Patent Application
20140315217
U.S. Pat. No. 6,811,967 which issued to Sitar et al. on Nov. 4, 2004, and the full disclosure of which is incorporated herein by reference, discloses a method for assaying activity of the enzyme spermidine/spermine N1-acetyltransferase (SSAT) using SSAT substrates by detecting acetylated forms of the SSAT substrates. The SSAT substrates may include amantadine wherein metabolism of amantadine occurs in part by the action of the inducible enzyme SSAT to produce the acetylated metabolite N-acetylamantadine. Disclosed also is the correlation of SSAT activity to pathological conditions.
SSAT is ubiquitously distributed in mammalian tissues and plays a role in catabolism and elimination of polyamines from cells. SSAT is an inducible enzyme that catalyzes the transfer of an acetyl group from an acetyl-coenzyme A to the aminopropyl moiety of the polyamines. This action by SSAT facilitates polyamine degradation, excretion, and cycling and/or intracellular cycling. In this manner SSAT participates in the maintenance of polyamine homeostasis in mammalian cells. However, in normal or uninduced mammalian tissues SSAT is present at very low levels.
Induction of SSAT expression can be caused by different drugs, growth factors, polyamines, polyamine analogues, toxic substances, hormones and physiological stimuli. Although all of the aforementioned compounds could cause induction of SSAT expression, induction occurs at different times for each individual compound. The regulation of SSAT expression occurs at the levels of transcription, mRNA stability, mRNA translation and protein stability. Induction or over-expression of SSAT is usually required for there to be sufficient SSAT enzyme present in cells or 100,000×g supernatant before in-vitro experiments can be successfully undertaken.
While current literature teaches that SSAT is an acetylating enzyme specifically for substrates including spermine and spermidine or its analogues, SSAT activity, SSAT enzyme kinetics and assay methodology for non-spermine/spermidine substrates of SSAT has not been understood. Current methods exist to quantify SSAT activity. However these techniques are dependent on highly skilled personnel and complicated experimental methods. More specifically, there has been a need for assay methodology which quantifies the activity of SSAT through detection of acetylated forms of non-spermine/spermidine substrates of SSAT that may be used to detect various pathological conditions.
There is provided a method for determining the activity of spermine/spermidine N1-acetyltransferase (SSAT) in a mammal comprising the step of assaying a sample derived from the mammal for the level of an acetylated form of a non-spermine/spermidine, or analogues thereof, SSAT substrate in the sample.
In a first embodiment of the method the SSAT substrate is rimantadine and the acetylated form of the SSAT substrate is acetyl-rimantadine. The method may include incubating the SSAT substrate with mammalian tissue or cells at a specific SSAT substrate dosage level in the range of 1-10 mg/kg or, alternatively, at 3-6 mg/kg. Samples to be assayed may be urine, blood and/or saliva samples from the mammal, which may be collected at 2-24 hours following substrate incubation and, alternatively, at 2-4 hours following incubation.
In a second embodiment of the method the SSAT substrate is tocainide and the acetylated form of an SSAT substrate is acetyl-tocainide. The method may include incubating the SSAT substrate with mammalian tissue or cells at a specific SSAT substrate dosage level in the range of 1-10 mg/kg or, alternatively, at 3-6 mg/kg. Samples to be assayed may be urine, blood and/or saliva samples from the mammal which may be collected at 2-24 hours following substrate incubation and, alternatively, at 2-4 hours following incubation.
In a third embodiment of the method, SSAT activity is detected in hepatocytes and the method comprises the steps of:
The drug may be rimantadine present in the range of 0-220 μM. The step of correlating the presence of the acetylated metabolite in the sample comprises correlating the amount of acetylated metabolite to a standard curve to determine the level of SSAT activity in the mammal.
In a fourth embodiment of the method, SSAT activity is assayed in mammal cells. The SSAT substrate is rimantadine and the acetylated form of the SSAT substrate is acetylated-rimantadine. The method comprises the steps of:
The cell culture may be a mammal cell culture and the test sample may be a hepatocyte. The step of contacting the test sample obtained from the cell culture with the drug may include incubating the sample with the substrate for about 24 hours.
In a fifth embodiment of the method, SSAT activity is detected in a mammal. The method comprises the steps of:
The biological fluids may be, but are not limited to, blood, saliva and urine.
In a sixth embodiment of the method, SSAT activity is detected in a mammal. The method comprises the steps of:
The biological fluids may be, but are not limited, to blood, saliva and urine.
In embodiments of the method, the relative level of the non-spermine/spermidine substrate in the sample may be correlated to a standard curve representing known activity levels and may be assayed by a variety of techniques including but not limited to gas chromatography, radio-labelling, high pressure liquid chromatography (HPLC), thin layer chromatography; mass spectroscopy may be coupled with chromatography and affinity chromatography with specific antibody or antibodies.
The assay method disclosed herein may be used to correlate SSAT activity to a pathological condition in the mammal including but not limited to lung cancer, gastric carcinoma, ovarian cancer, acute myelocytic leukemia, lymphoma, breast cancer, renal cancer, colorectal cancer and/or prostate cancer.
The invention will be more readily understood from the following description of the embodiments thereof given, by way of example only, with reference to the accompanying Figures, in which:
A method for assaying spermidine/spermine N1-acetyltransferase (SSAT) activity in vitro and in vivo models is described herein.
SSAT is an important enzyme in polyamine metabolism. SSAT is highly regulated and its role in regulating neoplastic growth, obesity, stress response and oxygen homeostasis has been proposed. SSAT utilizes N1-acetylspermine as a substrate in forming N1,N12-diacetylspermine. In vivo, SSAT is a cytosolic enzyme and N1-acetylspermine is the preferred substrate compared with spermine, although the Km value for spermine is actually lower than that for N1-acetylspermine. In addition to SSAT, arylamine N-acetyltransferases (NATs) are also cytosolic enzymes important in the N-acetylation of drugs and xenobiotics containing aromatic amine and hydrazine groups. In humans, two functional NAT isoforms (NAT1 and NAT2) and over 25 alleles of the two NAT isoforms have been identified.
In vitro and in vivo assays for evaluating SSAT activity based on liver homogenate derived from CD2F1 transgenic mice over-expressing SSAT have been published. Likewise, in vitro assays of NAT1 and NAT2 activities have also been described based on the use of human liver cytosol and human recombinant NAT1 and NAT2 isozymes. In both in vitro SSAT and NAT assays, acetyl-coenzyme A (co-factor) is required to provide activated acetyl group acetylation activity.
Rat and human primary hepatocytes in vitro assays have also been described in studies on SSAT and NAT activities. Since intact primary hepatocytes possess all of the required native drug metabolism co-factors, the use of an intact primary hepatocyte assay often offers a higher level of in vitro versus in vivo correlation of drug metabolism compared with alternative in vitro models based on microsome or subcellular cytosolic enzyme fractions.
The following plateable primary cryopreserved rat hepatocytes were used in this study:
Both lots were used in the pilot experiment. The confirmatory experiment was performed only with lot SKN.
A pilot experiment was initially performed to screen for the suitable testing ranges of substrate concentrations, and to determine the concentrations that would result in significant cytotoxicity (relative viability <50%). Based on these preliminary results, a confirmatory experiment was performed with the adjusted substrate concentrations. Data generated from substrate concentrations that resulted in significant cytotoxicity were excluded from enzyme kinetic analysis. Refer to the table below for the testing concentrations. Metabolites collected from the incubation reactions were measured by LC/MS/MS analysis.
1Not tested in pilot experiment.
In general, the experimental procedures for the pilot and the confirmatory experiments were the same.
The substrates were accurately weighed, dissolved and further diluted with the appropriate solvent (10% dimethyl sulfoxide in distilled water (pilot) or 100% dimethyl sulfoxide (confirmatory) for tocainide; and deionized water for amantadine, rimantadine and spermidine) into a series of solutions at 100× of their testing concentrations outlined in the table above.
Both lots of female rat hepatocytes (Lots ASM and SKN; corresponding to BRIVAL ID: STM-1351 (TSY) and STM-1352 (TSY), respectively) were used in the pilot experiment. The confirmatory experiment was performed with lot SKN only (corresponding to BRIVAL ID: STM-1407 (TSY)). The preparation procedures outlined below were performed for both the pilot and the confirmatory experiments.
Immediately before use, cryopreserved primary rat hepatocytes were thawed in a water bath at 37° C. and re-suspended in pre-warmed InVitroGRO™ CP Rat Medium. The viability of hepatocytes was confirmed to be above 70% based on Trypan Blue exclusion. Hepatocyte concentrations were adjusted by addition with InVitroGRO™ CP Rat Medium to achieve the target plating concentration of 0.70×106 cells/mL. Aliquots of hepatocytes were plated (0.5 mL/well) in 24-well CellAffix culture plates and the plates were placed in an incubator maintained at 37° C. with a highly humidified atmosphere of 95% air and 5% carbon for 4 hours to allow hepatocyte attachment before dosing with the selective substrate solutions.
Following cell attachment, the culture medium was aspirated from each well, and replaced with pre-warmed InVitroGRO™ HI Rat Medium (added with Torpedo antibiotic mix in the confirmatory experiment) and the substrate solution at the appropriate concentration. The treated cells were returned to incubation for 24 hours. Upon completion of incubation, the medium from each well was collected into 1.7-mL vials containing ice-cold methanol and stored at nominal −80° C. (−72° C. to −88° C.) prior to LC/MS/MS analysis. Hepatocytes remaining in the wells were subjected to MTT assay to evaluate the cytotoxic potential of the substrates at the testing concentrations.
Upon collection of the reaction medium, an aliquot of 0.5 mg/mL MTT in KHB was added immediately to the remaining hepatocytes in each well and then incubated for approximately 30 minutes. Following incubation, the medium was replaced with dimethyl sulfoxide (DMSO) to dissolve the formazan. An aliquot from each well was measured for absorbance at 540 nm on a 96-well flat bottom plate with a microplate reader and DMSO for background absorbance correction.
Stability controls were tested to monitor any non-enzymatic N-acetylation of the substrates under the experimental conditions employed. In parallel to the hepatocyte samples, a set of stability controls consisting of only the substrate solutions at the testing concentrations in the InVitroGRO™ HI Rat Medium without hepatocytes was incubated for 24 hours under the same conditions as the hepatocyte samples. Upon completion of incubation, stability control samples were collected for LC/MS/MS analysis following the same procedures for the hepatocyte samples.
Four LC/MS/MS assays were employed to individually quantitate the four metabolites in the incubation samples.
Refer to the table below for the reference standard used for the assay of each metabolite. Reference standard stock solutions were used for the preparation of calibration standards and quality control samples.
Deuterated N-acetyl-d3 amantadine was added as an internal standard for all the assays.
In general, assays of the different metabolites shared the same sample preparation procedures described below:
Calibration Standards and Quality Control Samples:
The reference standard stock solution was diluted and added with aliquots of an assay matrix of methanol to blank incubation reaction buffer (1:1 v/v) and the internal standard to afford a series of calibration standards and quality control samples for LC/MS/MS analysis.
Incubation Samples:
The supernatant of each thawed incubation sample was added with an aliquot of the assay matrix and an aliquot of the internal standard prior to LC/MS/MS analysis.
Exceptions:
Thawed incubation samples for assays of N-acetyl tocainide and N-acetyl spermidine were acidified with formic acid (final formic acid at 0.5% v/v) prior to further preparation as described above. In addition, an acidified assay matrix was used for preparations of calibration standards, quality control samples, and incubation samples. This was to minimize potential metabolite binding to the preparation containers.
Calibration standards, quality control samples and incubation samples for quantitation of N-acetyl spermidine were diluted 10× with 0.1% formic acid in diH2O prior to addition of the internal standard and LC/MS/MS analysis.
N-Acetyl Amantadine, N-Acetyl Rimantadine, and N-Acetyl Tocainide
N-Acetyl Spermidine
MassLynx™ v4.1 and Microsoft Excel 2007 were used for data analysis.
Analytical data were printed on hardcopy and processed according to BRIVAL Standard Operating Procedures. Electronic data backup was performed via BRIPHARM Windows Server 2008 following BRIVAL Standard Operating Procedures.
All experimental raw data, related documentation, and the study report will be archived at BRIVAL's archives (103-8898 Heather Street, Vancouver, BC, Canada) following procedures described in BRIVAL Standard Operating Procedures for a period of at least five years.
A summary of the relative enzyme kinetic parameters (Km and Vmax) of the SSAT-mediated N-acetylation of amantadine, rimantadine, tocainide and spermidine in plateable cryopreserved primary rat hepatocytes is shown in
The Km value of spermidine acetylation estimated from the confirmatory experiment was 287 μM, which is comparable to the literature reference of 267±46 μM derived from SSAT in cytosolic liver fraction of transgenic mice. Vmax cannot be compared to the literature as the values were presented in different units. From all stability controls, only negligible amounts of N-acetyl metabolites were observed, indicating that non-enzymatic N-acetylation was generally absent under the experimental conditions employed.
Results for each of the substrates from the confirmatory experiments are summarized in
The cytotoxic potential of the substrates to rat hepatocytes was evaluated by MTT cytotoxicity assays. The assays were performed in both the pilot and the confirmatory experiments. Results from the pilot experiments are summarized in
MTT assay results from the confirmatory experiment are presented in
Results from all calibration standards and quality control samples met the general batch acceptance criteria as per BRIVAL SOP-GP-011 (v7.0) and SOP-QA-025 (v1.0), established in accordance with FDA Bioanalytical Method Validation Guidelines. See, for example, “Guidance for Industry: Bioanalytical Method Validation” U.S. Department of Health and Human Services, FDA, CDER and CVM, May 2001, the full disclosure of which is incorporated herein by reference. All assays were quantitated with an internal standard approach, except for N-acetyl tocainide which was quantitated without the use of the internal standard. Representative calibration curves and LC/MS/MS chromatograms are presented in
The enzyme kinetic parameters (Km and Vmax) of spermidine/spermine N1-acetyltransferase (SSAT) to mediate N-acetylation of amantadine, rimantadine, tocainide and spermidine were characterized. Among the substrates tested, spermidine acetylation was observed to have the lowest Km value and the highest Vmax value at 287 μM and 7.21 pmol/min/million cells, respectively. Therefore, it has the highest relative maximum reaction rate. The Km values for the other substrates tested, in the ascending order, were 1659 μM for amantadine; 1835 μM for rimantadine; and 5033 μM for tocainide. The Vmax values of the other substrates tested, in the descending order (corresponding to descending order of relative maximum reaction rate), were 0.617 pmol/min/million cells for tocanide, 0.364 pmol/min/million cells for rimantadine, and 0.00197 pmol/min/million cells for amantadine.
It is concluded that determining the activity of spermine/spermidine N1-acetyltransferase (SSAT) in a mammal by assaying a sample derived from the mammal for the level of an acetylated form of a non-spermine/spermidine SSAT substrate in the sample may be used to correlate SSAT activity to a pathological condition in the mammal as shown in
It will be understood by a person skilled in the art that many of the details provided above are by way of example only, and are not intended to limit the scope of the invention which is to be determined with reference to the following claims.
This application claims the benefit of provisional application 61/560,700 filed in the United States Patent and Trademark Office on Nov. 16, 2012, the disclosure of which is incorporated herein by reference and priority to which is claimed.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2012/050828 | 11/16/2012 | WO | 00 | 5/15/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61560700 | Nov 2011 | US |
