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 .
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on July 7, 2025 has been entered.
Claims 1, 3-5, 7-12, 15-24 are pending. Claims 1, 17, 18, 20 and 21 are amended. Claims 23 and 24 are new.
Claim Interpretation for Claim 1
2. Claim 1 recites: A method for determining that a subject suffers or is at risk of suffering from a liver dysfunction, comprising the steps of:
obtaining a serum sample from the subject;
measuring in the serum sample a plurality of ligand binding capacities to serum albumin, wherein the plurality of ligand binding capacities is measured for gold (Au), copper (Cu), cadmium (Cd), L-thyroxine and/or dansylsarcosine;
wherein a predetermined amount of the plurality of ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand, and wherein the ratio for Cu is in the range of 1/5 to 1/10, the ratio for L-thyroxine is in the range of 1/5 to 1/10, the ratio for Au is in the range of 1/50 to 1/100, the ratio for cadmium 1/5 and the ratio for dansylsarcosine is 1/5;
determining that the subject suffers or is at risk of suffering from a liver dysfunction when the plurality of ligand binding capacities is decreased compared to the corresponding ligand binding capacities measured in serum of normal controls; and
administering a suitable therapy to the subject, wherein the therapy is selected from the group consisting of surgery to remove a gallbladder, an operation to treat portal hypertension and an immunosuppressive treatment.
The newly amended phrase of “wherein a predetermined amount of the plurality of ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand… and the ratio for dansylsarcosine is 1/5” is not considered a physical step, but rather, is an extraneous phrase describing how to express a predetermined amount of the plurality of ligands as ratios. There is no requirement for the claimed method to use the predetermined amounts of the plurality of ligands.
The step of determining that the subject suffers or is at risk of suffering from a liver dysfunction only occurs when the plurality of ligand binding capacities is decreased compared to the corresponding ligand binding capacities measured in serum of normal controls, therefore, there is no requirement to identify the subject tested as having a liver dysfunction before administering the claimed suitable therapy. The claims as currently constituted require administering the suitable therapy to every subject tested, regardless of measured ligand binding capacity and regardless of determination for suffering from or being at risk of liver dysfunction.
Claim Interpretation for Claim 17
3. Claim 17 recites: A method for determining whether a subject suffers from a liver dysfunction and treating the subject, comprising
obtaining a serum sample from the subject, wherein the biological sample is selected from the group consisting of blood and serum;
identifying in the serum sample human serum the presence of at least one albumin (HSA) isoform associated with the impaired ligand binding capacity selected form the group consisting of HAS-Cys, HAS-Gly, HAS-NO3, and HAS-Cys/NO3;
measuring in the biological sample form the subject, a plurality of ligand binding capacities to serum albumin, wherein the plurality of ligand binding capacities is measured for gold (Au), copper (Cu), cadmium (Cd), L-thyroxine and/or dansylsarcosine;
wherein the binding capacity is determined by mass spectrometry;
wherein predetermined amounts of the plurality of ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand, and wherein the ratio for Cu is in the range of 1/5 to 1/10, the ratio for L-thyroxine is in the range of 1/5 to 1/10, the ratio for Au is in the range of 1/50 to 1/100, the ratio for cadmium 1/5 and the ratio for dansylsarcosine is 1/5;
wherein a difference between the plurality of ligand binding capacities and a corresponding predetermined reference value indicates that the subject suffers or is at risk of suffering from the liver dysfunction;
treating the subject with a suitable therapy, wherein the therapy is selected from the group consisting of surgery to remove a gallbladder, an operation to treat portal hypertension and an immunosuppressive treatment.
The claim requires the option of obtaining a blood OR serum biological sample from the subject. The newly amended step of “identifying in the serum sample human serum the presence of at least one albumin (HAS) isoform associated with impaired ligand binding capacity” only occurs if the method obtains a serum sample from the subject. This step does not occur if a blood sample is obtained in the method for testing.
The newly amended phrase of “wherein predetermined amounts of the plurality of ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand… and the ratio for dansylsarcosine is 1/5” is not considered a physical step, but rather, is an extraneous phrase describing how to express predetermined amounts of the plurality of ligands as ratios. There is no requirement for the claimed method to use the predetermined amounts of the plurality of ligands.
The claim further recites a phrase describing that a result of any difference (increased or decreased) between the plurality of ligand binding capacities and a corresponding predetermined reference value indicates that the subject suffers from or is at risk of suffering from the liver dysfunction. This phrase does not identify what the corresponding predetermined reference value is. This phrase does not require an active step of comparing the subject’s resulting ligand binding capacities to a corresponding predetermined reference value. There is no requirement to identify the subject tested as having a liver dysfunction or risk of suffering from the liver dysfunction before administering the claimed suitable therapy. The claims as currently constituted require administering the suitable therapy to every subject tested, regardless of the resulting measured ligand binding capacity.
Claim Interpretation for Claim 18
4. Claim 18 recites: A method for identifying and treating a subject who suffers from or is at risk of suffering from a liver dysfunction, comprising the steps of
obtaining a serum sample from the subject;
exposing the serum sample to predetermined amounts of at least three ligand selected from the group consisting of gold (Au), copper (Cu), cadmium (Cd), L-thyroxine and dansylsarcosine;
wherein a predetermined amount of each of the at least two ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand, and wherein the ratio for Cu is in the range of 1/5 to 1/10, the ratio for L-thyroxine is in the range of 1/5 to 1/10, the ratio for Au is in the range of 1/50 to 1/100, the ratio for cadmium 1/5 and the ratio for dansylsarcosine is 1/5;
centrifuging and/or filtering at least one aliquot to separate a ligand-bound albumin fraction from a serum fraction of the serum sample;
measuring levels of free unbound ligand in the serum fraction for each of the at least three ligands;
comparing the levels of free unbound ligands with reference values obtained from measuring corresponding free unbound ligands in serum fraction sample form normal control subjects, wherein the serum samples form the normal control subjects have been subjected to the corresponding obtaining, exposing, and centrifuging and/or filtering steps;
determining that the subject suffers form or is at risk of suffering from a liver dysfunction when the levels of the at least three free unbound ligands are increased compared to the corresponding reference values; and
administering a suitable therapy to the subject, wherein the therapy is selected from the group consisting of surgery to remove a gallbladder, an operation to treat portal hypertension and an immunosuppressive treatment.
The newly amended phrase “wherein a predetermined amount of each of the at least two ligands is expressed as a ratio…” describes how to express a predetermined amount of the plurality of ligands as ratios. There is no requirement for the claimed method to expose the serum sample to the predetermined amounts of ligands, therefore the actual predetermined amounts of ligands exposed to the serum sample are not limited.
The step of determining that the subject suffers from or is at risk of suffering from a liver dysfunction only occurs when the levels of the at least three free unbound ligands are increased compared to the corresponding reference values. There is no requirement to identify the subject tested as having a liver dysfunction or risk of suffering from the liver dysfunction before administering the claimed suitable therapy. The claims as currently constituted require administering the suitable therapy to every subject tested, regardless of the resulting measured free unbound ligands.
Claim Objections
5. Claims 1, 17, and 18 are objected to because of the following informalities: The claims contain a typo where the word “is” is missing in the phrase: “the ratio for cadmium is 1/5.” Appropriate correction is required.
6. Claim 18 is objected to because of the following informalities: The claim contains a grammatical typo and should be corrected as: “wherein predetermined amounts of the plurality of ligands [[is]] are expressed…” Appropriate correction is required.
New Rejections
(necessitated by amendments)
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.
7. Claims 18-22 and 24 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.
Claim 19 recites:
“…exposing the serum sample to predetermined amounts of at least three ligands selected from the group consisting of gold (Au), copper (Cu), cadmium (Cd), L-thyroxine and/or dansylsarcosine; wherein predetermined amounts of each of the at least two ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand…”
There is insufficient antecedent basis for the limitation of “the at least two ligands” in the claim.
Examiner Suggestion: Amend claim 18 to recite “the at least three ligands.”
8. Claim 17 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 17 recites the phrase: “identifying in the serum sample human serum the presence of at least one albumin (HSA) isoform”. The phrase is grammatically unclear as to what “serum sample human serum” is. Clarification is required.
9. Claims 1, 3- 5, 7-12, 15-24 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.
Claim 1 and 17 recite the phrase: wherein a predetermined amount, or predetermined amounts, of the plurality of ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand, and wherein the ratio for Cu is in the range of 1/5 to 1/10, the ratio for L-thyroxine is in the range of 1/5 to 1/10, the ratio for Au is in the range of 1/50 to 1/100, the ratio for cadmium 1/5 and the ratio for dansylsarcosine is 1/5;
Claim 18 recites a similar phrase: wherein a predetermined amount of each of the at least two ligands is expressed as a ratio of 1 molecule of albumin to X atoms or molecules of each ligand, and wherein the ratio for Cu is in the range of 1/5 to 1/10, the ratio for L-thyroxine is in the range of 1/5 to 1/10, the ratio for Au is in the range of 1/50 to 1/100, the ratio for cadmium 1/5 and the ratio for dansylsarcosine is 1/5.
The claims are unclear with regard to what the scope of the predetermined amounts of ligands are. The claims recite the predetermined amounts are “expressed as” various ratios of 1 molecule of albumin to X atoms or molecules of ligand, however, there is no indication in the claims as to what the actual predetermined amount of ligand is, or how many albumins are present in the predetermined amount in order to determine how many ligands are present. One cannot identify the actual predetermined amount of albumin:ligand being claimed or potentially used. The claims recite expressing the predetermined amounts as ratios, but do not recite what source of albumin the ratios are based on, and what the resulting predetermined amounts of ligands are. What amount of albumin and what source of albumin is the ratio based on – the amount of albumin in the subject biological sample or some other source? How much ligand is in the claimed predetermine amount? In claim 18, does the predetermined amount exposed to the biological sample comprise both ligand and albumin present at the claimed ratio expressed? How does the expressed ratio related to the amount of ligand exposed to the sample? The metes and bounds of the claims cannot be determined.
Examiner Suggests: Amend claims 1, 17, and 18 to recite a step of contacting a specific volume of serum or biological sample with a specific concentration of each ligand. For example, as stated in paragraph [0077] below (see bold font).
The specification discloses inventors optimized the concentration of each ligand for assessing albumin binding capacity, so that a specific concertation of ligand was added to a specific volume of sample to achieve an optimal ratio of expected albumin present in the sample to ligand contacted with the sample:
[0067] Study of the Binding Capacities of HAS in Patients with No Hepatic Dysfunction:
[0068] In a first step, we have evaluated separately the global capacity of serum to bind Cu, Au, L-thyroxine, Cd and dansylsarcosine in patients with no liver dysfunctions. Increasing concentrations of each ligand were added to patient serum samples in order to obtain HSA/ligand theoretical ratios (mol/mol) of 1/1, 1/5, 1/10, 1/20, 1/50, 1/100, 1/500 and 1/1000 when possible. These theoretical ratios were calculated on the basis of HSA blood concentration of 0.6 mM, which is an average concentration in healthy subjects.
[0069] Six different serums (from six different patients) per ratio and per ligand were used for this evaluation. After incubation for 30 min of the serum samples spiked with a ligand, they were ultracentrifugated to measure the unbound ligand in the ultrafiltrates. In details,
[0070] Serum (200 μL) was incubated for 30 min at 4° C. with the abovementioned ligands with different solutions concentrations (500 μL of solutions at increasing concentrations of ligands),
[0071] The incubated serum was ultrafiltrated on Amicon® filters with a 30 kDa cut-off,
[0072] The ultrafiltrate (10 μL) was then diluted in HNO3 0.1 M before analysis using a multi-element ICP-MS method for the determination of free (unbound) ligand concentrations. The ICP-MS method measured Cu, Au, Cd, iodine (for L-Thyroxine) and sulfur (for dansylsarcosine) separately or simultaneously, depending on the sample content.
[0073] Percentages of retained ligands quantity as well as the real ratios of HSA/bound ligands (mol/mol) were then calculated, since the actual HSA concentration in each serum was known.
[0074] This allowed us to determine the maximum capacity of the serum to bind each ligand. These ceilings are at the basis of the SEB test to discriminate serum with modified HSA forms from serum with mostly native HSA.
[0075] Comparison of Binding Capacities of HSA in Cirrhotic Patients and Controls (Patients without Hepatic Dysfunction)
[0076] Discovery Cohort
[0077] After setting thresholds corresponding to the retention of more than 90% of each ligand, we performed the SEB test on the serum of patients with diagnosed cirrhosis (n=18) as compared to patients with no liver dysfunctions (n=18). The SEB test was then performed as described above but with the different solutions containing ligands, at specific concentrations proportional to the ligands' binding threshold. Briefly, solutions of Cu, Au, dansylsarcosine and L-thyroxine were prepared at 5950 μM, 23800 μM, 11900 μM and 150 μM, respectively. The solutions were incubated separately with 200 μL of serum for Cu, Au, and dansylsarcosine and with 50 μL of serum for L-thyroxine.
[0078] Albumin isoforms were determined in all serum samples of these two groups as described below.
[0079] In another experiment, we analyzed serum samples from 12 cirrhotic patients and 12 controls in order to study the discrimination power of the test when using solutions of ligands at lower concentrations. For this, Cu, Cd and Au solutions concentrations were set at 1190 μM for Cu, 1190 μM for Cd, 11900 μM for Au and 75 for L-thyroxine 75 μM.
[0090] Results
[0091] Enhanced Binding Capacity of Serum/HSA
[0092] By adding increasing concentrations of Cu to serum, we observed that up to 12 Cu atoms per albumin molecule were retained on the ultracentrifugation filter with an average retention of 95%. This percentage dropped to 40% or less when more Cu was added (FIG. 1A to 1D). Serum samples were able to bind with 100% retention up to 150 atoms of Au, 50 atoms of Cd and 2.5 molecules of dansylsarcosine per molecule of albumin. Serum samples were able to bind at least 10 molecules of L-thyroxine with 100% retention, but L-thyroxine could not be tested above the 1/10 ratio (HSA/L-thyroxine) because of dissolution problems. In order to confirm that the binding is only due to HAS and that there is no unspecific binding to other serum proteins, we performed the same tests with the Vialebex® commercial albumin solution at 200 mg/mL (supplemental data). The binding capacities of commercial solution of pure HSA were equivalent or higher than those of patient serum: for instance, the Cu/HSA retention ratio was 40 and the Au/HSA 150.
[0093] Based on these results we set thresholds best able to discriminate native HSA from modified HSA. Solutions of Au, Cu, dansylsarcosine and L-thyroxine were then prepared to obtain theoretical ratios of 1/100, 1/10, 1/5, and 1/10, respectively. The solutions were then incubated with serums samples from cirrhotic and control patients, as described above.
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.
10. Claim(s) 1, 3, 7, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Klammt 2007 (European J of Gastroenterology & Hepatology, 2007, 19:257-263) in view of WO2018/193087, Klammt et al, published October 25, 2018, claiming priority to April 2017; Klammt 2008 (Liver Transplantation, 2008, 14:1333-1339); Vierling (Clinical Gastroenterology and Hepatology 2015;13:2088–2108); and Mansour et al (Hepatology, April 2018, Vol. 18, Suppl. s60-s65).
Klammt 2007 teaches a method for determining that a subject suffers or is at risk of suffering from liver dysfunction, comprising the steps of:
obtaining a blood or plasma sample from a subject having liver cirrhosis (p. 258, col. 1);
adjusting the samples to the same albumin concentration of 150 µmol/l (p. 258, col. 2);
c) measuring the binding capacities of serum albumin to a plurality of dansylsarcosine (DS) ligands in the adjusted samples by contacting the samples with a predetermined volume/concentration of labeled DS ligands; separating unbound DS by ultrafiltration; separating several aliquots of albumin-free ultrafiltrate; measuring levels of free unbound DS ligand in the aliquots (p. 258, col. 2);
d) conducting the same adjusting and measurement of steps b) and c) in a normal healthy individual’s samples to obtain a level of free unbound DS ligand as a reference value (p. 258, col. 2);
e) comparing the subject’s levels of free unbound DS ligands with the reference value to determine the albumin binding capacity (ABiC) score (p. 259, col. 1-2; Figure 1-2):
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f) determining that a subject having higher levels of unbound free DS (lower ligand binding capacity) compared to the reference value identifies the subject as having liver dysfunction (Figures 1-2; p. 261).
Klammt also determined that levels of free unbound DS ligands increase with increasing severity of liver disease (Figures 1-3; Table 2; p. 259, col. 2; Discussion p. 259-261).
Klammt recognizes liver disease in patients with alcoholic hepatitis, cirrhosis, and patients awaiting liver transplant (p. 261, col. 2). Klammt suggests assaying for ABiC in response to therapeutics such as albumin substitution or extracorporeal liver support therapies (p. 261, col. 2).
Klammt does not teach testing serum samples from the blood samples.
Klammt does not teach expressing a quantity of DS and albumin as a ratio of 1:5 for albumin molecule:DS molecule.
Klammt does not teach further treating the subject having cirrhosis or a subject at risk of developing cirrhosis with suitable therapy such as immunosuppressive treatment, including prednisolone and azathioprine.
WO2018/193087, Klammt teaches a similar method to Klammt above for testing and comparing levels of free unbound ligand in patient samples and comparing to normal reference levels, wherein patients having liver dysfunction have higher levels of free unbound ligands than normal reference levels due to the impaired ability of albumin to bind ligands in patients with liver dysfunction (Examples; [40]). Klammt suggests biological samples or testing include any of blood, serum, and plasma ([29]). Klammt teaches that acute-on-chronic liver failure patients being treated with extracorporeal albumin dialysis (ECAD) had improvement of impaired albumin binding capacity (ABiC) and cites Klammt 2008 (below) for the study ([20]). Klammt teaches determining albumin binding function in liver dysfunction patients to identify the function of their albumin binding capacity so that they receive appropriate therapy (i.e., point of care diagnostics) ([22-24]; [55]).
Klammt 2008, cited by WO2018/193087, Klammt, teaches determining the ABiC of patients before and after treatment with ECAD, wherein ECAD improved ABiC (Table 1; Figures 1-3; Table 2 and 3; p. 1338, col. 2). ABiC was determined as described in Klammt 2007, wherein unbound levels of DS ligands were measured and compared to references to determine increases or decreases in levels, and wherein all samples were diluted to the same albumin concentration of 150 µmol/L and contacted with the same volume and same concentration of fluorescent DS (p. 1334, col. 2 to p. 1335, col. 1). Kalmmt suggests determining ABiC to characterize therapeutic effects of different liver support systems or other therapeutic measures in liver failure (p. 1339, col. 1).
Testing serum samples from the blood samples:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to test serum sample in the method of Klammt 2007. One would have been motivated to, and have a reasonable expectation of success to, because all of the cited references demonstrate the serum albumin tested came from patient blood samples; and WO2018/193087, Klammt suggests any of blood, serum and plasma can be assayed for albumin binding function.
Expressing a quantity of DS and albumin as a ratio of 1:5 for albumin molecule:DS molecule:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed for Klammt 2007 to express a ratio of albumin:DS as a ratio of 1:5. One would have been motivated to, and have a reasonable expectation of success to, because Klammt 2007 and 2008 demonstrate adjusting (diluting) the biological samples to comprise a set amount of albumin in order to add a predetermined volume/concentration of DS ligands to assess ligand binding capacity. Given the demonstrated success for Klammt 2007 and 2008 to standardize the amount of albumin in the samples and to administer a predetermines concentration of DS ligand to the samples in order to determine relevant ligand binding capacity in each sample, and given the demonstration of accurately determining relative amounts of albumin in the samples and concentration of ligand added to the samples, one of ordinary skill in the art would readily be able to determine and express ratios of albumin:DS in the sample in any amount, including 1/5, with a reasonable expectation of success.
It is noted that none of the rejected claims recite or require a limitation of contacting or exposing the biological/serum samples with a specific predetermined amount or concentration of ligand. See claim interpretations above.
Administering immunosuppressive therapy such as prednisolone and azathioprine:
Vierling teaches it is established in the art that patients having chronic liver disease and cirrhosis are successfully treated with immunosuppression therapy including prednisone/prednisolone + azathioprine (Figure 1; p. 2097, col. 1-2; p. 2100-2101).
Mansour teaches success in the treatment and management of liver dysfunction by administering prednisolone (p. S62, col. 2 to p. s63, col. 2).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer immunosuppressive therapy, such as prednisone/prednisolone and azathioprine, to the patients having liver dysfunction, including cirrhosis, in the method of Klammt 2007. One would have been motivated to, and have a reasonable expectation of success to, because: (1) all of the cited references recognize patients having liver dysfunction are in need to treatment, and Vierling and Mansour teach and demonstrate prednisone/prednisolone and azathioprine is an established treatment of patients having liver dysfunction including cirrhosis; (2) WO2018/193087, Klammt suggests assaying albumin binding function using ligands for the purpose of assessing therapeutic need or therapeutic efficacy; and (3) Klammt 2008 successfully demonstrates determining ABiC to characterize therapeutic effects of a treatment in patients having liver dysfunction, wherein ABiC values improved during therapy. Thus, the cited references recognize the need for patients having liver dysfunction to receive treatment, and demonstrate liver dysfunction/ cirrhosis patients are successfully treated with immunosuppressive therapy including prednisone/prednisolone and azathioprine.
11. Claim(s) 1, 3, 5, 7, 9-12, 17, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication 2009/0280519, Jalan et al, published November 2009; in view of Kumar et al (Journal of Clinical and Diagnostic Research, 2016, 10:BC09-BC12); Eom et al (Analytical Sciences, 2014, 30:985-990); and Leblanc et al (Journal of Chromatography B, September 15, 2018, 1095:87-93); Ramond et al (NELM, 1992, 326:507-512); Mansour et al (Hepatology, April 2018, Vol. 18, Suppl. s60-s65); and Lagetta (2016 "Post-Transcriptional Changes in Serum Albumin: Role in the Pathogenesis of Bacterial Infections in Cirrhosis." Dissertation thesis, Alma Mater Studiorum University of Bologna).
Jalan teaches a method for determining whether a subject suffers from liver dysfunction and is in need of treatment, the method comprising:
(a) obtaining a serum sample from the subject ([63]; [65]; Example 2);
(b) contacting the serum sample with a predetermined amount of a plurality of albumin ligand cobalt (Co) to allow albumin to bind to the Co, wherein 200 μL of patient serum is added to 50 μL of a solution of 1 g/L of cobalt chloride (Example 2; [139]);
(c) measuring the amount of unbound/free ligand (cobalt) using a colorimetric assay to provide a score for ischemia modified albumin (IMA);
(d) measuring total albumin in the serum sample;
(e) making a ratio of ischemia modified albumin (IMA) : albumin (ischemia modified albumin ratio (IMAR));
(f) providing a classification algorithm to that identifies subjects having high IMA or high IMAR scores as having liver dysfunction and worse prognosis, as compared to reference sample scores and subjects with better prognosis who have lower scores ([7-9]; [41-57]; [65-66]; [72-87]; [121]; Figures 8-11; Example 2; claims 1-8, 11-14, 16, 19, and 21);
(g) treating the subject identified as having liver dysfunction by administering an agent for the treatment of liver disease ([13-15]; [92-99]; [103]; claim 19);
wherein the subject has alcoholic cirrhosis ([4-5]; [46]; [48]; [57]; [122]; Figure 8; Example 2);
wherein the subject underwent a liver transplant ([45]; [61]).
Jalan does not teach utilizing cupper (Cu) as the ligand for measuring levels of ischemia modified albumin (IMA) (claims 1, 17).
Jalan does not teach the treatment is administering immunosuppressive treatment such as prednisolone/prednisone (claims 1, 17).
Jalan does not teach utilizing mass spectrometry to measure binding capacity (claim 17).
Jalan does not teach further identifying the presence of at least one albumin isoform associated with impaired ligand binding in the serum sample (claims 17, 23).
Jalan does not teach expressing the ligand as a ratio of 1 molecule albumin/X atoms or molecules of ligand, including Cu as 1/5 or 1/10 (claims 1, 17).
Copper as the albumin ligand:
Kumar teaches chronic liver disease (CLD) is characterized by gradual destruction of liver tissue over time. Ischemia Modified Albumin (IMA) is a biomarker elevated in conditions associated with ischemia and oxidative stress (abstract; Introduction). Kumar teaches that CLD patients are often plagued by ischemia and free radical damage, wherein oxidative stress is central to the pathogenesis of CLD, even more than with alcoholic disease. Kumor teaches IMA gives a measure of overall level of ischemic damage and oxidative stress in the body, and teach measuring IMA to quantity this in CLD patients and aid in determining severity of liver diseases (Introduction).
Kumar teaches it is known that metallic ions cobalt or copper bind to the N-terminal end of albumin, however IMA has a conformation change wherein the albumin is modified, so the IMA fails to bind to the metal ions (Introduction).
Kumar measured the IMA and IMA/albumin ratio in patients with CLD and correlated them with liver function and Model for End Stage Liver Disease (MELD). Kumar successfully exemplifies a method comprising:
(a) obtaining serum samples from patients with CLD;
(b) measuring serum IMA using the Albumin Cobalt Binding Test (ABT), wherein the test is based on the principle that a sample containing IMA will bind less cobalt and have more free cobalt ions (p. 10, col. 1); the measuring done by adding a predetermined amount of a plurality of cobalt ligands (50 µL of 1g/L cobalt solution) to a 200 µL patient serum sample; measuring the free, unbound cobalt in the sample (Materials and Methods; Table/Fig-3);
(c) estimating albumin and IMA in both patient and control groups;
(d) applying statistical analysis and comparing levels of IMA and IMA/albumin ratio for patient and control groups (Materials and Methods; Table/Fig-1, Fig-2 and Fig-3));
(e) determining that there is a significant increase in serum IMA levels and IMA/albumin ratio in CLD patients compared to healthy subjects (Discussion; Table/Fig-1).
Kumar concludes IMA serves as a biomarker that can be easily quantified to assess the disease severity and prognosis of CLD patients (Conclusion).
Eom teaches successfully making and using an assay to quantify IMA in serum samples using copper as the albumin ligand, named “albumin copper binding (ACuB) assay” (abstract; Experimental; Figure 5). Eom teaches that IMA is a biomarker of ischemia and oxidative stress (abstract; Introduction). Albumin is known to bind ligands cadmium (Cd2+), cobalt, and copper (p. 986, col. 1, first paragraph). Metal ions of cobalt and copper bind to the same N-terminus region of albumin, whereas IMA has reduced binding affinity to metal ions (p. 986, col. 1, first paragraph). Eom demonstrates testing binding of several metal ions to albumin including cobalt and copper, and demonstrates combining a set volume of serum sample (40 µL) with a predetermined amount of copper solution (Experimental p. 986-987). Eom demonstrates that albumin had a stronger affinity to copper than cobalt, teaching copper demonstrated suitability for IMA assay because of its strong binding capacity to albumin (p. 987, Results and Discussion; Figure 2). Eom concludes that ACuB is a reliable and sensitive method for the detection of IMA levels and ischemic states (abstract; Conclusions).
Leblanc teaches and demonstrates successfully utilizing copper as a ligand to detect isoforms of albumin in human serum. Leblanc also recognizes that copper binds to the N-terminal site of albumin (abstract). Leblanc teaches that identifying isoforms of albumin in serum is of great interest for diagnosis and treatment of patients with diseases of the liver, like for instance cirrhosis (Introduction). Leblanc demonstrates utilizing mass spectrometry to measure isoforms of albumin bound to copper ligand (section 3.3; Table 1; Figure 5). Leblanc teaches they used this method directly on albumin in raw plasma samples (p. 92, col. 1) and suggests using the assay for direct analysis of patient’s raw plasma in studies using individual human samples under pathological conditions versus controls (p. 92, col. 2; Conclusions section 4). Leblanc successfully identified modified forms of HSA including HSA+CYS and HAS+CYS-GLY (Figures 1-5; Table 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to substitute the cobalt ligand of Jalan for a copper ligand. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Kumar and Eom teach cobalt and copper are established albumin ligands binding to the N-terminus of albumin; (2) Eom teaches and demonstrates copper had higher affinity to albumin and was preferential to cobalt for measuring IMA in serum; (3) Eom and Leblanc demonstrated successfully utilizing copper as a ligand for measuring albumin binding in serum; and (4) Leblanc suggests assaying liver dysfunction patients for albumin isoforms, including diagnosing those with cirrhosis, and suggests using copper as an albumin ligand for assessing and comparing patient pathological samples versus normal controls.
Mass spectrometry:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to utilize mass spectrometry in assessing copper ligand binding of albumin. One would have been motivated to, and have a reasonable expectation of success to, because LeBlanc suggests and successfully demonstrates doing so.
Identifying the presence of at least one albumin isoform associated with impaired ligand binding in the serum sample:
Leblanc successfully identified modified forms of HSA including HSA+CYS and HAS+CYS-GLY by mass spectrometry, and recognized HSA is modified at the N-terminal site which prevents binding of HSA to metal ions including copper, as set forth above.
Laggetta recognizes liver cirrhosis is frequently caused by alcoholic liver disease, and can be caused by progression of non-alcoholic steatohepatitis (p. 3 and 9; Table 2). Laggetta teaches it is known that ischemia modified albumin (IMA) is an isoform of albumin with an altered amino terminal site which causes loss of scavenging ability leading metals to circulate and participate into reactions that produce determinantal metabolites for patients (p. 23). Laggetta teaches (p. 23-24):
“Jalan et al 72 have measured plasmatic levels of IMA in patients with different grades of cirrhosis severity. In this study the authors found that the ratio IMA/serum albumin concentration (IMAR) increased with the severity of cirrhosis, a result that mirrored the increase of oxidative stress which characterizes the more advanced phases of the disease. IMAR was also found to be an eligible biomarker to predict mortality and to determine survival in patients with acute decompensation of cirrhosis72.
During the last ten years, new proteomic techniques as High-performance liquid
chromatography (HPLC) coupled to mass spectrometry (ESI-TOF MS) allowed the better identification and characterization of HSA structural alterations73. This method was initially used from Bar-Or and colleagues74 to evaluate post-transcriptional changes of HSA in different commercial preparations of albumin and to compare them to that found in circulating HSA from healthy volunteers. Cysteinylation and nitrosylation of the cysteine 34 residue were described together with a S-nitrosoalbumin isoform74. It’s well known that NO bound to albumin has a prolonged half-life moreover experimental studies conferred to this isoform anti-inflammatory action as in case of lung acute injury due to endotoxemia75 and a protective effect on liver cells in animal models of ischemia/reperfusion injury76. Otherwise, nitrosylation of cys34 causes a decrease in allosterical affinity to copper ions and to other molecules30. An additional modifications described by Bar-or and colleagues was the truncation at the N-terminal portion of HSA74. This change consists in a loss of the last two aminoacidic residue (Asp-Ala) from the N-terminal portion of HSA, the site of the molecule involved in the binding of transition metals ions71. The truncation and further loss of this site means a permanent
loss of HSA scavenging ability.
More recently, our group revisited the method initially described by Bar-Or and
colleagues in order to render this approach reliable to be applied in clinics for the
assessment of circulating albumin microheterogeneity73. With this approach several
HSA isoforms were identified in plasma samples from healthy voluntaries and from a
small series of cirrhotic patients. Namely, isoforms characterized by reversible
(HSA+CYS) or irreversible (HSA+SO2H) oxidation of the Cys-34 residue, truncation of
the N-terminal (HSA-DA) and C-terminal (HSA-L) portion of the molecule,
glycosylation (HSA+GLYC), and the co-occurrence of such structural alterations
(HSA+CYS-DA: N-terminal truncated and reversibly oxidized; HSA+CYS+GLYC:
reversibly oxidized and glycosylated) have been identified73 (Figure 6).”
Laggetta teaches evaluating IMA and IMAR in human plasma samples utilizing the Albumin Cobalt binding test (ACB) (section 4.1, p. 34). Laggetta teaches: “The evaluation of the residual functional capacity of the N-terminal portion of the HSA
molecule was carried out through the measurement of the circulating level of Ischemia
Modified Albumin (IMA). The term Ischemia-Modified Albumin (IMA) identify an
HSA isoform with a reduced ability to chelate cobalt at the N-terminal region which is
is a strong binding site for transition metals, including also copper and nickel” (p. 69).
Laggetta demonstrates successfully identifying and quantifying modified forms of HSA in serum samples from subjects with cirrhosis utilizing mass spectrometry, including HSA+CYS and HSA+GLY, wherein patients hospitalized with cirrhosis has statistically higher percentage of HSA+CYS and HSA+GLY than healthy controls, and increasing levels of modified HAS correlated with worse prognosis (p. 27-48; Tables 4, 5, 9). Laggetta teaches: “The first important finding of the present study is that the molecular structure of HSA is extensively altered in patients with cirrhosis” (p. 65). Laggetta teaches: “In the present study we found a significant increase of both absolute IMA levels and IMA to albumin ratio (IMAr) in patients with cirrhosis with respect to healthy controls” (p. 69).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to further identify in the serum sample of Jalan, the presence of at least one HSA modified isoform associated with impaired ligand binding capacity, including HSA+CYS and HSA+GLY. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Laggetta teaches successfully identifying the presence of HSA modified isoforms HSA+CYS and HSA+GLY in cirrhosis patient samples in addition to testing HSA binding to metal ion, (2) Leblanc demonstrates successfully quantifying HSA modified isoforms in subject samples by mass spectrometry, including HSA+CYS and HSA+ CYS-GLY; (3) Laggetta and Leblanc teach the presence of the HSA modifications are the reason for impaired ability of HSA to bind metal ions, and (4) Laggetta teaches increased percentages of HSA+CYS and HSA+GLY compared to healthy controls corelates with the presence of cirrhosis and worse prognosis for patients as another biomarker of liver dysfunction.
Expressing the ligand as a ratio of 1 molecule albumin/X atoms or molecules of ligand including ratios of 1/5 to 1/10 of Cu:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed for Jalan to express a ratio of albumin:Cu as a ratio of 1:5 or 1:10. One would have been motivated to, and have a reasonable expectation of success to, because Jalan, Kumar, Eom, and Laggetta demonstrate utilizing a set volume of patient biological sample to contact with a predetermined amount/concentration of cobalt or copper ligand to assess ligand binding capacity. Given the demonstrated success for Jalan, Kumar, Eom, and Laggetta to standardize the volume of patients sample comprising albumin and to contact the samples with a predetermined concentration of cobalt or copper ligand to the samples in order to determine relevant ligand binding capacity in each sample, and given the demonstration of accurately determining relative amounts of albumin in the samples and concentration of ligand added to the samples, one of ordinary skill in the art would readily be able to determine and express ratios of albumin:Cu in the sample in any amount, including 1/5 or 1/10, with a reasonable expectation of success.
It is noted that none of the rejected claims recite or require a limitation of contacting or exposing the biological/serum samples with a specific predetermined amount or concentration of ligand. See claim interpretations above.
Treating patients by administering prednisolone:
Mansour teaches success in the treatment and management of liver dysfunction in alcoholic patients by administering prednisolone (p. S62, col. 2 to p. s63, col. 2).
Ramond also teaches treating alcoholic patients having hepatitis and cirrhosis by administering prednisolone, wherein treatment improved short-term survival (abstract).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to treat the alcoholic liver disease patients of Jalan with prednisolone. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Jalan suggests treating their patients; and (2) Ramond and Mansour teach administering prednisolone was beneficial in treatment.
12. Claim(s) 4 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication 2009/0280519, Jalan et al, published November 2009; Kumar et al (Journal of Clinical and Diagnostic Research, 2016, 10:BC09-BC12); Eom et al (Analytical Sciences, 2014, 30:985-990); and Leblanc et al (Journal of Chromatography B, September 15, 2018, 1095:87-93); Ramond et al (NELM, 1992, 326:507-512); Mansour et al (Hepatology, April 2018, Vol. 18, Suppl. s60-s65); and Lagetta (2016 "Post-Transcriptional Changes in Serum Albumin: Role in the Pathogenesis of Bacterial Infections in Cirrhosis." Dissertation thesis, Alma Mater Studiorum University of Bologna); as applied to claims 1, 3, 5, 7, 9-12, 17, and 23 above, and further in view of Ge et al (Gastroenterology Research and Practice, 2016, 2016; article ID 5120760, 7 pages).
Jalan, Kumar, Eom, Leblanc, Ramond, Mansour, and Lagetta (the combined references) teach a method for determining whether a subject is at risk or suffering from a liver dysfunction, such as cirrhosis, utilizing methods of measuring albumin binding capacity to a plurality of copper ligands, as set forth above.
Jalan recognizes non-alcoholic fatty liver disease (NAFLD) as a class of liver dysfunction in need of identification ([3]) and teaches using a liver inflammation scoring system similar to that used for non-alcoholic steatohepatitis (NASH) ([122]).
Laggetta recognizes liver cirrhosis is frequently caused by alcoholic liver disease, and can be caused by progression of non-alcoholic steatohepatitis, as stated above.
The combined references do not teach practicing the method on subjects suffering from non-alcoholic fatty liver disease such as NASH.
Ge teaches that NAFLD is becoming a common chronic disease in both developed and developing countries with increasing incidence, and NASH may also cause cirrhosis and eventually lead to hepatocyte carcinoma. Ge suggests testing albumin binding function to its ligands as a marker for NAFLD (section 1. Introduction; section 4. Discussion first paragraph). Ge demonstrates successfully testing albumin binding capacity to cobalt ligand (sections 1.4.1; 3.1) and fatty acid ligand (section s 3.2; 3.3; Table 3), and correlating a reduced albumin binding affinity to ligand to the presence of NAFLD and cirrhosis (Table 3; section 3.1). Ge concludes reduced albumin binding activity is an early biomarker for liver function during disease development for NAFLD and cirrhosis (p. 5, col. 1-2).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to test albumin binding capacity for ligands of subjects having NAFLD and NASH in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Jalan, Laggetta, and Ge recognize and suggest that NALFD and NASH result in liver dysfunction in need of diagnosis and detection; and (2) Ge demonstrates successfully practicing the method on NALFD patients, providing a correlation between reduced albumin binding capacity to ligands and the presence of liver dysfunction in NAFLD and cirrhosis patients.
13. Claim(s) 16 is rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication 2009/0280519, Jalan et al, published November 2009; Kumar et al (Journal of Clinical and Diagnostic Research, 2016, 10:BC09-BC12); Eom et al (Analytical Sciences, 2014, 30:985-990); and Leblanc et al (Journal of Chromatography B, September 15, 2018, 1095:87-93); Ramond et al (NELM, 1992, 326:507-512); Mansour et al (Hepatology, April 2018, Vol. 18, Suppl. s60-s65); and Lagetta (2016 "Post-Transcriptional Changes in Serum Albumin: Role in the Pathogenesis of Bacterial Infections in Cirrhosis." Dissertation thesis, Alma Mater Studiorum University of Bologna); as applied to claims 1, 3, 5, 7, 9-12, 17, and 23 above, and further in view of Escobosa et al (Anal Bioanal. Chem. 2015, 407:1149-1157); Catalani et al (Hematology, 2011, Article ID 690620, 6 pages); and Ordonez et al (Journal of Analytical Atomic Sepctriometry, 2009, 24:1037-1043).
Jalan, Kumar, Eom, Leblanc, Ramond, Mansour, and Lagetta (the combined references) teach a method for determining whether a subject is at risk or suffering from a liver dysfunction, such as cirrhosis, utilizing methods of measuring albumin binding capacity to a plurality of copper or cobalt ligands and employing mass spectrometry analysis, as set forth above.
The combined references do not teach using ICP-MS methods for measuring copper binding capacity to albumin.
Escobosa demonstrates successfully using ICP-MS to measure various forms of albumin binding to ligand copper (abstract; Figure 1 and 3). Escobosa suggests applying their method to detect copper binding in vivo under pathological conditions (