring multiple metabolites provides an entire fingerprint of histamine metabolism (page 2, par 3).
It would have been obvious to one of ordinary skill in the art to determine a ratio of relative amounts of histamine and one or more histamine inactivation products, such as histamine/methylhistamine or histamine/imidazole acetic acid, in the modified method of claim 1 because Baste teaches evaluating a distribution profile of histamine and metabolites for histamine intolerance diagnosis, and Nelis teaches that measurement of histamine metabolites allows calculation of histamine turnover and provides a fingerprint of histamine metabolism. Determining a ratio of the measured amount of remaining histamine to the measured amount of a histamine inactivation product would have been a routine and predictable way to express the relative conversion of histamine into its metabolites and thereby evaluate histamine inactivation activity.
Furthermore, because the modified method of claim 1 administers stable-isotope-labeled histamine, a person of ordinary skill would have reasonably expected the methylhistamine, imidazole acetic acid, and methyl imidazole acetic acid generated from the administered stable-isotope-labeled histamine to also be isotopically labeled. It would have been obvious to determine isotope-labeled ratios, such as labeled histamine/labeled methylhistamine or labeled histamine/labeled imidazole acetic acid, in order to distinguish the metabolism of the administered histamine load from endogenous histamine and endogenous metabolites.
Regarding claim 12, Komericki teaches a defined histamine oral load in the form of a solution because Komericki teaches oral provocation of patients suspected of histamine intolerance with 75 mg of liquid histamine, and further teaches open provocation with 75 mg of pure histamine in 100 mL of peppermint tea (page 17).
Nelis teaches stable-isotope-labeled histamine in the form of deuterated histamine, histamine-d4, for LC-MS/MS analysis (page 4). As discussed above with respect to claims 1 and 5, it would have been obvious to use stable-isotope-labeled histamine as the defined histamine oral load in the modified diagnostic method so that the administered histamine and its corresponding inactivation products could be distinguished by mass spectrometry from endogenous histamine and endogenous metabolites.
Nelis further teaches aqueous stock and working solutions containing histamine and histamine metabolites of interest, including methylhistamine, imidazole acetic acid, and methyl imidazole acetic acid (page 3). Nelis also teaches that the endogenous nature of histamine and its metabolites presents a quantitation challenge and that stable-isotope-labeled internal standards are useful in such multicomponent analyses, although Nelis used standard addition because labeled internal standards for each compound were unavailable (page 2).
It would have been obvious to one of ordinary skill in the art to implement the modified diagnostic method of claim 7 using a kit comprising a defined stable-isotope-labeled histamine oral load and stable-isotope-labeled standard solutions for histamine catabolites because Komericki teaches a standardized oral histamine provocation test for suspected histamine intolerance, Nelis teaches HILIC-MS/MS measurement of histamine and its metabolites and recognizes the analytical value of stable-isotope-labeled internal standards.
A person of ordinary skill would have been motivated to provide the oral load and internal standard solutions in kit form to standardize the amount of histamine administered, standardize sample analysis, reduce operator preparation error, and improve reproducibility across diagnostic testing sites. Use of stable-isotope-labeled standards would have predictably improved LC-MS/MS quantitation by compensating for matrix effects, sample preparation variability, and ionization variability when measuring endogenous histamine-related analytes.
Claim(s) 4 and 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Basté in view of Komericki and Nelis, and further in view of applicant admitted prior art.
Regarding claim 4, the combination of Baste, Komericki, and Nelis teaches the method of claim 1, as discussed above.
Baste further teaches that diamine oxidase (DAO) is a secretory protein responsible for scavenging extracellular histamine and is the main enzyme for intestinal histamine metabolism (page 379, par 2). Baste also teaches that current identification of individuals with histamine intolerance is based on plasmatic DAO activity through a biochemical assay that measures the amount of histamine that can be degraded by this enzyme (abstract).
However, Baste, Komericki, and Nelis do not expressly teach that the method further comprises an immunological determination of secreted human diamine oxidase in serum or plasma.
Applicant admitted prior art teaches that immunoassays, including ELISA, for directly measuring the amount of DAO enzyme in human serum, as well as dried blood spots, were available from Immundiagnostik AG, including IDK® DAO ELISA. Applicant admitted prior art further teaches that DAO activity assays in human serum and plasma were known (par [007]).
It would have been obvious to one of ordinary skill in the art to further include an immunological determination of secreted human diamine oxidase in serum or plasma in the modified method of claim 1 because Baste teaches that histamine intolerance is mainly associated with DAO deficiency and that existing identification of histamine-intolerant individuals was based on plasmatic DAO testing, while the admitted prior art teaches a known ELISA immunoassay for directly measuring DAO enzyme in human serum. A person of ordinary skill would have been motivated to include such serum/plasma DAO immunological determination as a complementary diagnostic measurement to the urinary histamine/metabolite determination, in order to provide additional information regarding whether the subject’s histamine intolerance is associated with reduced secreted DAO amount or activity.
Regarding claim 11, Baste teaches the relevance of diamine oxidase (DAO) to histamine intolerance (abstract). In particular, Baste teaches that DAO is a secretory protein responsible for scavenging extracellular histamine and is the main enzyme for metabolism of intestinal histamine (abstract). Baste further teaches that histamine intolerance is associated with reduced intestinal degradation of histamine mainly caused by DAO enzyme deficiency (abstract), and that current identification of individuals with histamine intolerance is based on plasmatic DAO activity through a biochemical assay that measures the amount of histamine that can be degraded by DAO (abstract).
Applicant admitted prior art teaches immunological determination of soluble human diamine oxidase in serum or plasma. Specifically, applicant admitted prior art teaches that immunoassays, including ELISA, for directly measuring the amount of DAO enzyme in human serum were available (par [007]).
Applicant admitted prior art also teaches determination of enzyme activity of DAO in serum or plasma. Specifically, applicant admitted prior art teaches DAO activity assays, including 3H-putrescine/pyrroline REA, and further identifies quantitative determination of diamine oxidase activity in human serum and plasma (par [007]).
It would have been obvious to one of ordinary skill in the art to further include both an immunological determination of soluble human DAO in serum or plasma and a determination of DAO enzyme activity in serum or plasma in the modified diagnostic method of claim 7 because Baste teaches that histamine intolerance is associated with DAO deficiency and that plasmatic DAO activity was already used for identifying individuals with histamine intolerance, while the admitted prior art teaches known immunological DAO assays and known DAO activity assays in human serum/plasma. A person of ordinary skill would have been motivated to include both measurements as complementary diagnostic indicators, because the immunological assay provides the amount/concentration of soluble DAO enzyme while the enzyme activity assay provides functional information regarding whether the DAO present in serum/plasma is active or inhibited.
Claim(s) 9 and 13-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Basté in view of Komericki and Nelis, and further in view of Frost (WO 2019/158718, IDS).
Regarding claim 9, the combination of Baste, Komericki, and Nelis teaches the method of claim 7, as discussed above.
Frost teaches determining total histamine degradation capacity in biological samples. Frost teaches that the biological sample may include whole blood, serum, plasma, cerebrospinal fluid, saliva, tear fluid, urine and/or a homogenate of a biopsy or stool sample, and further teaches that the biological sample is preferably selected from whole blood, serum, or plasma.
Frost further teaches spiking a biological sample with histamine by adding and mixing a first partial sample with a defined amount of histamine provocation solution containing histamine, incubating the histamine-spiked sample under conditions in which histamine can be degraded by mediators in the sample, and determining the histamine remaining in the sample. Frost therefore teaches spiking a serum/plasma sample with histamine for determining histamine degradation capacity.
Frost further teaches that the histamine provocation solution contains histamine in a stabilizing buffer in a concentration between 1 and 300 ng/ml, or between 1 and 100 ng/ml, preferably between 3 and 60 ng/ml, or between 6 and 30 ng/ml, and particularly preferably between 17 and 23 ng/ml (page 6, line 1-3). The claimed concentration of 200 to 500 nmol/litre serum corresponds to approximately 22 to 56 ng/ml histamine, based on histamine having a molecular weight of about 111 Da. This range falls within Frost’s preferred concentration range of 3 to 60 ng/ml.
Frost does not expressly teach that the spiked histamine is isotopically labelled. However, Nelis teaches using deuterium-labelled histamine, histamine-d4, in an LC-MS/MS histamine analysis. As discussed above for claims 1, 5, and 7, it would have been obvious to use stable-isotope-labelled histamine in the modified method so that the spiked/test histamine and its corresponding inactivation products could be distinguished by mass spectrometry from endogenous histamine and endogenous metabolites.
It would have been obvious to one of ordinary skill in the art to further modify the method of claim 7 by spiking a serum sample with isotopically labelled histamine to achieve a concentration of 200 to 500 nmol/litre serum because Frost teaches serum/plasma histamine challenge testing for determining total histamine degradation capacity and teaches histamine provocation concentrations overlapping the claimed range, while Nelis teaches stable-isotope-labelled histamine for LC-MS/MS histamine analysis. A person of ordinary skill would have been motivated to use an overlapping, low histamine challenge concentration in serum to provide a defined amount of test substrate for evaluating histamine degradation capacity, and would have been motivated to use an isotope label to distinguish the added substrate and its metabolites from endogenous histamine and endogenous metabolites.
Regarding claim 13, Frost teaches determining total histamine degradation capacity in a biological sample, including serum or plasma. Frost teaches that, rather than externally provoking the patient, the method performs an in-vitro provocation of the tested liquid biological sample, such as a serum sample (page 3). Frost further teaches adding and mixing a first partial sample with a defined amount of histamine provocation solution containing histamine, incubating the histamine-spiked sample under conditions in which histamine can be degraded by mediators present in the sample, and determining histamine degradation capacity (page 3).
Frost further teaches implementing the histamine degradation-capacity test as a kit (page 8). In particular, Frost teaches a kit for determining total histamine degradation capacity in a liquid biological sample, the kit comprising sample-receiving vessels, a histamine provocation solution containing histamine in a stabilizing buffer, a histamine modification reagent, means for determining modified histamine, and optional standards/reagents (page 8). Therefore, Frost teaches a kit for diagnosis/testing in a serum or plasma sample, comprising a histamine spiking/provocation solution containing a defined amount of histamine.
Frost does not expressly teach that the histamine spiking solution contains stable-isotope-labelled histamine. However, Nelis teaches stable-isotope-labelled histamine in the form of deuterated histamine, histamine-d4, for LC-MS/MS analysis. Histamine-d4 corresponds to a histamine deuterium labelled at one or more hydrogen positions, as recited in claim 13 (page 4).
It would have been obvious to one of ordinary skill in the art to modify Frost’s histamine spiking/provocation solution to contain stable-isotope-labelled histamine, such as histamine-d4, because the modified method of claim 7 uses LC-MS/MS to determine histamine and histamine inactivation products, and stable-isotope labelling would allow the added/spiked histamine and its corresponding conversion products to be distinguished from endogenous histamine and endogenous metabolites already present in the serum or plasma sample. Such a modification would have predictably improved quantitation and attribution of measured histamine conversion to the defined spiked substrate.
Nelis further teaches aqueous working solutions containing histamine metabolites of interest, including methylhistamine, imidazole acetic acid, and methyl imidazole acetic acid (page 3). Nelis also teaches that the endogenous nature of histamine and its metabolites presents quantitation challenges and that stable-isotope-labelled internal standards are useful in multicomponent analyses, although Nelis used standard addition because labeled internal standards for each compound were unavailable (page 2).
It would have been obvious to one of ordinary skill in the art to include solutions containing stable-isotope-labelled standards in the modified Frost/Nelis kit because Nelis concern LC-MS/MS/HILIC analysis of histamine and histamine metabolites. A person of ordinary skill would have been motivated to include the internal standard solutions in kit form to standardize the LC-MS/MS analysis, compensate for matrix effects and sample-preparation variability, and improve reproducibility of histamine/metabolite quantitation in serum or plasma samples.
Regarding claims 14-15, Frost teaches implementing a histamine degradation-capacity diagnostic method using a kit (page 8). Frost teaches that the kit includes, among other components, a histamine provocation solution containing histamine in a stabilizing buffer, reagents for modifying histamine, means for determining modified histamine, optional buffers and standards, and optionally a peroxidase or catalase, preferably in lyophilized form (page 8). Frost further teaches adding peroxidase or catalase, preferably catalase, in an amount of 0.1 to 10 units, preferably 0.2 to 5 units, more preferably 0.5 to 2 units, and particularly 1 unit per 100 µL sample (page 8).
It would have been obvious to one of ordinary skill in the art to further include an enzyme solution of catalase and/or peroxidase in the kit used in the modified method of claim 7 because Frost teaches providing catalase or peroxidase as optional kit components for a histamine degradation-capacity assay. A person of ordinary skill would have been motivated to include these enzyme reagents in the diagnostic kit to standardize the enzymatic/sample-treatment conditions used during the histamine degradation assay and to provide the reagents needed for carrying out the assay in a reproducible manner.
Claim(s) 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Basté in view of Komericki, Nelis, and Frost, and further in view of applicant admitted prior art.
Regarding claim 10, Frost teaches determining total histamine degradation capacity in a biological sample, preferably whole blood, serum, or plasma. Frost teaches adding a defined amount of histamine provocation solution to a biological sample (page 3), incubating the histamine-spiked sample for a defined period under conditions in which histamine can be degraded by mediators in the sample (page 3), determining the remaining histamine (page 3), and calculating the percent total histamine degradation capacity (page 3). Frost further teaches that the histamine-degrading mediators include diamine oxidase (DAO) and/or histamine N-methyltransferase (NMT/HNMT) (page 2, 4).
Frost also teaches incubation conditions suitable for enzymatic histamine degradation. In particular, Frost teaches an incubation period of 1 to 50 hours, preferably 12 to 36 hours, more preferably 20 to 28 hours, and teaches incubation temperatures of 27°C to 42°C, preferably 30°C to 40°C, more preferably 34°C to 38°C (page 5). These conditions overlap physiological conditions and promote or optimize the histamine degradation/conversion activity of mediators in the sample, such as DAO and NMT/HNMT.
Nelis teaches the specific histamine inactivation routes and pathway-specific metabolites (Fig. 1). Specifically, Nelis teaches that histamine is converted by DAO to imidazole acetaldehyde and then to imidazole acetic acid, and that histamine is converted by HNMT to methylhistamine and then to methyl imidazole acetic acid (Fig. 1). Nelis further teaches that measurement of histamine metabolites in body specimens provides a way to calculate histamine turnover and that determining multiple metabolites provides an entire fingerprint of histamine metabolism (page 1).
It would have been obvious to one of ordinary skill in the art to calculate the activity of DAO and HNMT in the sample from the amount of isotopically labeled histamine substrate converted per unit time because Frost teaches incubating a histamine-spiked serum/plasma sample for a defined time and calculating histamine degradation capacity, while Nelis teaches measuring the specific DAO-pathway and HNMT-pathway histamine metabolites. A person of ordinary skill would have recognized that the amount of labeled histamine consumed, or the amount of labeled pathway-specific metabolite formed, over a known incubation time provides a conversion rate, and that such conversion rate corresponds to enzymatic histamine inactivation activity under the applied physiological incubation conditions.
It would further have been obvious to determine histamine inactivation or half-life of histamine in the patient because Frost teaches determining histamine degradation capacity based on the difference in histamine concentration before and after incubation, and a half-life is a routine way of expressing the time-dependent disappearance of a substrate. Applying the known time-dependent conversion data to calculate half-life would have predictably provided another expression of the same histamine degradation/inactivation capacity.
Frost further teaches using the histamine degradation capacity result diagnostically. In particular, Frost teaches that patients with diagnosed histamine intolerance have low or insufficient total histamine degradation capacity, that a degradation range of 0-25% characterizes insufficient or pathologically low histamine degradation capacity, that 25-40% characterizes restricted/borderline total histamine degradation capacity, and that greater than 40% characterizes sufficient histamine degradation capacity and a histamine-intolerance-negative result (page 10). Frost also teaches that a histamine degradation activity of about less than 3 U/mL is not efficiently detectable over the applied time period (page 4).
Applicant admitted prior art teaches that immunoassays, including ELISA, for directly measuring the amount of DAO enzyme in human serum and dried blood spots, as well as DAO activity assays in human serum and plasma, were available. Thus, to the extent claim 10 recites diagnosis based on DAO activity measured by immunoassay, the use of a known DAO immunoassay/ELISA would have been obvious as a complementary measurement in the same diagnostic context (par [007]).
It would have been obvious to one of ordinary skill in the art to diagnose histamine intolerance when the patient’s histamine degradation activity or histamine half-life deviates from a healthy-subject threshold because Baste teaches comparing suspected histamine-intolerant individuals with healthy individuals based on urinary histamine/metabolite profiles, Nelis teaches that histamine metabolites can be measured to calculate histamine turnover, and Frost teaches classifying subjects into healthy, borderline, and histamine-intolerant groups based on histamine degradation capacity measured in the patient sample. A person of ordinary skill would have been motivated to compare the patient’s conversion rate, DAO/HNMT pathway activity, and/or histamine half-life against a healthy-subject threshold in order to provide an objective diagnostic criterion for histamine intolerance.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Frost (WO 2019/158718, IDS) in view of Nelis et al. (Talanta, 2020) (Nelis).
Regarding claim 8, Frost teaches a method of diagnosis of histamine intolerance syndrome in a subject suspected of suffering from insufficient secreted DAO activity and/or deficient HNMT activity (abstract), comprising the steps of:
obtaining a sample of plasma or serum from said subject suspected of suffering from histamine intolerance (page 3);
adding to said sample a predefined amount of histamine to produce a defined concentration of histamine in said sample (page 3),
incubation of said sample for a predefined period under physiological conditions to obtain inactivation of said substrate by the activity of DAO and/or enzyme contained in said sample of plasma or serum (page 3);
Frost further teaches determining the amount of histamine remaining after incubation and calculating the total histamine degradation capacity of the sample (page 3). Thus, Frost teaches obtaining a serum/plasma sample from a subject suspected of histamine intolerance, adding a defined amount of histamine to the sample, incubating the sample under conditions that permit degradation by DAO and/or HNMT, and calculating histamine degradation/inactivation activity.
Frost does not expressly teach that the added histamine is stable-isotope-labelled, that the sample is analyzed by HILIC LC-MS/MS, or that one or more isotopically labelled histamine inactivation products are determined.
Nelis teaches an LC-MS/MS method for separating and quantifying histamine and its main metabolites, including imidazole acetaldehyde, imidazole acetic acid, methyl imidazole acetic acid, methylhistamine, and acetylhistamine (Fig. 3). Nelis teaches that histamine is converted by DAO to imidazole acetaldehyde and then to imidazole acetic acid, and that histamine is converted by HNMT to methylhistamine and then to methyl imidazole acetic acid (Fig. 1). Nelis further teaches that measurement of histamine metabolites in body specimens provides a way to calculate histamine turnover and that measuring multiple histamine metabolites provides a fingerprint of histamine metabolism (page 1).
Nelis teaches using hydrophilic interaction chromatography with tandem mass spectrometry because histamine and its metabolites are highly polar, reverse-phase chromatography is insufficient, and HILIC is well suited for MS detection (page 2). Nelis further teaches sample preparation using acetonitrile, including dilution of urine with acetonitrile and protein precipitation by adding acetonitrile (page 4). Acetonitrile is an aprotic solvent miscible with aqueous samples. Nelis also teaches use of stable-isotope-labelled histamine, specifically histamine-d4, in an LC-MS/MS analysis (page 4).
Nelis also teaches adding standards/internal standards in the analytical method, including histamine-d4 as a deuterated internal standard (page 4). To the extent the claim requires adding an aqueous buffer containing stable-isotope-labelled internal standards, it would have been obvious to provide the isotope-labelled internal standard in an aqueous buffer compatible with the LC-MS/MS method, such as an aqueous ammonium formate/formic acid buffer, because Nelis uses aqueous LC-MS-compatible buffered phases and stable-isotope-labelled histamine for LC-MS/MS quantitation.
It would have been obvious to one of ordinary skill in the art to modify Frost’s in-vitro serum/plasma histamine degradation-capacity method by using stable-isotope-labelled histamine, such as histamine-d4, as taught by Nelis, because endogenous histamine and endogenous histamine metabolites are naturally present in biological samples, and stable-isotope labelling would allow the added/test histamine and its corresponding conversion products to be distinguished by mass spectrometry from endogenous histamine and endogenous metabolites. This would predictably improve the accuracy of the in-vitro degradation assay by attributing measured histamine disappearance and metabolite formation to the defined spiked substrate.
It would further have been obvious to analyze the modified Frost sample using the HILIC LC-MS/MS method and acetonitrile sample preparation taught by Nelis because Nelis provides a sensitive and specific method for separating and quantifying histamine and multiple histamine inactivation products, including imidazole acetic acid, methylhistamine, and methyl imidazole acetic acid. A person of ordinary skill would have been motivated to use HILIC LC-MS/MS in Frost’s assay to obtain more complete information regarding the histamine degradation pathways, rather than only determining remaining histamine.
It would further have been obvious to calculate histamine inactivation activity and/or DAO activity in plasma or serum based on the amounts of labelled histamine and labelled histamine inactivation products because Frost teaches calculating total histamine degradation capacity from the histamine consumed during a defined incubation period, while Nelis teaches measuring the pathway-specific histamine metabolites needed to calculate histamine turnover. A person of ordinary skill would have recognized that the amount of stable-isotope-labelled histamine converted, and the amount of corresponding labelled metabolites formed, over a predefined incubation time provides the histamine inactivation activity of the serum/plasma sample and provides information regarding DAO and/or HNMT pathway activity.
Accordingly, claim 8 is unpatentable over Frost in view of Nelis.
Conclusion
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/XIAOYUN R XU, Ph.D./Primary Examiner, Art Unit 1797
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