Compositions and systems for renal function assays

Abstract

The present application provides compositions and systems for renal function assays. The present disclosure relates to systems and methods for determining glomerular filtration rate or assessing renal function in a patient in need thereof. The system includes a computing device, a power source, one or more sensors, and at least one tracer that fluoresces when exposed to electromagnetic radiation. The electromagnetic radiation is detected using a sensor, and the glomerular filtration rate of the patient is calculated using the rate of fluorescence reduction in the patient.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880009623.1, entitled “Compositions and systems for renal function determination”, filed on October 26, 2018. Technical Field

[0002] The present invention relates to compositions and systems for measuring renal function. Background Art

[0003] The field disclosed herein relates generally to methods and pharmaceutical compositions for assessing renal function in a patient in need thereof, the pharmaceutical compositions comprising pyrazine derivatives.

[0004] Acute renal failure (ARF) is a common disease among patients admitted to general surgery hospitals. Approximately half of patients who develop ARF die directly from ARF or from complications related to the underlying medical condition, while survivors experience significantly increased morbidity and prolonged hospital stays. Early diagnosis is generally considered important because renal failure is often asymptomatic and careful tracking of renal function markers in the blood is often required. Dynamic monitoring of a patient's renal function is required to minimize the risk of acute renal failure caused by a variety of clinical, physiological, and pathological conditions. In the case of critically ill or injured patients, such dynamic monitoring is often particularly important because most of these patients are often at risk of multiple organ failure (MOF), which may lead to death. MOF is a sequential failure of the lungs, liver, and kidneys and is triggered by one or more of acute lung injury (ALI), adult respiratory distress syndrome (ARDS), hypermetabolism, hypotension, persistent inflammatory lesions, and sepsis syndrome. Common histological features of hypotension and shock that lead to MOF typically include tissue necrosis, vascular congestion, interstitial and cellular edema, hemorrhage, and microthrombi. These changes typically affect the lungs, liver, kidneys, intestines, adrenal glands, brain, and pancreas in descending order of frequency. The transition from the early stages of trauma to clinical MOF typically corresponds to a certain degree of liver and kidney failure and a change in the risk of death from approximately 30% to approximately 50%.

[0005] Traditionally, crude measurements of urine output and plasma creatinine levels have been used to determine a patient's renal function. These values ​​are often misleading because they are affected by age, hydration status, renal perfusion, muscle mass, dietary intake, and many other clinical and anthropometric variables. In addition, a single value obtained a few hours after sampling may be difficult to correlate with other physiological events, such as blood pressure, cardiac output, hydration status, and other specific clinical events (e.g., bleeding, bacteremia, ventilator settings, etc.).

[0006] Chronic kidney disease (CKD) is a medical condition characterized by a gradual loss of kidney function over time. It involves conditions that damage the kidneys and reduce their ability to properly remove waste products from a person's blood. In addition to an increased risk of heart disease, complications of CKD include high blood pressure, anemia (low blood cell counts), bone weakness, malnutrition, and nerve damage. According to the National Kidney Foundation, approximately two-thirds of all CKD cases are caused by diabetes or high blood pressure. In addition to a family history of kidney disease, other risk factors include age, race, high blood pressure, and diabetes. Glomerular filtration rate (GFR) is the best test to determine the level of kidney function and assess a patient's CKD stage.

[0007] GFR is an important test to determine the level of kidney function, which determines the stage of CKD. Figure 28 As shown, the lower the GFR, the more severe the CKD. GFR can be estimated based on a blood test that measures blood creatinine levels, combined with other factors. A more accurate and therefore more useful method requires injecting an endogenous substance into the patient's body and then carefully monitoring urine output over a period of time. These substances are usually contrast agents (CAs), which can cause kidney problems on their own. Radioactive isotopes or iodinated aromatic rings are two common types of CAs used for GFR determination.

[0008] Table 1

[0009]

[0010] Contrast-induced nephropathy (CIN) is a serious complication associated with the use of radioisotope or iodinated CAs. CIN is thought to be caused by CA-induced renal vasoconstriction or tubular damage. The definition of CIN varies between studies, but the most common definition, based on the symptoms experienced by the patient, includes an increase in serum creatinine of at least 25% above baseline, occurring 2 to 5 days after CA exposure in the absence of other causes of acute renal failure. Due to these complications, CIN can be fatal in up to 20% of hospitalized patients. The more severe the CKD, the greater the risk of CIN. Therefore, the patients who most need accurate GFR data are those who are at highest risk for the sometimes fatal complications of obtaining this data.

[0011] With respect to conventional renal monitoring procedures, an approximation of a patient's glomerular filtration rate (GFR) can be made by a 24-hour urine collection procedure, which (as the name implies) typically requires approximately 24 hours of urine collection, followed by analysis over several hours, and meticulous bedside collection techniques. Unfortunately, the undesirable late timing and significant duration of this conventional procedure can reduce the likelihood of effectively treating the patient and / or saving the kidney. As another disadvantage of this type of procedure, duplicate data is often as difficult to obtain as the originally acquired data.

[0012] Occasionally, changes in a patient's serum creatinine must be adjusted based on measurements, such as the patient's urine electrolytes and osmolality, and derived calculations, such as the "renal failure index" and / or "fractional excretion of sodium." Such adjustments of serum creatinine undesirably tend to require the simultaneous collection of additional serum and / or urine samples and further calculations after some delay. Typically, the dosage of medications adjusted for renal function can therefore be similarly inaccurate, similarly delayed, and difficult to reassess the measurements and calculations on which the medication is based. Ultimately, clinical decisions in critically ill populations are often as important in their timing as in their accuracy.

[0013] It is known that hydrophilic anionic substances are generally excreted by the kidneys. Renal clearance generally occurs via two pathways: glomerular filtration and tubular secretion. Tubular secretion can be characterized as an active transport process; therefore, substances eliminated via this pathway typically exhibit specific properties in terms of size, charge, and lipophilicity.

[0014] Most substances that pass through the kidneys are filtered through the glomeruli (small interwoven groups of capillaries in the kidney's Malpighian corpuscles). Examples of foreign substances that can clear the kidneys by glomerular filtration (hereinafter referred to as "GFR agents") are shown in Figure 1 and includes creatinine (1), sodium o-iodine hippurate (2) and 99m Tc-DTPA (3). Examples of exogenous substances that can be cleared by the kidneys via tubular secretion include 99m Tc-MAG3(4) and others known in the art. 99m Tc-MAG3(4) is also widely used to assess renal function by gamma scintigraphy and by renal blood flow measurement. Figure 1 A disadvantage of the substances shown in , sodium o-iodine hippurate (2), 99m Tc-DTPA(3) and 99m Tc-MAG3(4) includes a radioactive isotope to enable its detection. Even if non-radioactive analogs (e.g., analogs of sodium o-iodine hippurate(2)) or other non-radioactive substances are used for renal function monitoring, such monitoring generally requires the use of undesirable ultraviolet radiation to excite these substances.

[0015] Pyrazine derivatives are known in the art for use in renal function monitoring and include those disclosed in US 8,155,000, US 8,664,392, US 8,697,033, US 8,722,685, US 8,778,309, US 9,005,581, US 9,114,160, US 9,283,288, US 9,376,399 and US 9,480,687, the entire contents of which are incorporated by reference. Summary of the Invention

[0016] In one aspect, disclosed herein are compounds of Formula I, wherein

[0017]

[0018] X 1 and X 2 Independently -CO2R 1 、-CONR 1 R 2 、-CO(AA) or -CONH(PS); Y 1 and Y 2 Each independently selected from -NR 1 R 2 and

[0019]

[0020] Z 1 For single bond, -CR 1 R 2 -、-O-、-NR 1 -、-NCOR 1 -、-S-、-SO- or -SO2-;R 1 to R 2 Each independently selected from H, -CH2(CHOH) a H, -CH2(CHOH) a CH3, -CH2(CHOH) a CO2H, -(CHCO2H) a CO2H, -(CH2CH2O) c H, -(CH2CH2O) c CH3, -(CH2) a SO3H, -(CH2) a SO3 - 、-(CH2) a SO2H, -(CH2) a SO2 - 、-(CH2) a NHSO3H, -(CH2) a NHSO3 - 、-(CH2) a NHSO2H, -(CH2) a NHSO2 - ,-(CH2) a PO4H3, -(CH2) a PO4H2 - 、-(CH2) a PO4H2- 、-(CH2) a PO4 3- 、-(CH2) a PO3H2, -(CH2) a PO3H - and -(CH2) a PO3 2- ; AA is a peptide chain comprising one or more amino acids selected from natural and unnatural amino acids, the amino acids being linked together by peptide or amide bonds, and each instance of AA may be the same as or different from every other instance; PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units linked by glycosidic bonds; and "a" is a number from 1 to 10, "c" is a number from 1 to 100, and each of "m" and "n" is independently a number from 1 to 3.

[0021] In another aspect, disclosed herein is a system for determining the GFR of a patient in need thereof. The system includes a computing device, a display device communicatively coupled to the computing device, a power supply operably coupled to the computing device and electrically isolated from an external power source, one or more sensor heads operably coupled to the computing device, and at least one tracer configured to emit spectral energy when exposed to electromagnetic radiation. The computing device is configured to operate and control the sensor heads, record one or more measurements sent from the sensor heads, and calculate the patient's GFR based on the measurements. The one or more sensor heads include at least one electromagnetic radiation source and are configured to generate and deliver electromagnetic radiation, detect and measure spectral energy emitted by the tracer, and transmit the measurements emitted by the tracer to the computing device. The tracer is configured to be administered to the patient and, when exposed to electromagnetic radiation, emits spectral energy detectable by the sensor heads.

[0022] In yet another aspect, disclosed herein is a system for transdermal determination of a patient's body-size normalized GFR. The system includes a computing device, a display device communicatively coupled to the computing device, a power supply operably coupled to the computing device and electrically isolated from an external power source, one or more sensor heads operably coupled to the computing device, and at least one tracer configured to emit spectral energy when exposed to electromagnetic radiation. The one or more sensor heads include at least one electromagnetic radiation source and are configured to generate and deliver electromagnetic radiation, detect and measure spectral energy emitted by the tracer, and transmit the measured value emitted by the tracer to the computing device. The tracer is configured to be administered to the patient and, when exposed to electromagnetic radiation, emit spectral energy detectable by the sensor head. The computing device is configured to operate and control the sensor head, record one or more measurements sent from the sensor head, determine an attenuation parameter from spectral energy measured during a measurement time window, determine a quality metric associated with the spectral energy measured during the measurement time window, evaluate the attenuation parameter using the quality metric to determine whether it is sufficiently accurate, and if not, increase the measurement time window until the quality metric evaluation indicates sufficient accuracy, and convert the attenuation parameter to a volume corrected for body size or tracer distribution volume (V d ) normalized GFR measurements and report the results on a display device.

[0023] In another aspect, disclosed herein is a method for determining the glomerular filtration rate (GFR) of a patient in need thereof. The method comprises administering a compound of Formula I or a pharmaceutically acceptable salt thereof to the patient, measuring the concentration of the compound of Formula I in the patient during a measurement time window, and determining the GFR in the patient, wherein in the compound of Formula I, X 1 and X 2 Each independently

[0024]

[0025] Formula I

[0026] -CO2R 1 、-CONR 1 R 2 、-CO(AA) or -CONH(PS); Y 1 and Y 2 Each independently selected from -NR 1 R 2 and

[0027]

[0028] Z 1 For single bond, -CR1 R 2 -、-O-、-NR 1 -、-NCOR 1 -、-S-、-SO- or -SO2-;R 1 to R 2 Each independently selected from H, -CH2(CHOH) a H, -CH2(CHOH) a CH3, -CH2(CHOH) a CO2H, -(CHCO2H) a CO2H, -(CH2CH2O) c H, -(CH2CH2O) c CH3, -(CH2) a SO3H, -(CH2) a SO3 - 、-(CH2) a SO2H, -(CH2) a SO2 - 、-(CH2) a NHSO3H, -(CH2) a NHSO3 - 、-(CH2) a NHSO2H, -(CH2) a NHSO2 - ,-(CH2) a PO4H3, -(CH2) a PO4H2 - 、-(CH2) a PO4H 2- 、-(CH2) a PO4 3- 、-(CH2) a PO3H2, -(CH2) a PO3H - and -(CH2) a PO3 2- ; AA is a peptide chain comprising one or more amino acids selected from natural and unnatural amino acids, the amino acids being linked together by peptide or amide bonds, and each instance of AA may be the same as or different from every other instance; PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units linked by glycosidic bonds; and "a" is a number from 1 to 10, "c" is a number from 1 to 100, and each of "m" and "n" is independently a number from 1 to 3.

[0029] In yet another aspect, disclosed herein is a method for assessing renal function in a patient. The method comprises administering a fluorescent compound or a pharmaceutically acceptable salt thereof to the patient; exposing the fluorescent compound to electromagnetic radiation, thereby emitting spectral energy from the fluorescent compound; detecting the spectral energy emitted from the fluorescent compound; and assessing the patient's renal function based on the detected spectral energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Several known contrast agents used for renal function monitoring are shown.

[0031] Figure 2 is a diagram of a system for monitoring GFR in a patient.

[0032] Figure 3A 、 Figure 3B 、 Figure 3C and Figure 3D is a graph of MB-102 clearance showing a two-compartment pharmacokinetic model for four different patients with varying GFR values ​​ranging from 120 mL / min (3A) to 25 mL / min (3D).

[0033] Figure 4 Comparison of MB-102 and Omnipaque Graph of measured GFR.

[0034] Figure 5 is a bar graph showing the percent recovery of MB-102 in human patient urine after 12 hours.

[0035] Figure 6 is a bar graph of the plasma concentration half-life of MB-102 in human patients.

[0036] Figure 7 The GFR is 117 mL / min / 1.73 m 2 Figure 2 shows the correlation between MB-102 plasma concentration and transdermal fluorescence intensity over time in patients with .

[0037] Figure 8 The GFR is 61 mL / min / 1.73 m 2 A graph showing the correlation between MB-102 plasma concentration and transcutaneous fluorescence intensity over time in patients with .

[0038] Figure 9 The GFR is 23 mL / min / 1.73 m 2 A graph showing the correlation between MB-102 plasma concentration and transcutaneous fluorescence intensity over time in patients with .

[0039] Figure 10is a plot relating transcutaneously predicted GFR to plasma-measured GFR using the MB-102 assay and normalized to the subject's body surface area (outlier exclusion method 1; hybrid offset method).

[0040] Figure 11 is a plot relating transdermal predicted GFR to plasma measured GFR using the MB-102 assay and normalized to the volume of distribution of the tracer in the subject (outlier rejection method 1; mixing bias method).

[0041] Figure 12 is a graph relating plasma-measured GFR to transcutaneous fluorescence clearance: GFR by iohexol, unnormalized (no outlier exclusion; fixed offset fitting method).

[0042] Figure 13 Figure 2 is a graph relating plasma-measured GFR to transcutaneous fluorescence clearance: GFR by iohexol, BSA-normalized (no outlier exclusion; fixed-offset fitting method).

[0043] Figure 14 is a graph relating plasma-derived GFR to transcutaneous fluorescent clearance: GFR via iohexol, V d - Normalization (Method 1) (no outlier rejection; fixed-offset fitting method).

[0044] Figure 15 Figure 2 is a graph relating plasma-measured GFR to transcutaneous fluorescence clearance: GFR by MB-102, unnormalized (no outlier exclusion; fixed-offset fitting method).

[0045] Figure 16 Figure 2 is a graph relating plasma-measured GFR to transcutaneous fluorescence clearance: GFR by MB-102, BSA-normalized (no outlier exclusion; fixed-offset fitting method).

[0046] Figure 17 is a graph correlating plasma-derived GFR with transcutaneous fluorescence clearance: GFR by MB-102, V d - Normalization, method 1 (no outlier rejection; fixed-offset fitting method).

[0047] Figure 18 is a graph correlating plasma-derived GFR with transcutaneous fluorescence clearance: GFR by MB-102, V d- Normalization, method 2 (no outlier rejection; fixed-offset fitting method).

[0048] Figure 19 is a graph relating plasma-derived GFR to transcutaneous fluorescence clearance: variable offset method (no outlier exclusion; using V d Normalization method 2 GFR determination by MB-102).

[0049] Figure 20 is a plot relating plasma-derived GFR to transcutaneous fluorescence clearance: mixed bias method (no outlier exclusion; using V d Normalization method 2 GFR determination by MB-102).

[0050] Figure 21 Figure 2 is a graph correlating plasma-measured GFR with transcutaneous fluorescence clearance: Outlier Rejection Method 1 (hybrid bias method; GFR measurements normalized to BSA by MB-102).

[0051] Figure 22 is a plot correlating plasma-measured GFR with transcutaneous fluorescence clearance: Outlier exclusion method 1 (hybrid bias method; using V d Normalization method 2 GFR determination by MB-102).

[0052] Figure 23 Figure 2 is a graph correlating plasma-measured GFR with transcutaneous fluorescence clearance: outlier rejection method 2 (hybrid bias method; GFR measurements normalized to BSA by MB-102).

[0053] Figure 24 is a plot correlating plasma-measured GFR with transcutaneous fluorescence clearance: Outlier exclusion method 2 (hybrid bias method; using V d Normalization method 2 GFR determination by MB-102).

[0054] Figure 25 is a graph summarizing the optimization of the RDTC conversion to determine the offset method for fitting the RDTC.

[0055] Figure 26 is a graph summarizing the optimization of the outlier error threshold for the fluorescence decay rate constant.

[0056] Figure 27 is a graph summarizing the optimization of the outlier error threshold for plasma-measured GFR.

[0057] Figure 28 It is a graphical depiction of GFR for stage 5 chronic kidney disease.

[0058] Figure 29A This is a plot of eGFR versus plasma PK-derived GFR, normalized for subject body surface area (nGFR). Superimposed on the plot is an error grid, which represents the diagnostic accuracy of the number of CKD stages. eGFR correctly diagnoses measurements that fall within the grid with only green edges. eGFR incorrectly diagnoses measurements that fall within grids with both green and yellow edges by one CKD stage. eGFR incorrectly diagnoses measurements that fall within grids with both yellow and red edges by two CKD stages.

[0059] Figure 29B This graph plots transdermally measured GFR (tGFR) versus plasma PK-measured GFR, normalized for subject body surface area (nGFR). Superimposed on the graph is an error grid that indicates the diagnostic accuracy of the number of CKD stages. tGFR correctly diagnoses measurements that fall within the grid with only green edges. tGFR incorrectly diagnoses measurements that fall within the grid with both green and yellow edges by one CKD stage. tGFR incorrectly diagnoses measurements that fall within the grid with both yellow and red edges by two CKD stages.

[0060] Figure 29C This graph plots transdermally measured GFR (tGFR) versus plasma PK-measured GFR, normalized to the volume of distribution of the tracer in the subject (nGFR). Superimposed on the graph is an error grid that represents the diagnostic accuracy of the number of CKD stages. tGFR correctly diagnoses measurements that fall within the grid with only green edges. tGFR incorrectly diagnoses measurements that fall within the grid with both green and yellow edges by one CKD stage. tGFR incorrectly diagnoses measurements that fall within the grid with both yellow and red edges by two CKD stages.

[0061] Figure 30 is a graph of transdermal GFR measured automatically in real time at two body sites. DETAILED DESCRIPTION

[0062] All references herein to "pyrazine," "pyrazine derivative," "pyrazine molecule," "pyrazine compound," or "pyrazine analog" apply to all compounds of Formula I. In addition, unless otherwise indicated, each reference to a pyrazine includes all pharmaceutically acceptable salts thereof. Salt forms may be charged or uncharged and may be protonated to form an appropriate cation or deprotonated to form an appropriate anion. All aspects and embodiments disclosed herein apply to compounds of Formula I, and the specific examples are illustrative only and do not limit the scope of the disclosure.

[0063] In one aspect, disclosed herein is a pyrazine derivative of Formula I or a pharmaceutically acceptable salt thereof,

[0064]

[0065] where X 1 and X 2 Each independently is -CO2R 1 、-CONR 1 R 2 、-CO(AA) or -CONH(PS); Y 1 and Y 2 Each independently selected from -NR 1 R 2 and

[0066]

[0067] Z 1 For single bond, -CR 1 R 2 -、-O-、-NR 1 -、-NCOR 1 -、-S-、-SO- or -SO2-;R 1 to R 2 Each independently selected from H, -CH2(CHOH) a H, -CH2(CHOH) a CH3, -CH2(CHOH) a CO2H, -(CHCO2H) a CO2H, -(CH2CH2O) c H, -(CH2CH2O) c CH3, -(CH2) a SO3H, -(CH2) a SO3 - 、-(CH2) a SO2H, -(CH2) a SO2 - 、-(CH2) a NHSO3H, -(CH2) a NHSO3 - 、-(CH2) a NHSO2H, -(CH2) a NHSO2 - ,-(CH2) a PO4H3, -(CH2) a PO4H2 - 、-(CH2) a PO4H 2- 、-(CH2) a PO4 3- 、-(CH2)a PO3H2, -(CH2) a PO3H - and -(CH2) a PO3 2- AA is a peptide chain comprising one or more amino acids selected from natural and unnatural amino acids, the amino acids linked together by peptide or amide bonds, and each instance of AA may be the same as or different from every other instance; PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units linked by glycosidic bonds; and "a" is a number from 0 to 10, "c" is a number from 1 to 100, and each of "m" and "n" is independently a number from 1 to 3. In another aspect, "a" is a number from 0 to 10. In yet another aspect, "a" is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0068] In some respects, X 1 and X 2 At least one of is -CO(PS) or -CO(AA). In another aspect, X 1 and X 2 They are all -CO(AA).

[0069] (AA) is a peptide chain comprising one or more natural or non-natural amino acids linked together by peptide bonds or amide bonds. A peptide chain (AA) can be a single amino acid, a homopolypeptide chain (homopolypeptide chain) or a heteropolypeptide chain (heteropolypeptide chain), and can be any suitable length. In some embodiments, a natural or non-natural amino acid is an α-amino acid. On the other hand, an α-amino acid is a D-α-amino acid or an L-α-amino acid. In a polypeptide chain comprising two or more amino acids, each amino acid is selected independently of one another in all respects, including but not limited to side chain structure and stereochemistry. For example, in some embodiments, a peptide chain can comprise 1 to 100 amino acids, 1 to 90 amino acids, 1 to 80 amino acids, 1 to 70 amino acids, 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, or even 1 to 10 amino acids. In some embodiments, the peptide chain may include 1 to 100 α-amino acids, 1 to 90 α-amino acids, 1 to 80 α-amino acids, 1 to 70 α-amino acids, 1 to 60 α-amino acids, 1 to 50 α-amino acids, 1 to 40 α-amino acids, 1 to 30 α-amino acids, 1 to 20 α-amino acids, or even 1 to 10 α-amino acids. In some embodiments, the amino acids are selected from D-alanine, D-arginine, D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine, glycine, D-histidine, D-homoserine, D-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-proline, D-serine, D-threonine, D-tryptophan, D-tyrosine, and D-valine. In some embodiments, the α-amino acid of the peptide chain (AA) is selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, homoserine, lysine, and serine. In some embodiments, the α-amino acid of the peptide chain (AA) is selected from aspartic acid, glutamic acid, homoserine, and serine. In some embodiments, the peptide chain (AA) refers to a single amino acid (e.g., D-aspartic acid or D-serine).

[0070] In some embodiments, (AA) is a single amino acid selected from the 21 essential amino acids. In other aspects, AA is selected from D-arginine, D-asparagine, D-aspartic acid, D-glutamic acid, D-glutamine, D-histidine, D-homoserine, D-lysine, and D-serine. Preferably, AA is D-aspartic acid, glycine, D-serine, or D-tyrosine. Most preferably, AA is D-serine.

[0071] In some embodiments, (AA) is a β-amino acid. Examples of β-amino acids include, but are not limited to, β-phenylalanine, β-alanine, 3-amino-3-(3-bromophenyl)propionic acid, 3-aminobutyric acid, cis-2-amino-3-cyclopentene-1-carboxylic acid, trans-2-amino-3-cyclopentene-1-carboxylic acid, 3-aminoisobutyric acid, 3-amino-2-phenylpropionic acid, 3-amino-4-(4-biphenyl)butyric acid, cis-3-amino-cyclohexanecarboxylic acid, trans-3-amino-cyclohexanecarboxylic acid, 3-amino-cyclopentanecarboxylic acid, 3-amino-2-hydroxy-4-phenylbutyric acid, 2-(aminomethyl)phenylacetic acid, 3-amino-2-methylpropionic acid, 3-amino-4-(2-naphthyl)butyric acid, 3-amino-5-phenylpentanoic acid, 3-amino- 2-phenylpropionic acid, 4-bromo-β-Phe-OH, 4-chloro-β-homophe-OH, 4-chloro-β-Phe-OH, 2-cyano-β-homophe-OH, 2-cyano-β-homophe-OH, 4-cyano-β-homophe-OH, 3-cyano-β-Phe-OH, 4-cyano-β-Phe-OH, 3,4-dimethoxy-β-Phe-OH, γ,γ-diphenyl-β-homoala-OH, 4-fluoro-β-Phe-OH, β-Gln-OH, β-homoala-OH, β-homoarg-OH, β-homogln-OH, β-homoglu-OH, β-homohyp-OH, β-homoleu-OH, β-homo lys-OH, β-homomet-OH, β2-homophenylalanine, β-homophe-OH, β3-homopro-OH, β-homoser-OH, β-homothr-OH, β-homotrp-OH, β-homotrp-OMe, β-homotyr-OH, β-Leu-OH, β-Leu-OH, β-Lys(Z)-OH, 3-methoxy-β-Phe-OH, 3-methoxy-β-Phe-OH, 4-methoxy-β-Phe-OH, 4-methyl-β-homophe-OH, 2-methyl-β-Phe-OH, 3-methyl-β-Phe-OH, 4-methyl-β-Phe-OH, β-Phe-OH, 4-(4-pyridyl)-β-homoala-OH, 2-(trifluoromethyl)-β-homophe-OH, 3-(trifluoromethyl)-β-homophe-OH, 4-(trifluoromethyl)-β-homophe-OH, 2-(trifluoromethyl)-β-Phe-OH, 3-(trifluoromethyl)-β-Phe-OH, 4-(trifluoromethyl)-β-Phe-OH, β-Tyr-OH, ethyl 3-(benzylamino)propionate, β-Ala-OH, 3-(amino)-5-hexenoic acid, 3-(amino)-2-methylpropionic acid, 3-(amino)-2-methylpropionic acid, 3-(amino)-4-(2-naphthyl)butanoic acid, 3,4-difluoro-β-homophe-OH, γ,γ-biphenyl-β-high ala-OH, 4-fluoro-β-high phe-OH, β-Gln-OH, β-high ala-OH, β-high arg-OH, β-high gln-OH, β-high glu-OH, β-high hyp-OH, β-high ile-OH, β-high leu-OH, β-high lys-OH, β-high met-OH, β-high phe-OH, β3-homoproline, β-high thr-OH, β-high trp-OH, β-high tyr-OH, β-Leu-OH, 2-methyl-β-high phe- OH, 3-methyl-β-homophe-OH, β-Phe-OH, 4-(3-pyridyl)-β-homoala-OH, 3-(trifluoromethyl)-β-homophe-OH, β-glutamic acid, β-homoalanine, β-homoglutamic acid, β-homoglutamine, β-homohydroxyproline, β-homoisoleucine, β-homoleucine, β-homomethionine, β-homophenylalanine, β-homoproline, β-homoserine, β-homothreonine, β-homotryptophan, β-homotyrosine, β-leucine, β-phenylalanine, pyrrolidine-3-carboxylic acid, and β-Dab-OH.

[0072] (PS) is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units connected by glycosidic bonds. Polysaccharide chain (PS) can be any suitable length. For example, in some embodiments, the polysaccharide chain may comprise 1 to 100 monosaccharide units, 1 to 90 monosaccharide units, 1 to 80 monosaccharide units, 1 to 70 monosaccharide units, 1 to 60 monosaccharide units, 1 to 50 monosaccharide units, 1 to 40 monosaccharide units, 1 to 30 monosaccharide units, 1 to 20 monosaccharide units, or even 1 to 10 monosaccharide units. In some embodiments, the polysaccharide chain (PS) is a homopolysaccharide chain consisting of pentose or hexose monosaccharide units. In other embodiments, the polysaccharide chain (PS) is a heteropolysaccharide chain consisting of one or two of a pentose and hexose monosaccharide unit. In some embodiments, the monosaccharide unit of the polysaccharide chain (PS) is selected from glucose, fructose, mannose, xylose, and ribose. In some embodiments, the polysaccharide chain (PS) refers to a single monosaccharide unit (e.g., glucose or fructose). In another aspect, the polysaccharide chain is an amino sugar, wherein one or more hydroxyl groups on the sugar have been replaced by an amine group. The attachment to the carbonyl group can be through the amine group or the hydroxyl group.

[0073] In some embodiments, for the pyrazine derivative of Formula I, Y 1 or Y 2 At least one of

[0074]

[0075] where Z 1 Is a single bond, -CR 1 R 2-、-O-、-NR 1 -、-NCOR 1 -, -S-, -SO- or -SO2-; and R 1 to R 2 Each independently selected from H, -CH2(CHOH) a H, -CH2(CHOH) a CH3, -CH2(CHOH) a CO2H, -(CHCO2H) a CO2H, -(CH2CH2O) c H, -(CH2CH2O) c CH3, -(CH2) a SO3H, -(CH2) a SO3 - 、-(CH2) a SO2H, -(CH2) a SO2 - 、-(CH2) a NHSO3H, -(CH2) a NHSO3 - 、-(CH2) a NHSO2H, -(CH2) a NHSO2 - ,-(CH2) a PO4H3, -(CH2) a PO4H2 - 、-(CH2) a PO4H 2- 、-(CH2) a PO4 3- 、-(CH2) a PO3H2, -(CH2) a PO3H - and -(CH2) a PO3 2- ; a, c, m and n are as described elsewhere herein.

[0076] In another aspect, Y 1 and Y 2 At least one of them is -NR 1 R 2 , and R 1 to R 2 As described above. In another aspect, Y 1 and Y 2 Both are -NR 1 R 2 , and R 1 to R 2 As above. Alternatively, R1 and R 2 are independently selected from H, -CH2(CHOH) a CH3, -(CH2) a SO3H, -(CH2) a NHSO3H and -(CH2) a PO3H2. On the other hand, R 1 and R 2 It's all hydrogen.

[0077] In any aspect of the pyrazine compound, one or more atoms may be replaced by an isotopically labeled atom of the same element. For example, hydrogen atoms may be labeled with deuterium or tritium isotopes; carbon atoms may be labeled with 13 C or 14 C isotope labeling; nitrogen atoms can be 14 N or 15 N isotope labeling. The isotope labeling can be a stable isotope or can be an unstable isotope (i.e., radioactive). The pyrazine molecule can contain one or more isotope labels. The isotope labeling can be partial or complete. For example, the pyrazine molecule can be labeled with 50% deuterium, thereby giving the molecule a characteristic that can be easily monitored by mass spectrometry or other techniques. As another example, the pyrazine molecule can be labeled with tritium, thereby giving the molecule a radioactive characteristic that can be monitored in vivo and in vitro using techniques known in the art.

[0078] Pharmaceutically acceptable salts are known in the art. In any aspect herein, the pyrazine may be in the form of a pharmaceutically acceptable salt. By way of example and not limitation, pharmaceutically acceptable salts include those described by Berge et al., J. Pharm. Sci., 66(1), 1 (1977), the entire contents of which are incorporated herein by reference for their teachings. The salt may be cationic or anionic. In some embodiments, the counterion of the pharmaceutically acceptable salt is selected from acetate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glucoheptonate, gluconate, glutamate, glycolyllarsanilate, hexylresorcinate, hydrilamine penicillin, hydrobromide, hydrochloride, xinafoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methyl bromide, methylnitrate, methylsulfate, mucate, naphthenate, nitrate, pamoate, pantothenate Salt, phosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theocyanate, triethyl iodide, adipate, alginate, aminosalicylate, dehydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, jemisulfate, hydrofluoride (judroflu) oride), hydroiodide (judroiodide), methylenebis (salicylate), naphthalene disulfonate, oxalate, pectinate, persulfate, phenylethyl barbiturate (phenylethylbarbarbiturate), picrate, propionate, thiocyanate, toluenesulfonate, undecanoate, benzathine penicillin, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, phenethylbenzylamine, clemizole, diethylamine, piperazine, tromethamine, aluminum, calcium, lithium, magnesium, potassium, sodium zinc, barium and bismuth. Any functional group in the pyrazine derivative capable of forming a salt can be optionally formed using methods known in the art. As an example and not limitation, amine hydrochlorides can be formed by adding hydrochloric acid to pyrazine. Phosphates can be formed by adding phosphate buffer to pyrazine.Any acidic functional groups present, such as sulfonic, carboxylic, or phosphonic acids, can be deprotonated with a suitable base and the resulting salt. Alternatively, amine groups can be protonated with a suitable acid to form amine salts. The salt forms can be singly, doubly, or even triply charged, and when more than one counterion is present, each counterion can be the same as or different from each of the others.

[0079] In yet another aspect, disclosed herein is a method for measuring the glomerular filtration rate (GFR) of a patient in need thereof. The method comprises administering a pyrazine compound or a pharmaceutically acceptable salt thereof to the patient, measuring the patient's transdermal fluorescence over a period of time, and determining the patient's GFR. The time period for determining a single GFR measurement is referred to herein as a measurement time window. In many cases, it would be clinically useful to assess GFR in real time over time. Therefore, in some aspects of the present disclosure, multiple sequential assessments of GFR are provided. In some aspects, multiple sequential GFR estimates are provided after a single administration of a tracer. The total length of time for providing GFR measurements after a single injection is referred to herein as a single injection reporting period. In some aspects, there is a time overlap between measurement time windows. In this case, the time interval for reporting GFR (reporting time interval) is not necessarily the same as the measurement time window. For example, in one embodiment, adjacent measurement time windows overlap by 50%, and the reporting time interval is half of the measurement time window. In some aspects, the measurement time window has a variable length. In a preferred embodiment, if temporally adjacent measurement time windows have different lengths, the overlapping period is selected to be 50% of the smaller of the two measurement time windows.In some aspects, a patient's GFR is determined using a system disclosed elsewhere herein.

[0080] On the other hand, according to the metric relevant to signal quality (hereinafter referred to as quality metric) automatic adjustment measurement time window.Quality metric can be based on the estimation of fluorescence signal-to-noise ratio (SNR), signal to background ratio (SBR), goodness of fit (good-of-fit) metric, correlation coefficient or its any combination.In one aspect, line fitting is the logarithm vs. time of the fluorescence intensity in the measurement time window (or equivalently, single exponential is fitted to fluorescence intensity vs. time). The difference (" fitting residual ") between fitted line and data is used to estimate " noise ".In one aspect, noise is the root mean square (RMS) of fitting residual.On the other hand, noise is the absolute median difference (MAD, median absolute deviation) of fitting residual. " signal " can be defined as the amplitude of the single exponential obtained from fitting.On the other hand, signal is selected as the difference between the fitting fluorescence at the beginning and end of the measurement time window.On the other hand, before injection, fluorescence signal level is used to determine " background ", and calculates SBR by dividing background into signal level. When using SNR or SBR as the quality metric, a minimum threshold can be defined, and the fit is considered valid for the purpose of determining GFR only if the quality metric exceeds the threshold. On the other hand, the estimated error of the time or rate constant determined by fitting to fluorescence vs. time is used as the quality metric. In this case, the fit is considered valid only if the calculated quality metric is below a predetermined threshold. In some other aspects, the fitting time or rate constant is defined as the signal, and the estimated error from the fit is defined as the noise, and this form of SNR is used as the quality metric. On the other hand, the correlation coefficient is used as the quality metric. Any of the various methods known in the art for calculating the correlation coefficient can be used, such as the Pearson correlation coefficient or the concordance correlation coefficient. On the other hand, a combination of different quality metrics is combined into a single metric, or the fitting result is considered valid for the purpose of determining GFR only if all selected quality metrics are passed.

[0081] In another aspect, a minimum measurement time window is defined, and the quality metric is used to determine whether to report the GFR, or to extend the length of the measurement time window. In one such embodiment, the length of the measurement time window is automatically increased until the quality metric reaches a threshold, at which point the GFR is reported. In another aspect, a preliminary fit is applied to the time or rate constant or predicted GFR and is used to set the measurement time window to a predetermined length. In one embodiment, the minimum measurement time is set to 60 minutes, at which point the fit is performed and a preliminary estimate of the GFR is made. If the preliminary estimate of the GFR is equal to 75 mL / min / 1.73 m 2 If the initial estimate of GFR is below 75 mL / min / 1.73 m 2, the results are not reported to the user and the measurement time window is increased to 120 minutes.

[0082] On the other hand, estimate remaining single injection reporting period (remaining single injection reporting period) and regularly offer to user.The basis of estimating remaining single injection reporting period can be SNR, SBR or estimation fitting error, for example the above-mentioned method for measuring quality metric, but the quality metric for measuring measurement time window and the quality metric for measuring remaining single injection reporting period can be identical or different. In some aspects, except quality metric, use the fluorescence decay time or rate constant of fitting to estimate remaining single injection reporting period. In one embodiment, the fluorescence decay time constant and SNR of fitting are combined to predict remaining single injection reporting period. SNR is adjusted proportionally (scale) to the scope between minimum and maximum value 0 and 1, and is multiplied with the fluorescence decay time constant. Then, product is adjusted proportionally to predict single injection reporting period. Proportional factor is the calibration factor determined by analyzing the data previously collected on human patients, animals, in vitro studies, simulation or its any combination.

[0083] In another aspect, filtering and / or outlier rejection is applied to the fluorescence data prior to fitting within the measurement time window. Examples of suitable filters include: boxcar average, infinite response function filter, median filter, trimmed mean filter. Examples of outlier rejection methods include all of the above quality metrics, but applied to a subset of the measurement time window.

[0084] In some aspects, a quality metric is calculated based on the tracer's emission energy as a function of time measured over a measurement time window. The quality metric can be used to determine whether to report the calculated GFR. In other aspects, the quality metric is used to determine whether to extend the measurement time window. For example, the measurement time window can be automatically extended until the quality metric exceeds a predetermined threshold, at which point the GFR is reported.

[0085] The GFR normalized to the patient's body size is determined by fitting the measured tracer's emission energy as a function of time to a decay parameter over a measurement time window. In some aspects, the decay parameter is a rate constant (or its reciprocal, referred to as a time constant) from a single exponential fit. In certain aspects, the offset of the fitting function is fixed to zero; in other aspects, the offset is a variable term (variable term) in the fit; in other aspects, whether the offset is fixed or allowed to vary depends on a preliminary assessment of the decay parameter. The measurement time window is selected to begin after the tracer has equilibrated into the body, during the time when the decay in emission intensity is due to renal clearance of the tracer. The fitted rate constant is multiplied by the calibration slope to determine the GFR normalized to the patient's body size. The calibration slope is determined by analyzing data previously collected on human patients, animals, in vitro studies, simulations, or any combination thereof.

[0086] Because the patient's body size can affect the assessment of renal function, in some aspects, body size measurements are used to normalize GFR calculations to further improve the measurement. In some aspects, the body size measurement used to normalize GFR is body surface area (BSA). In other aspects, the body size measurement is the volume of distribution (V) of the tracer. d ).

[0087] The methods and systems disclosed herein also allow for real-time monitoring of a patient's GFR. Furthermore, multiple GFR measurements or determinations can be performed with a single tracer administration. In some aspects, a single GFR measurement is determined after tracer administration. In other aspects, multiple GFR measurements are determined after tracer administration, providing a real-time GFR trend. In some such aspects, an estimate of the remaining time during which the tracer concentration will be sufficient to continue determining GFR is provided.

[0088] In yet another aspect, disclosed herein is a method for measuring the glomerular filtration rate (GFR) of a patient in need thereof. The method comprises administering a pyrazine compound or a pharmaceutically acceptable salt thereof to the patient, measuring the patient's transdermal fluorescence during a measurement time window, and determining the patient's GFR. In some aspects, the patient's GFR is determined using a system disclosed elsewhere herein.

[0089] In another aspect, disclosed herein is a method for determining GFR in a patient in need thereof, comprising administering a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable formulation thereof, to the patient, measuring the concentration of the compound of Formula I in the patient during a measurement time window, and determining the GFR in the patient.

[0090] In some aspects and still with reference to the above methods, measuring the concentration of pyrazine comprises monitoring the patient's transdermal fluorescence. In another aspect, measuring the concentration of pyrazine comprises taking an aliquot of blood from the patient and measuring the concentration of pyrazine by HPLC or other methods known in the art. For example, the pyrazine can contain a quantifiable radioactive isotope. In another aspect, measuring the concentration of pyrazine can comprise collecting the patient's urine over a period of time to determine the rate at which the kidneys eliminate the compound from the patient's body.

[0091] On the other hand and still with reference to the above method, the concentration of pyrazine in the patient is monitored by transdermal fluorescence. This can include contacting a medical device with the patient's skin, wherein the medical device is configured to cause a fluorescent reaction in the compound of Formula I, and detecting the reaction. The medical device can contact the patient's skin at any suitable position. Specific positions known to be suitable are the sternum, lower sternum, pectoralis major, occipital triangle, forehead, chin, upper hip, and lower hip. Other positions available on the patient can be determined based on convenience, medical device design, and / or medical necessity. In some aspects, the method uses a system disclosed elsewhere herein.

[0092] In one aspect of the above method, a display device is used to prompt the user to attach the sensor to one or more specific body parts. In one such embodiment, a touch screen interface is used, and the user is instructed to touch a rendition of the body part location to which the sensor is attached in order to move to the next step in the measurement setup process. This has the benefit of preventing the sensor from being placed on a body part that is inappropriate or suboptimal for GFR determination.

[0093] On the other hand, the next step is to set the light source output level (output level) and the detector gain level (detector gain level). In one such aspect, the detector gain level and the light source level are initially set to a low state, and then the light source level is sequentially increased until the target signal level is reached. In one embodiment, the light source is an excitation source of a fluorescent GFR agent, and the source drive current increases until the target fluorescence signal is reached or a predefined maximum current is reached. When the maximum source current is reached and the required fluorescence signal level (fluorescence signal level) is not obtained, the detector gain is then sequentially increased until the target fluorescence signal is reached, or the maximum detector gain setting is reached.

[0094] In some aspects, in addition to measuring the fluorescence of the skin and the GFR agent, the diffuse reflectance of the skin is also measured. In these aspects, the diffuse reflectance signal can be used to determine the optimal source output and detector gain level. In other aspects, diffuse reflectance measurements are performed within a wavelength band to excite and emit the fluorescent GFR agent. In these aspects, the setting of the LED source level and the detector gain can be performed by using diffuse reflectance rather than the fluorescence signal level to guide the setting. In one such aspect, the target level or diffuse reflectance signal is 15% to 35% of the signal level at which the detector or amplifier saturation effect is observed. This provides headroom for signal fluctuations that may be associated with patient movement or other physiological changes. The procedures for optimizing light source output and / or detection gain have the following benefits: they provide a means of compensating for physiological variations in different patients or different body parts on the same patient. In one aspect, the primary factor for compensation is the melanin content of the skin. Other physiological factors that may need to be compensated for include blood content, water content, and scattering within the tissue volume optically interrogated by the sensor. On the other hand, if the required signal target is not achieved, the user is prevented from taking a measurement. In this way, the reporting of inaccurate results is prevented.

[0095] Once the desired signal level is successfully achieved, a baseline signal is recorded. In one aspect, the stability of the baseline is assessed, for example, by fitting the slope of the signal over time, and the baseline is not considered valid unless the slope over time falls below a predetermined threshold. In some aspects, the display device instructs the user not to continue administering the tracer until a stable baseline is achieved. In this way, if the sensor is not properly positioned or attached, measurements are prevented. Additionally, if the tracer from a previous injection has not been cleared from the body to the desired extent, the user can be prevented from continuing with the measurement.

[0096] Once a stable baseline is obtained, in another aspect of the above method, a tracer is injected into the patient's vascular space. As measured by one or more sensors, tracer administration is automatically detected as a rapid increase in the patient's transdermal fluorescence. For this purpose, a predetermined threshold value for rate of change, absolute signal change, or relative signal change can be adopted. Automatic reagent detection can be reported to the user on a display device such as a touch screen monitor. On the other hand, once the tracer is detected, a further threshold value is used to determine whether there is enough tracer to start GFR measurement. In one such aspect, the measurement of fluorescence (Fmeas) and diffuse reflectance (DR) is combined in a manner that reduces the influence of physiological changes on the combined result (referred to herein as endogenous fluorescence or IF), such that, for example, the influence of skin color on the measurement is compensated. The adequacy of the tracer is then assessed by comparing IF with a predetermined threshold value. In some aspects, IF is determined by using a formula of the following form:

[0097]

[0098] Wherein the subscript DR term refers to measurements collected within the tracer excitation (ex) and emission (em) wavelength bands, with filtered and unfiltered detectors, and the superscript DR term refers to a calibration factor that can be determined by analyzing previously collected data on human patients, animals, in vitro studies, simulations, or any combination thereof. In this manner, if the tracer that has been administered is insufficient for an accurate GFR assessment, the medical professional administering the measurement can be provided with the opportunity to administer additional tracer, or to interrupt the measurement.

[0099] Once the tracer has been administered, in another aspect, the equilibrium of the tracer into the extracellular space is monitored. In one aspect, the measurement time window does not begin until equilibrium has been determined to be sufficiently complete. The progress of equilibrium can be assessed using a fit to an exponential function. For example, the change in fluorescence intensity over time can be fit to a single exponential function, and equilibrium is considered complete only when the fitted time constant stabilizes. In one such aspect, a running estimate of when the first GFR determination will be available is provided to the user. In another aspect, the user is prevented from entering the measurement phase until and unless sufficient equilibrium has been achieved. In one such aspect, the equilibrium time is compared to a predetermined threshold, and if the equilibrium time exceeds the threshold, the user is prevented from continuing with the GFR determination. In this way, if the sensor is located in a position that is poorly communicated with the circulatory system, the assessment of GFR is prevented.

[0100] In some aspects, the reporting time interval, measurement time window and / or single injection reporting period are based on the specific medical assessment being performed and can vary accordingly. For example, for patients suffering from chronic renal failure, a single GFR determination may be sufficient. However, for patients suffering from or at risk of acute renal failure, real-time assessment or GFR trend provides huge potential benefits. In some aspects, the reporting time interval is about 15 minutes. In other aspects, the reporting time interval is about 30 minutes, about one hour, about two hours, about three hours, about five hours, about eight hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours or about 168 hours. In some aspects, the reporting time interval is 15 minutes to 168 hours. In some aspects, the single injection reporting period will be based on the elimination half-life of the pyrazine compound. The elimination half-life can be measured in the patient in advance, estimated based on the patient's medical condition, or measured transdermally using the methods described herein. In some aspects, the single injection reporting period is one elimination half-life, two elimination half-lives, three elimination half-lives, four elimination half-lives, five elimination half-lives, six elimination half-lives, eight elimination half-lives, or ten elimination half-lives. The maximum single injection reporting period makes it possible to no longer detect pyrazine in the patient's bloodstream. As used herein, "undetectable" refers to the concentration of pyrazine that can no longer be detected by the method for measuring. In some cases, when the detection level of the instrument makes this a very long period of time (e.g., more than one week), "undetectable" means that the concentration level has dropped to less than 0.39% (i.e., eight elimination half-lives). On the other hand, the reporting time interval is about 1 to 168 hours, and all one-hour increments therebetween.

[0101] In some aspects, the measurement time window is 15 minutes to 168 hours. In some aspects, the measurement time window can be changed according to the patient's specific medical needs, and can change accordingly. In some aspects, it will be about 15 minutes. In other aspects, the measurement time window is about 30 minutes, about one hour, about two hours, about three hours, about five hours, about eight hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours or about 168 hours. In some aspects, the measurement time window is 15 minutes to 168 hours. During each single injection reporting period, there may be one or more measurement time windows. In some aspects, the single injection reporting period is divided into multiple measurement time windows, and wherein each measurement time window is identical. On the other hand, the single injection reporting period is divided into multiple measurement time windows, and wherein each measurement time window is independent of other measurement time windows selection and can be identical or different with other measurement time windows.

[0102] The methods and systems disclosed herein have the benefit of automatically adjusting for skin melanin content, making GFR determination accurate on a variety of skin types and pigmentation levels. The Fitzpatrick scale is a numerical classification scheme for human skin color. It is widely considered to be a useful tool for dermatological research on human skin pigmentation. Scores range from Type I (very fair skin with minimal pigmentation) to Type VI (dark pigmentation and dark brown). The systems and methods disclosed herein are applicable to all six types of skin pigmentation on the Fitzpatrick scale. Specifically, the systems and methods disclosed herein are applicable to skin pigmentation of Type I, Type II, Type III, Type IV, Type V, and Type VI.

[0103] In another aspect, the pyrazine is combined with at least one pharmaceutically acceptable excipient selected from the group consisting of solvents, pH adjusters, buffers, antioxidants, tonicity adjusters, osmotic regulators, preservatives, antimicrobial agents, stabilizers, viscosity modifiers, surfactants, and combinations thereof.

[0104] Pharmaceutically acceptable solvents can be aqueous or non-aqueous solutions, suspensions, emulsions, or suitable combinations thereof. Non-limiting examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers are water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media.

[0105] By way of example and not limitation, pharmaceutically acceptable buffers include acetate, benzoate, carbonate, citrate, dihydrogen phosphate, gluconate, glutamate, glycinate, dihydrogen phosphate, lactate, phosphate, tartrate, Tris·HCl, or a combination thereof at a pH of 4 to 9, preferably 5 to 8, most preferably 6 to 8, and very most preferably 7.0 to 7.5. In another aspect, the pH is 6.7 to 7.7. Other buffers known in the art may be selected based on the specific salt form of the pyrazine derivative being prepared or the specific medical application. A preferred buffer is phosphate-buffered saline at physiological pH (about 7.2).

[0106] The example of tonicity modifying agent is glycerol, sorbitol, sucrose, or preferably sodium chloride and / or mannitol.The example of viscosity modifier includes bentonite, calcium magnesium silicate etc.The example of diluent includes ethanol, methanol etc.The example of antimicrobial includes benzalkonium chloride, benzethonium chloride, ethyl parahydroxybenzoate, methyl parahydroxybenzoate etc.The example of osmotic regulator includes aminoethanol, calcium chloride, choline, dextrose, diethanolamine, lactated Ringer's solution, meglumine, potassium chloride, Ringer's solution, sodium bicarbonate, sodium chloride, sodium lactate, TRIS or its combination.These examples are only for illustrative purposes, are not intended to be exhaustive or limitative.

[0107] Also disclosed herein is a method for assessing renal function in a patient in need thereof, comprising administering a compound of Formula I or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable formulation thereof to the patient, exposing the patient to electromagnetic radiation so that spectral energy is emitted from the compound of Formula I, detecting the spectral energy emitted from the compound, and assessing the patient's renal function based on the detected spectral energy.

[0108] In some aspects, the compound of formula I is not metabolized by the patient; on the contrary, it is completely eliminated by renal excretion without being metabolized (e.g., without oxidation, glucuronidation or other combination (conjugation)). In some aspects, before renal excretion, at least 95% of the compound of formula I is not metabolized by the patient. In some aspects, before renal excretion, at least 96% of the compound of formula I is not metabolized by the patient. In some aspects, before renal excretion, at least 97% of the compound of formula I is not metabolized by the patient. In some aspects, before renal excretion, at least 98% of the compound of formula I is not metabolized by the patient. In some aspects, before renal excretion, at least 99% of the compound of formula I is not metabolized by the patient. In some embodiments, the compound is completely eliminated by the patient in less than a predetermined time period. In some aspects, assessing the patient's renal function can also include determining the patient's GFR.

[0109] Pyrazine can be administered by any suitable method. The method will be based on the patient's medical needs and selected by the medical professional administering the pyrazine or performing the procedure. Examples of administration methods include, but are not limited to, transdermal, oral, parenteral, subcutaneous, enteral, or intravenous administration. Preferably, the pyrazine compound will be administered using an intravenous or transdermal method. In some embodiments, pyrazine is administered by a single bolus intravenous injection. In another embodiment, pyrazine is administered by multiple bolus intravenous injections. As used herein, transdermal and transdermal both refer to administration through the patient's skin and are used interchangeably.

[0110] As used herein, "enteral administration" refers to any method of administration that uses the gastrointestinal tract to deliver a drug directly or indirectly to a patient. Examples of enteral administration include, but are not limited to, oral, sublingual, buccal, and rectal administration. As used herein, "parenteral administration" refers to any method of administration that delivers a drug directly or indirectly to a patient by injection or infusion. Examples of parenteral administration include, but are not limited to, intravenous, intraarterial, intradermal, transdermal, subcutaneous, and intramuscular administration.

[0111] Also disclosed herein is a stable parenteral composition comprising a pyrazine derivative of Formula I and a pharmaceutically acceptable buffer. The composition has a tonicity suitable for administration to a patient by parenteral administration. The tonicity of the parenteral composition can be adjusted using a tonicity regulator as described elsewhere herein. The composition has a pH suitable for administration to a patient in need thereof, and can be adjusted using a buffer or other pH regulator as described elsewhere herein. The composition has an osmotic pressure suitable for administration to a patient in need thereof, and can be adjusted using an osmotic pressure regulator as described elsewhere herein. The composition is packaged in a sealed container and terminally sterilized to reduce or eliminate the microbial burden of the formulation. The composition is stable to degradation and other adverse chemical reactions and has a pharmaceutically acceptable shelf life.

[0112] As used herein, "stable" means maintained in a state or condition suitable for administration to a patient. The formulations according to the present invention were found to be stable when kept at room temperature for at least 12 months, and typically for 12 to 24 months.

[0113] As used herein, "sterile" compositions refer to compositions that have reached a sterile state and have not been exposed to microbial contamination thereafter, i.e., the container in which the sterile composition is kept is not damaged. Sterile compositions are typically prepared by pharmaceutical manufacturers according to the current good manufacturing practices ("cGMP") of the U.S. Food and Drug Administration. In some aspects, the compositions are packaged in heat-sterilized containers. The container can be any container suitable for use in a medical setting, examples including, but not limited to, vials, ampoules, bags, bottles, and syringes.

[0114] In some embodiments, the composition can be used for parenteral administration in the form of a sterile, ready-to-use formulation. This avoids the inconvenience of diluting the concentrated parenteral formulation into an infusion diluent before infusion or injection, and reduces the risk of microbial contamination during aseptic operation and any potential calculation or dilution errors. Alternatively, the formulation can be a solid formulation that is diluted before administration to the patient.

[0115] The aqueous sterile pharmaceutical composition disclosed herein is suitable for parenteral administration to patients in need thereof. For example, the composition can be administered in the form of a bolus injection or intravenous infusion. Suitable routes of parenteral administration include intravenous, subcutaneous, intradermal, intramuscular, intraarticular, and intrathecal. The ready-to-use formulation disclosed herein is preferably administered by bolus injection. In some embodiments, the composition is suitable for transdermal delivery to the epidermis or dermis of the patient. Transdermal delivery methods and devices are known in the art, and a variety of methods are used to deliver the pharmaceutical composition to the patient.

[0116] As discussed elsewhere herein, aqueous sterile pharmaceutical compositions are formulated in combination with one or more pharmaceutically acceptable excipients. Aqueous sterile pharmaceutical compositions are formulated so that they are suitable for administration to a patient in need thereof. Tonicity, osmotic pressure, viscosity, and other parameters can be adjusted using the reagents and methods described elsewhere herein.

[0117] In another aspect, disclosed herein is an aqueous sterile pharmaceutical composition for parenteral administration. The composition comprises about 0.1 to 50 mg / mL of a pyrazine compound of Formula I. It also comprises about 0.01 to 2 M of a buffer disclosed elsewhere herein. It also comprises about 0-500 mg / mL of an osmotic regulator and about 5-500 mg / mL of a tonicity regulator. The aqueous sterile pharmaceutical composition may optionally comprise one or more additional pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients may be selected from solvents, pH regulators, buffers, antioxidants, tonicity regulators, osmotic pressure regulators, preservatives, antimicrobial agents, stabilizers, viscosity regulators, surfactants, and combinations thereof. Specific examples of excipients are disclosed elsewhere herein.

[0118] The pyrazine compound used in the aqueous sterile pharmaceutical composition is any compound disclosed herein. Specific examples include, but are not limited to, all compounds prepared in the Examples. A preferred example is (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxy-propionic acid), which is the molecule shown in Example 12 (also known as MB-102 or 3,6-diamino-N2,N5-bis(D-serine)-pyrazine-2,5-dicarboxamide).

[0119] The pH of the aqueous sterile pharmaceutical composition is suitable for administration to a patient. In some aspects, the pH is between 4 and 9, preferably between 5 and 8, most preferably between 6 and 8, and very most preferably between 7.0 and 7.5. In another aspect, the pH is between 6.7 and 7.7. Even more preferably, the pH in phosphate buffered saline is about 7.2.

[0120] In some aspects, pyrazine is administered to a patient whose kidney is suspected or known to have at least one medical problem, and methods disclosed herein are used to determine the level of renal injury or defect present in the patient. In some aspects, the patient has an estimated GFR (eGFR) or a previously determined GFR less than 110, less than 90, less than 60, less than 30, or less than 15. Standard medical techniques are used to determine the patient's eGFR, and such methods are known in the art. In some aspects, the patient will not suffer from or is suspected of having a medical problem with their kidney. GFR monitoring can be performed as part of a patient's general or routine health assessment or as a preventive assessment.

[0121] As is known in the art, the rate at which a patient eliminates waste from their bloodstream (i.e., the elimination half-life) depends on the health and normal function of their renal system. "Complete elimination" used in this context means that their pyrazine levels in the bloodstream have dropped to less than 0.39% (i.e., eight half-lives). The elimination half-life will depend on the patient's GFR and is greatly slowed down as the function of the renal system decreases due to disease, age or other physiological factors. In the absence of the known risk factors associated with CKD, in patients with normal GFR and / or normal eGFR, the single injection reporting period is 24 hours. In some aspects, the single injection reporting period for patients with GFR or eGFR lower than 110 is 24 hours. For patients with GFR or eGFR lower than 90, the single injection reporting period is 24 hours. For patients with GFR or eGFR lower than 60, the single injection reporting period is 48 hours. For patients with GFR or eGFR lower than 30, the single injection reporting period is 48 hours. In some aspects, the reporting period for a single infusion is equal to 8 elimination half-lives for patients with a GFR or eGFR less than 110. For patients with a GFR or eGFR less than 90, the reporting period for a single infusion is equal to 8 elimination half-lives. For patients with a GFR or eGFR less than 60, the reporting period for a single infusion is equal to 8 elimination half-lives. For patients with a GFR or eGFR less than 30, the reporting period for a single infusion is equal to 8 elimination half-lives.

[0122] Since an increase in protein concentration in a patient's urine may indicate some form of renal damage or defect, the methods disclosed herein are applicable to patients whose urinalysis shows an increase in protein levels. In some aspects, the increase in protein levels in the patient's urine is determined by standard medical tests (e.g., test strip tests). As an example and not limitation, a patient's urinalysis may show an increase in albumin, an increase in creatinine, an increase in blood urea nitrogen (i.e., a BUN test), or any combination thereof.

[0123] Still referring to the above method, the pyrazine derivative is exposed to electromagnetic radiation, such as, but not limited to, visible light, ultraviolet light and / or infrared light. The exposure of the pyrazine to electromagnetic radiation can occur at any appropriate time, but preferably occurs when the pyrazine derivative is located in the patient's body. Due to the exposure of the pyrazine to electromagnetic radiation, the pyrazine emits spectral energy (e.g., visible light, ultraviolet light and / or infrared light) that can be detected by appropriate detection equipment. The spectral energy emitted from the pyrazine derivative tends to exhibit a wavelength range that is greater than the wavelength range absorbed. As an example but not limitation, if an embodiment of the pyrazine derivative absorbs light of about 440 nm, the pyrazine derivative can emit light of about 560 nm.

[0124] Detection of pyrazine (or more specifically, the spectral energy emitted therefrom) can be achieved by optical fluorescence, absorbance, or light scattering techniques. In some aspects, the spectral energy is fluorescence. In some embodiments, the detection of the emitted spectral energy can be characterized as the collection of the emitted spectral energy and the generation of an electrical signal indicative of the collected spectral energy. The mechanism for detecting the spectral energy of pyrazine derivatives present in the patient's body can be designed to detect only selected wavelengths (or wavelength ranges) and / or can include one or more appropriate spectral filters. Various catheters, endoscopes, ear clips, handbands, headbands, surface coils, finger probes, and other medical devices can be used to expose pyrazine derivatives to electromagnetic radiation and / or detect the spectral energy emitted therefrom. The devices for exposing pyrazine to electromagnetic radiation and detecting the spectral energy emitted therefrom can be the same or different. That is, one or two devices can be used. The detection of spectral energy can be done intermittently once or multiple times, or can be substantially continuous.

[0125] The patient's renal function or GFR is determined based on the detected spectral energy. This is achieved by using data indicating the detected spectral energy and generating an intensity / time curve (spectrum, profile) indicating that the pyrazine derivative is cleared from the patient's body. The curve can be related to a physiological or pathological condition. For example, the patient's clearance curve and / or clearance rate can be compared with a known clearance curve and / or clearance rate to assess the patient's renal function and diagnose the patient's physiological condition. In the case of analyzing the presence of pyrazine derivatives in body fluids, a concentration / time curve can be generated and analyzed (preferably in real time) to assess renal function. Alternatively, the patient's clearance curve can be compared with one or more previously measured clearance curves from the same patient to determine whether the patient's renal function changes over time. In some aspects, renal function assessment is performed using a system disclosed elsewhere herein.

[0126] Physiological function can be assessed by: (1) comparing the differences in the manner in which normal and damaged cells or organs eliminate pyrazine derivatives from the bloodstream; (2) measuring the rate of elimination or accumulation of pyrazines in an organ or tissue of a patient; and / or (3) obtaining a cross-sectional image of the organ or tissue having pyrazines associated therewith. For example, blood pool clearance can be measured non-invasively from surface capillaries such as in the earlobe or finger, or can be measured invasively using an appropriate instrument such as an intravascular catheter. Transcutaneous fluorescence can also be monitored non-invasively on the patient's body. Many locations on the patient's epidermis may be suitable for non-invasive monitoring of transcutaneous fluorescence. The site on the patient is preferably a site where vasculature and tissue equilibrium occur relatively quickly. Examples of suitable sites on the patient include, but are not limited to, the sternum, substernal region, pectoralis major, occipital triangle, forehead, chin, upper hip, and lower hip. The accumulation of pyrazine derivatives in target cells can be assessed in a similar manner.

[0127] Improved pulmonary artery catheters can also be used to make the desired measurements, particularly of the spectral energy emitted from pyrazine derivatives. The ability of a pulmonary catheter to detect the spectral energy emitted from the pyrazines is a significant improvement over current pulmonary artery catheters, which only measure intravascular pressure, cardiac output, and other derived blood flow measurements. Traditionally, critically ill patients have been managed using only the parameters listed above, and their treatment has tended to rely on intermittent blood sampling and tests to assess renal function. These traditional parameters provide discontinuous data and are often misleading in many patient populations.

[0128] The improvement of the standard pulmonary artery catheter only requires that its fiber optic sensor be wavelength specific. There are currently catheters that combine fiber optic technology to measure mixed venous oxygen saturation. In one feature, the improved pulmonary artery catheter incorporates a wavelength-specific optical sensor into the tip of the standard pulmonary artery catheter. This wavelength-specific optical sensor is used to monitor the renal function-specific elimination of a designed optically detectable chemical entity (e.g., a pyrazine derivative disclosed herein). Therefore, real-time renal function can be monitored by the disappearance / clearance of the optically detected compound.

[0129] In some aspects, the pyrazine compound is administered to a patient, wherein the patient has previously been diagnosed with at least 1 phase of CKD. In other aspects, the patient has previously been diagnosed with 2 phases of CKD, 3 phases of CKD, 4 phases of CKD, or 5 phases of CKD. In another aspect, the patient has not yet been diagnosed with CKD but has one or more risk factors associated with CKD. In another aspect, the patient has no known risk factors for CKD.

[0130] Based on the medical tests performed and the medical needs of the patient, the pyrazine compound is administered by any suitable method. Suitable methods are disclosed elsewhere herein. Preferably, the pyrazine is administered by transdermal or intravenous administration.

[0131] Also disclosed herein is a system for determining GFR or assessing renal function in a patient in need thereof. The system includes a computing device, a display device communicatively coupled to the computing device, a power supply operably coupled to the computing device and electrically isolated from an external power source, one or more sensor heads operably coupled to the computing device, and at least one tracer configured to emit light when exposed to electromagnetic radiation. The computing device is configured to operate and control the sensor heads, record one or more light measurements sent from the sensor heads, and calculate the patient's GFR based on the light measurements.

[0132] It should be noted that as used herein, the term "coupled" is not limited to direct mechanical, electrical, and / or communication connections between components, but may also include indirect mechanical, electrical, and / or communication connections between multiple components. The display device and the one or more sensor heads may communicate with the computing device using: a wired network connection (e.g., Ethernet or fiber optic), wireless communication means, such as radio frequency (RF), such as FM radio and / or digital audio broadcasting, Institute of Electrical and Electronics Engineers (IEEE) wireless communication technology. 802.11 standards (e.g., 802.11(g) or 802.11(n)), Worldwide Interoperability for Microwave Access Standard, short-range wireless communication channels such as Cellular telephone technology (e.g., Global Standard for Mobile Communications (GSM)), satellite communication links, and / or any other suitable communication means. IEEE is a registered trademark of the Institute of Electrical and Electronics Engineers, New York, NY. WIMAX is a registered trademark of the WiMax Forum, Beaverton, Oregon. BLUETOOTH is a registered trademark of Bluetooth SIG, Inc., Kirkland, WA.

[0133] In some aspects, one or more sensor heads include at least one electromagnetic radiation source, generate and deliver electromagnetic radiation to the patient's skin, detect and measure the electromagnetic radiation emitted by the tracer, and transmit the measured value of the electromagnetic radiation emitted by the tracer to the computing device. In a system with more than one sensor head, each sensor head can be the same or different, and the electromagnetic radiation emitted therefrom can be the same or different. In some aspects, the sensor head is configured to be attached to the patient's skin. As an example and not limitation, in a system with two sensor heads, one sensor head can emit and monitor electromagnetic radiation of one wavelength, while the second sensor head can emit and monitor a different wavelength. This enables data to be compared to increase the accuracy of the information available to medical professionals who measure and manage GFR assessments. In another non-limiting example, in a system with two sensor heads, the two sensor heads are used to separate local equilibrium kinetics from terminal phase kinetics. This enables medical professionals to determine when equilibrium is complete and reduces artifacts caused by local movement of the sensors.

[0134] In some aspects, the tracer is configured to be administered to the patient by intravenous or transdermal administration, to be eliminated solely by glomerular filtration in the patient's kidneys, and to emit light detectable by the sensor head when exposed to electromagnetic radiation. In some aspects, the tracer is a pyrazine compound of Formula I as disclosed elsewhere herein. Preferably, the tracer is a compound prepared in one embodiment. Most preferably, the tracer is (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropionic acid) (also known as MB-102 or 3,6-diamino-N2,N5-bis(D-serine)-pyrazine-2,5-dicarboxamide). In some aspects, the tracer is a pyrazine derivative in a formulation suitable for administration to a patient in need thereof. Such formulations are described elsewhere herein.

[0135] A system for determining a patient's GFR or assessing renal function can be configured to perform the methods disclosed herein on a patient in need thereof. The computing device in the system can be any standard computer with all the capabilities implied by it, specifically including but not limited to permanent memory, a processor capable of performing complex mathematical calculations, a user interface for interacting with the computer, and a display device communicatively coupled to the computing device. Thus, the permanent memory of the computing device can store any information, programs, and data necessary to perform the functions of the system for determining GFR or assessing a patient's renal function. Such information, programs, and data can be standards and / or controls that can be used to compare transdermal fluorescence values ​​collected by one or more sensor heads with known values. In some aspects, the computing device can save the results of previous assessments or GFR measurements in a patient so that results obtained at a later date can be compared. This allows medical professionals to monitor the health of a patient's kidneys over time. In some aspects, the computing device is a laptop computer.

[0136] In various aspects, the computing device includes a processor and / or a memory device. In various other aspects, the processor is coupled to one or more of a user interface, a display device, and a memory device via a system bus. In one aspect, the processor communicates with the user, for example, by prompting the user via a display device and / or receiving user input via a user interface. The term "processor" generally refers to any programmable system, including system and microcontrollers, reduced instruction set circuits (RISCs), application specific integrated circuits (ASICs), programmable logic circuits (PLCs), and any other circuit or processor capable of performing the functions described herein. The above examples are exemplary only and are not intended to limit the definition and / or meaning of the term "processor" in any way.

[0137] In various aspects, a memory device includes one or more devices that enable information, such as executable instructions and / or other data, to be stored and retrieved. Furthermore, a memory device includes one or more computer-readable media, such as, but not limited to, dynamic random access memory (DRAM), static random access memory (SRAM), a solid-state drive, and / or a hard disk. In various aspects, a memory device stores, but is not limited to, application source code, application object code, configuration data, additional input events, application state, assertion statements, verification results, and / or any other type of data.

[0138] The user interface is configured to receive at least one input from a user (such as an operator of a system for determining GFR or assessing a patient's renal function). In one aspect, the user interface includes a keyboard that enables the user to input relevant information. In various other aspects, the user interface includes, but is not limited to, a pointing device, a mouse, a stylus, a touch-sensitive panel (e.g., a touchpad, a touch screen), a gyroscope, an accelerometer, a position detector, and / or an audio input interface (e.g., including a microphone).

[0139] The display device is configured to display information such as input events and / or verification results to the user. The display device may also include a display adapter coupled to at least one display device. In one aspect, the display device can be a visual display device, such as a cathode ray tube (CRT), a liquid crystal display (LCD), an organic LED (OLED) display, and / or an "electronic ink" display. In various other aspects, the display device includes an audio output device (e.g., an audio adapter and / or a speaker) and / or a printer. In some aspects, the display device includes a touch screen.

[0140] In various aspects, computing devices generally include a processor. The processor can be programmed by encoding the operation using one or more executable instructions and providing executable instructions in a memory device. In one aspect, the processor is programmed to calculate the time constant of renal function decline within a predetermined time period. In one aspect, transdermal fluorescence data in the patient's body is collected within a predetermined time period, and a graph of time (x-axis) to fluorescence (y-axis) is made. The decay rate can be curved or linear, and the time constant of the decay rate is calculated. In one aspect, for a semi-logarithmic (y) graph, the decay rate is linear. The time constant is compared with a known value, thereby measuring the patient's GFR. In some aspects, the decay rate corresponds to standard first-order kinetics. On the other hand, the decay rate can show a multi-compartment pharmacokinetic model. Figures 3A to 3D Two-compartment pharmacokinetics are described, from which standard pharmacokinetic software can determine the time constant for the decline in renal function.

[0141] GFR determinations were performed using linear regression, outlier exclusion, calculation of correlation coefficients (R2), and calibrated standard errors and are described more fully in the Examples.

[0142] This written description uses examples to disclose the invention, including the best mode, and also to enable those skilled in the art to practice the invention, including making and using any device or system and performing any combined method. As will be understood by those skilled in the art, any aspect or embodiment disclosed herein may be used in combination with any other aspect or embodiment. If other embodiments have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that are not substantially different from the literal language of the claims, then these other embodiments are intended to fall within the scope of the claims.

[0143] Example 1

[0144] Preparation of 3,6-diamino-N2,N2,N5,N5-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

[0145]

[0146] A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (200 mg, 1.01 mmol), bis-2-(methoxyethyl)amine (372 μL, 335.5 mg, 2.52 mmol), HOBt·H2O (459 mg, 3.00 mmol) and EDC·HCl (575 mg, 3.00 mmol) was stirred together in DMF (20 mL) at room temperature for 1 hour. The mixture was concentrated to dryness and the residue was partitioned between EtOAc and water. The layers were separated and the EtOAc solution was washed with saturated NaHCO3 and brine. The solution was dried over anhydrous Na2SO4, filtered and concentrated. Purification by radial flash chromatography (SiO2, 10 / 1 CHCl3-MeOH) gave 228.7 mg (53% yield) of Example 1 as an orange foam: 1 H NMR (300 MHz, CDCl 3 ), 4.92 (s, 4H), 3.76 (apparent t, J = 5.4 Hz, 4H), 3.70 (apparent t, J = 5.6 Hz, 4H), 3.64 (apparent t, J = 5.4 Hz, 4H), 3.565 (apparent t, J = 5.4 Hz), 3.67 (s, 6H), 3.28 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 167.6 (s), 145.6 (s), 131.0 (s), 72.0 (t), 70.8 (t), 59.2 (q), 49.7 (t), 47.1 (t). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 3.14 minutes, (M+H) + =429.UV / vis (100 μM PBS solution)λ abs =394nm.Fluorescence (100nm)λ ex =394nmλ em =550nm.

[0147] Example 2

[0148] 3,6-Diamino-N 2 ,N 5 -Bis(2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

[0149]

[0150] Step 1. 3,6-Diamino-N 2 ,N 5 Synthesis of Bis((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)pyrazine-2,5-dicarboxamide

[0151]

[0152] A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (350 mg, 1.77 mmol), racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (933 μL, 944 mg, 7.20 mmol), HOBt·H2O (812 mg, 5.3 mmol), and EDC·HCl (1.02 g, 5.32 mmol) was stirred together in DMF (20 mL) for 16 hours at room temperature. The mixture was concentrated to dryness, and the residue was partitioned between EtOAc and water. The layers were separated, and the EtOAc solution was washed with saturated NaHCO3 and brine. The solution was dried over anhydrous Na2SO4, filtered, and concentrated to give 665 mg (88% yield) of the diastereometric pair of bisamides as a yellow solid: 1NMR (300 MHz, CDCl 3 ) δ 8.38 (t, J = 5.8 Hz, 2H), 6.55 (s, 4H), 4.21 (quintet, J = 5.8 Hz, 2H), 3.98 (dd, J = 8.4 Hz, 6.3 Hz, 2H), 3.65 (dd, J = 8.4 Hz, J = 5.8 Hz, 2H), 3.39 (apparent quartet - diastereotopic mixture, J = 5.9 Hz, 4H), 1.35 (s, 6H), 1.26 (s, 6H). 13 C NMR (75 MHz, CDCl 3 ) δ 165.7 (s), 146.8 (s), 126.8 (s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1 (t-diastereoisomer pair), 27.6 (q), 26.1 (q).

[0153] Step 2. The product of step 1 was dissolved in THF (100 mL) and treated with 1.0N HCl (2 mL). After hydrolysis was complete, the mixture was treated with K CO (1 g) and stirred for 1 hour, and filtered through a C18 plug using methanol. The filtrate was concentrated to dryness and the residue was ground with MeOH (50 mL). The solid was filtered and discarded, and the residue was treated with ether (50 mL). The precipitate was collected by filtration and dried under high vacuum. The substance was purified by radial flash chromatography to obtain 221 mg (36% yield) of Example 2 as an orange solid: 1 H NMR (300 MHz, DMSO-d6) δ 8.00 (bm, 6H), 5.39 (bs, 2H), 4.88 (bs, 2H), 3.63-3.71 (complex m, 2H), 3.40 (dd, J = 11.1, 5.10 Hz, 2H), 3.28 (dd, J = 11.1, 6.60 Hz, 2H), 2.92 (dd, J = 12.6, 3.3 Hz, 2H), 2.65 (dd, J = 12.6, 8.4 Hz, 2H). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 4.13 minutes, (M+H) + =345.UV / vis(100μM in H2O)λ abs =432nm. Fluorescence λex ex =432nm,λ em =558nm.

[0154] Example 3

[0155] (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis-(azanediyl))-bis(3-hydroxypropionic acid)

[0156]

[0157] Step 1. Synthesis of dimethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2'S)-bis(3-(benzyloxy)propionate

[0158]

[0159] A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24 mmol), L-Ser(OBn)-OMe·HCl salt (647 mg, 2.64 mmol), HOBt·H2O (570 mg, 3.72 mmol), and EDC·HCl (690 mg, 3.60 mmol) in DMF (25 mL) was treated with TEA (2 mL). The resulting mixture was stirred for 16 hours and concentrated. Workup was performed as in Example 1 to obtain 370 mg (51% yield) of the bisamide as a bright yellow powder: 1 NMR (300 MHz, CDCl 3 ): 8.47 (d, J = 8.74 Hz, 2H), 7.25-7.37 (complex m, 10H), 5.98 (bs, 4H), 4.85 (dt, J = 8.7, 3.3 Hz, 2H), 4.56 (ABq, J = 12.6 Hz, Δv = 11.9 Hz, 4H), 3.99 (d half of ABq, J = 8.7, 3.3, Δv unclear, 2H), 3.76-3.80 (ABq half - unclear, 2H), 3.78 (s, 6H). 13 C NMR (75 MHz, CDCl3) δ 170.5 (s), 165.1 (s), 146.8 (s), 138.7 (s), 128.6 (d), 128.1 (d), 127.8 (d), 126.9 (s), 73.5 (t), 69.8 (t), 53.0 (q), 52.9 (q). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 4.93 minutes, (M+H)+ = 581.

[0160] Step 2. Synthesis of (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)-bis(azanediyl))bis(3-(benzyloxy)propionic acid

[0161]

[0162] A solution of the product of step 1 (370 mg, 0.64 mmol) in THF (10 mL) was treated with 1.0 N sodium hydroxide (2.5 mL). After stirring at room temperature for 30 minutes, the reaction was judged to be complete by TLC. The pH was adjusted to about 2 by adding 1.0 N HCl, and the resulting solution was extracted with EtOAc (3x). The layers were combined, dried over sodium sulfate, filtered and concentrated to give 353 mg (100% yield) of the diacid as an orange foam: LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 minutes), retention time on a 30 mm column = 4.41 min, (M+H) + =553.

[0163] Step 3. To a solution of the product of step 2 (353 mg, 0.64 mmol) in methanol (20 mL) was added 5% Pd / C (300 mg) and ammonium formate (600 mg). The resulting reaction was heated at reflux for 2 hours. The reaction was cooled to room temperature, filtered through a diatomaceous earth (C salt, celite) plug and concentrated. The residue was recrystallized from methanol-ether to obtain 191 mg (80% yield) of Example 3 as a yellow foam: 1 NMR (300 MHz, DMSO-d6) 8.48 (d, J = 6.9 Hz, 2H), 6.72 (bs, 4H), 3.95 (apparent quartet, J = 5.1 Hz, 2H), 3.60 (apparent ABq doublet; downfield group centered at 3.71, J = 9.9, 5.1 Hz, 2H; upfield group centered at 3.48, J = 9.9, 6.3 Hz, 2H). 13 C NMR (75 MHz, CDCl3) δ 172.9 (s), 164.9 (s), 147.0 (s), 127.0 (s), 62.9 (d), 55.7 (t). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 1.45 minutes, (M+H) + =373.UV / vis (100 μM PBS solution)λ abs =434nm. Fluorescence λ ex =449nm,λ em =559nm.

[0164] Example 4

[0165] 3,6-bis(bis(2-methoxyethyl)amino)-N 2 ,N 2 ,N 5 ,N 5-Tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide bis(TFA) salt

[0166]

[0167] Step 1. Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic acid

[0168]

[0169] 3,6-diaminopyrazine-2,5-dicarboxylic acid (499 mg, 2.52 mmol) was dissolved in 48% hydrobromic acid (10 mL) and cooled to 0°C in an ice-salt bath. To this stirred mixture was added dropwise a solution of sodium nitrite (695 mg, 10.1 mmol) in water (10 mL), keeping the temperature below 5°C. The resulting mixture was stirred at 5-15°C for 3 hours, during which time the red mixture turned into a yellow solution. The yellow solution was poured into a solution of copper bromide (2.23 g, 10.1 mmol) in water (100 mL), and the resulting mixture was stirred at room temperature. After an additional 3 hours, the aqueous mixture was extracted with EtOAc (3x). The combined extracts were dried (Na2SO4), filtered and concentrated to give 440 mg (54% yield) of 3,6-dibromopyrazine-2,5-dicarboxylic acid as a light yellow solid: 13 C NMR (75 MHz, CDCl3) δ 164.3 (s), 148.8 (s), 134.9 (s). HPLC (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak on a 250 mm column, retention time = 2.95 min.

[0170] Step 2. 3-(bis(2-methoxyethyl)amino)-6-bromo-N 2 ,N 2 ,N 5 ,N 5 Synthesis of Tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

[0171]

[0172] The product of step 1 (440 mg, 1.36 mmol) was dissolved in DMF (25 mL), treated with HOBt·H2O (624 mg, 4.08 mmol) and EDC·HCl (786 mg, 4.10 mmol) and stirred at room temperature for 30 minutes. Bis(2-methoxyethyl)amine (620 mL, 559 mg, 4.20 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours and concentrated. The residue was partitioned with water and EtOAc. The EtOAc layer was separated and the aqueous layer was extracted again with EtOAc. The combined organic layers were washed with 0.5N HCl, saturated sodium bicarbonate and brine. The organic layer was dried (Na2SO4), filtered and concentrated to give 214 mg of 3-(bis(2-methoxyethyl)amino)-6-bromo-N 2 ,N 2 ,N 5 ,N 5 -Tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide (26% yield) as a brown oil: LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 3.85 min, (M+H) + =608.

[0173] Step 3. To the product of Step 2 (116 mg, 0.19 mmol) was added bis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and a "spatula tip" of Pd(PPh3)4. The resulting mixture was heated to 140°C for 2 hours. The reaction was cooled and concentrated. The residue was purified by flash chromatography (SiO2, 10 / 1 CHCl3-MeOH). The resulting material was further purified by reverse phase medium pressure chromatography (C18, 10-50% manual gradient of acetonitrile in 0.1% TFA) to give 12 mg (10% yield) of Example 4 as an orange-brown film: LCMS (15-95% gradient of acetonitrile in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 3.85 min, (M+H) + =661.UV / vis (100 μM PBS solution)λ abs =434nm. Fluorescence λ ex =449nm,λ em =559nm.

[0174] Example 5

[0175] 3,6-Diamino-N 2 ,N 5 -Bis(2-aminoethyl)pyrazine-2,5-dicarboxamide bis(TFA) salt

[0176]

[0177] Step 1. 3,6-Diamino-N 2 ,N 5 Synthesis of Bis[2-(tert-Butoxycarbonyl)-aminoethyl]pyrazine-2,5-dicarboxamide

[0178]

[0179] A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (500 mg, 2.07 mmol), tert-butyl 2-aminoethylcarbamate (673 mg, 4.20 mmol), HOBt·H2O (836 mg, 5.46 mmol), and EDC·HCl (1.05 g, 5.48 mmol) in DMF (25 mL) was stirred for 16 hours and concentrated. Workup was performed as in Example 1 to give 770 mg (76% yield) of the bisamide as an orange foam: 1 NMR (300 MHz, DMSO-d6) major conformer, δ 8.44 (t, J = 5.7 Hz, 2H), 6.90 (t, J = 5.7 Hz, 2H), 6.48 (bs, 4H), 2.93-3.16 (complex m, 8H), 1.37 (s, 9H), 1.36 (s, 9H). 13 C NMR (75 MHz, DMSO-d6), conformer δ 165.1 (s), 155.5 (bs), 155.4 (bs), 146.0 (s), 126.2 (s), 77.7 (bs), 77.5 (bs), 45.2 (bt), 44.5 (bt), 28.2 (q).

[0180] Step 2. To a solution of the product from Step 1 (770 mg, 1.60 mmol) in dichloromethane (100 mL) was added TFA (25 mL) and the reaction (mixture) was stirred at room temperature for 2 hours. The mixture was concentrated and the residue was taken up in methanol (15 mL). Diethyl ether (200 mL) was added and the orange solid precipitate was isolated by filtration and dried under high vacuum to afford 627 mg (77% yield) of Example 5 as an orange powder: 1 NMR (300MHz, DMSO-d6) δ8.70 (t, J = 6Hz, 2H), 7.86 (bs, 6H), 6.50 (bs, 4H), 3.46-3.58 (m, 4H), 3.26-3.40 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 166.4 (s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 3.62 min, (M+H) + =283.UV / vis (100 μM PBS solution)λ abs =435nm.Fluorescence (100nM)λ ex =449nm,λ em =562nm.

[0181] Example 6

[0182] 3,6-Diamino-N 2 ,N 5 -Bis(D-aspartic acid)-pyrazine-2,5-dicarboxamide

[0183]

[0184] Step 1. 3,6-Diamino-N 2 ,N 5 Synthesis of Bis(Benzyl DO-Benzyl-Aspartic Acid)-Pyrazine-2,5-Dicarboxamide

[0185]

[0186] A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (600 mg, 2.48 mmol), Asp(OBn)-OMe-p-TosH salt (2.43 g, 5.00 mmol), HOBt H2O (919 mg, 6.00 mmol) and EDC HCl (1.14 g, 5.95 mmol) in DMF (50 mL) was treated with TEA (4 mL). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was partitioned with water and EtOAc. The EtOAc layer was separated and washed with saturated sodium bicarbonate, water, and brine. The EtOAc solution was dried (Na2SO4), filtered, and concentrated. The residue was purified by flash chromatography (SiO2, 50 / 1CHCl3-MeOH to 10 / 1) to give 1.15 g of bisamide (58% yield) as a yellow foam: 1NMR (500MHz, CDCl3) δ8.61 (d, J = 8.4Hz, 2H), 7.29-7.9 (m, 20H), 5.85 (bs, 4H), 5.22 (A Bq, J=10.0Hz, △v=17.3Hz, 4H), 5.10 (ABq, J=12.2Hz, △v=34.3Hz, 4H), 5.06-5.09(obs m, 2H), 3.11 (d of ABq, J = 17.0, 5.14Hz Δv = 77.9Hz, 4H). 13 C NMR (75 MHz, CDCl3) δ 170.7 (s), 170.7 (s), 165.4 (s) 135.7 (s), 135.6 (s), 129.0 (d), 128.9 (d), 128.8 (d), 128.75 (d), 128.7 (d), 126.9 (s), 68.0 (t), 67.3 (t), 49.1 (d), 37.0 (t). LCMS (50-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 5.97 min, (M+H) + =789.

[0187] Step 2. THF (20 mL) and water (10 mL) were added to the product of step 1 (510 mg, 0.65 mmol). 10% Pd (C) (500 mg) and ammonium formate (1 g) were added to the stirred mixture. The resulting mixture was heated to 60 ° C for 2 hours and then allowed to cool to room temperature. The mixture was filtered through celite and concentrated. The resulting material was purified again by reverse phase medium pressure chromatography (C18, 10-70% manual gradient acetonitrile in 0.1% TFA solution) to obtain 137.8 mg (54% yield) of Example 6 as an orange solid: 1 NMR (300 MHz, DMSO-d6 = 8.4 Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J = 8.4, 5.4 Hz, 2H), 2.74-2.88 (complex m, 4H). 13 C NMR (75 MHz, DMSO-d6) δ 172.6 (s), 165.2 (s), 147.0 (s), 126.6 (s), 60.8 (t), 49.1 (d). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 4.01 min, (M+H) + =429.UV / vis (100 μM PBS solution)λ abs =433nm.Fluorescence (100nM)λ ex =449nm,λ em=558nm.

[0188] Example 7

[0189] 3,6-Diamino-N 2 ,N 5 -Bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl)pyrazine-2,5-dicarboxamide

[0190]

[0191] To a solution of Example 5 (77.4 mg, 0.15 mmol) in DMF (5 mL) was added TEA (151 mg, 1.49 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11-tetraoxatetradecane-14-oate (113 mg, 0.34 mmol) and the reaction mixture was stirred at room temperature for 16 hours. The reaction was concentrated and the residue was purified by medium pressure reverse phase chromatography (LiChroprep RP-18 Lobar (B) 25x310 mm-EMD chemicals 40-63 μm, ~70 g, 90 / 10 to 80 / 20 0.1% TFA-ACN) to give 37.4 mg (35% yield) of Example 7 as an orange film: 1 NMR (300 MHz, DMSO-d6) δ 8.47 (t, J = 5.7 Hz, 2H), 7.96 (t, J = 5.4 Hz, 2H), 3.20-3.60 (complex m, 36H), 3.47 (s, 3H), 3.46 (s, 3H), 2.30 (t, J = 6.3 Hz, 4H). 13 C NMR (75 MHz, DMSO-d6) δ 170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 69.4 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 4.01 min, (M+H) + =719,(M+Na) + =741.UV / vis (100 μM PBS solution)λ abs =437nm.Fluorescence (100nM)λ ex =437nm,λ em =559nm.

[0192] Example 8

[0193] 3,6-Diamino-N 2 ,N 5 -Bis(26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-yl)pyrazine-2,5-dicarboxamide

[0194]

[0195] To a solution of Example 5 (50.3 mg, 0.10 mmol) in DMF (5 mL) was added TEA (109 mg, 1.08 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxahexacosa-26-oate (128 mg, 0.25 mmol) and the reaction (mixture) was stirred at room temperature for 16 hours. The reaction was concentrated and the residue was purified by medium pressure reverse phase chromatography (LiChroprep RP-18 Lobar (B) 25 x 310 mm-EMD chemicals 40-63 μm, ~70 g, 90 / 10 to 80 / 20 0.1% TFA-ACN) to give 87.9 mg (82% yield) of Example 8 as an orange film: 1 NMR (300 MHz, DMSO-d6) δ 8.46 (t, J = 5.7 Hz, 2H), 7.96 (t, J = 5.4 Hz, 2H), 3.16-3.73 (complex m, 74H), 2.28-2.32 (m, 2H). 13 C NMR (75 MHz, DMSO-d6) - multiple conformation-δ 170.1 (s), 169.9 (s) 169.8 (s), 165.1 (s), 146.0 (s), 126.2 (s). 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 5.90 min, (M+H) + =1071,(M+2H) 2+ =536.UV / vis (100 μM PBS solution)λ abs =438nm.Fluorescence (100nM) λ ex =438nm, λem em =560nm.

[0196] Example 9

[0197] 3,6-Diamino-N 2 ,N 5-Bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39-azahexadecane-41-yl)pyrazine-2,5-dicarboxamide

[0198]

[0199] To a solution of Example 5 (53.1 mg, 0.10 mmol) in DMF (5 mL) was added TEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioctadecanoate (144 mg, 0.21 mmol) in DMF (2.0 mL), and the resulting mixture was stirred for 16 hours. The reaction was concentrated and the residue was purified by medium pressure reverse phase chromatography (LiChroprep RP-18 Lobar (B) 25 x 310 mm - EMD chemicals 40-63 μm, ~70 g, 90 / 10 to 80 / 20 0.1% TFA-ACN) to give 87.5 mg (61% yield) of Example 9 as an orange film: 1 NMR (300 MHz, DMSO-d6) δ 8.48 (t, J = 5.7 Hz, 2H), 7.96 (t, J = 5.4 Hz, 2H), 7.80-7.86 (m, 2H), 5.94 (bm, 2H), 3.30-3.60 (complex m, 106H), 2.26-2.33 (m, 4H). 13 C NMR (75 MHz, DMSO-d6) ξ 170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 5.90 min, (M+2H) 2+ =712.UV / vis (100 μM PBS solution)λ abs =449nm.Fluorescence (100nM)λ ex =449nm,λ em =559nm.

[0200] Example 10

[0201] Bis(2-(PEG-5000)ethyl)6-(2-(3,6-diamino-5-(2-aminoethylcarbamoyl)pyrazine-2-carboxamido)ethylamino)-6-oxohexane-1,5-diyldicarbamate

[0202]

[0203] A solution of Example 5 (25 mg, 0.049 mmol) in DMF (30 mL) was treated with TEA (1 mL) and m-PEG2-NHS (1 g, 0.1 mmol), and the resulting mixture was stirred at room temperature for 48 hours. The mixture was concentrated, and the residue was partially purified by gel filtration chromatography (G-25 resin, water). The product was concentrated and further purified by reverse phase medium pressure chromatography (C18, 10-70% manual gradient of acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield) of Example 10 as a tan waxy solid: MaldiMS m / z = 11393.

[0204] Example 11

[0205] (R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinic acid

[0206]

[0207] Step 1. Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine.

[0208]

[0209] A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in MeOH (500 mL) was treated with NBS (34.3 g, 193.1 mmol) in batches over 1 hour. The resulting mixture was then stirred for 16 hours. TLC analysis at this point showed that a small amount of starting material remained. Another 1.4 g of NBS was added and the reaction was heated to 50 ° C for 2 hours. The mixture was then cooled to 38 ° C and treated with NCS (25.8 g, 193.1 mmol). The reaction mixture was then heated to 50 ° C for 16 hours. The mixture was then cooled to room temperature and treated with water (500 mL). The precipitate was collected by filtration and dried in a vacuum dessicator to give 45.4 g (97% yield) of 2-amino-5-bromo-3,6-dichloropyrazine as a white solid: 13C NMR (75 MHz, CDCl3) δ 149.9 (s), 145.6 (s), 129.6 (s), 121.5 (s). LCMS (15-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 30 mm column, retention time = 4.51 min, (M+H) + =244,(M+H+ACN) + =285.

[0210] Step 2. Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile.

[0211]

[0212] A mixture of CuCN (8.62 g, 96.3 mmol) and NaCN (4.72 g, 96.3 mmol) was heated to 90 ° C under high vacuum. The resulting mixture was subjected to three argon / vacuum cycles and placed under a final positive pressure of argon. The mixture was cooled to room temperature and DMF (150 mL) was added. The heterogeneous mixture was heated to 130 ° C and maintained for 2.5 hours. A solution of the product of step 1 (15.6 g, 64.2 mmol) dissolved in DMF (150 mL) was added dropwise to the resulting homogeneous mixture of sodium dicyanocuprate over a period of 1 hour. The temperature was gradually raised to 150 ° C, and the resulting mixture was then stirred at this temperature for 10 hours. The reaction was then cooled to room temperature and poured into water (1 L). The resulting mixture was extracted with EtOAc (3x), and the combined extracts were filtered to remove the flocculent dark solid, washed with brine, dried (Na2SO4), filtered again, and concentrated. Purification by flash column chromatography (SiO2, 10 / 1 hexanes-EtOAc to 3 / 1) afforded 6.70 g (55% yield) of the nitrile product as a tan solid: 13 C NMR (75 MHz, CDCl3) δ 153.9 (s), 149.1 (s), 131.7 (s), 115.4 (s), 111.0 (s). GCMS (injection temperature = 280°C, helium flow rate of 1.0 mL / min, temperature program: 100°C (hold for 2 minutes), heating to 300°C, at 10°C / min (hold for 2 minutes), main peak retention time = 16.556 min, m / z (EI) = 188, 190.

[0213] Step 3. Synthesis of 5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyrazine-2-carbonitrile

[0214]

[0215] To a solution of the product (1.00 g, 5.29 mmol) in ACN (20 mL) of step 2, bis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) was added and the reaction mixture was heated to 70 ° C for 16 hours. The reaction (mixture) was cooled and concentrated. The residue was distributed with EtOAc and water. The organic layer was separated and the aqueous layer was extracted with EtOAc again. The combined organic extracts were washed with brine, dried (Na2SO4), filtered and concentrated. Purified by flash column chromatography (SiO2, 10 / 1 hexane-EtOAc to 1 / 1) to obtain 950 mg (63% yield) of the desired adduct as a yellow solid: 1 NMR (300MHz, CDCl3) δ7.47 (bs, 2H), 3.77 (t, J = 5.7Hz, 4H), 3.52 (t, J = 5.4Hz, 4H), 3.25 (s, 6H). 13 C NMR (75 MHz, CDCl3) δ 154.7 (s), 152.0 (s), 120.9 (s), 119.5 (s), 95.8 (s), 71.0 (t), 59.1 (q), 50.0 (t). LCMS (50-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 4.91 min, (M+H) + =286,(M+Na) + =308,(M+Na+ACN) + =349.

[0216] Step 4. Synthesis of 3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile.

[0217]

[0218] At 0 ° C (ice-salt bath), a solution of sodium nitrite (673 mg, 9.75 mmol) in 48% hydrobromic acid (20 mL) was added dropwise over 30 minutes. The resulting mixture was stirred at 0-5 ° C for 1 hour and poured into a stirred solution of CuBr2 (1.64 g, 7.34 mmol) in water (100 mL). Then, the resulting mixture was stirred at room temperature for 16 hours. The mixture was extracted with EtOAc (3x). The combined organic layers were dried (Na2SO4), filtered and concentrated. Purified by flash column chromatography (SiO2, 50 / 1CHCl3-MeOH) to obtain 1.00 g (58% yield) of bromide as an orange-brown solid: 1NMR (300MHz, CDCl3) δ3.99 (t, J=5.4Hz, 4H), 3.64 (t, J=5.4Hz, 4H), 3.35 (s, 6H). 13 C NMR (75 MHz, CDCl3) δ 152.8 (s), 140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4 (t), 59.1 (t), 50.5 (q). LCMS (50-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 4.55 min, (M+H) + =349,351.

[0219] Step 5. Synthesis of 3-(bis(2-methoxyethyl)amino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile.

[0220]

[0221] A mixture of the product of step 4 (1.0 g, 2.87 mmol), 2-furylboronic acid (643 mg, 5.75 mmol), Cs2CO3 (3.31 g, 10.2 mmol), TFP (35 mol%, 236 mg, 1.02 mmol) and Pd2dba3-CHCl3 (5 mol%, 10 mol% Pd, 150 mg) was subjected to three vacuum / argon cycles and placed under a positive pressure of argon. Anhydrous dioxane (50 mL) was added, and the reaction mixture was heated to 75°C and maintained for 16 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc (100 mL), and filtered through a medium frit. The residue was concentrated and purified by flash chromatography (SiO2, 50 / 1 CHCl3-MeOH) to give 757 mg of the furan adduct (78% yield) as a tan powder: LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 6.41 min, (M+H) + =337.

[0222] Step 6. Synthesis of 6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxylic acid.

[0223]

[0224] To a well-stirred mixture of ACN (11 mL), CCl₄ (7 mL), and water (11 mL) were added sodium periodate (1.07 g, 5.00 mmol) and RuO₂·H₂O (13.3 mg, 0.10 mmol) in sequence. The resulting mixture was stirred vigorously at room temperature for 30 minutes and treated with sodium bicarbonate (2.10 g, 25.0 mmol), followed by water (5 mL). The mixture was stirred vigorously for an additional 15 minutes, after which a solution of the product from Step 5 (276 mg, 0.82 mmol) dissolved in ACN (1 mL) was added. The green mixture was stirred at room temperature for 5.5 hours. The mixture was transferred to a separatory funnel and extracted with EtOAc. The aqueous layer was adjusted to pH ~3.5 and extracted again with EtOAc (2x). The combined extracts were washed with 20% sodium bisulfite and brine and dried (Na₂SO₄). Filtration and concentration gave 140 mg (54% yield) of the carboxylic acid as a light yellow solid: LCMS (5-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak on a 250 mm column, retention time = 5.05 min, (M+H) + =315.

[0225] Step 7. Synthesis of (R)-dibenzyl-2-(6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamido)succinate.

[0226]

[0227] A mixture of the product from step 6 (140 mg, 0.45 mmol), EDC·HCl (128 mg, 0.67 mmol), and HOBt·H2O (102 mg, 0.67 mmol) in anhydrous DMF (25 mL) was stirred together at room temperature for 30 minutes. To this stirred mixture was added (R)-dibenzyl 2-aminosuccinate p-TsOH salt (213 mg, 0.44 mmol), followed by TEA (1 mL). The resulting mixture was then stirred for 16 hours. The reaction mixture was concentrated and partitioned between EtOAc and saturated sodium bicarbonate solution. The EtOAc layer was separated, washed with saturated sodium bicarbonate and brine, dried (Na2SO4), filtered, and concentrated to give 240 mg (88% yield) of pyrazinamide as an orange foam: LCMS (15-95% acetonitrile gradient in 0.1% TFA over 10 minutes), single peak retention time on a 250 mm column = 8.76 min, (M+H) + =610,(M+Na) + =632.

[0228] Step 8. (R)-Dibenzyl-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinate.

[0229]

[0230] To the product of step 7 (240 mg, 0.39 mmol) was added morpholine (5 mL). The reaction mixture was heated to 70 ° C and maintained for 2 hours. The mixture was cooled and concentrated. The residue was partitioned with EtOAc and water. The EtOAc layer was separated and washed with saturated sodium bicarbonate and brine. The EtOAc layer was dried (Na2SO4), filtered and concentrated. Purification by flash column chromatography (SiO2, 3: 1 to 1: 1 hexane-EtOAc) gave 199 mg (75% yield) of morpholine adduct as an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 minutes), single peak retention time on a 250 mm column = 8.76 min, (M+H) + =661,(M+Na) + =683.

[0231] Step 9. Synthesis of Example 11:

[0232]

[0233] To a solution of the dibenzyl ester (115 mg, 0.17 mmol) in THF (10 mL) was added 1.0 N sodium hydroxide (4 mL). The mixture was stirred at room temperature for 1 hour. The pH was adjusted to ~2 with 1.0 N HCl and the solution was concentrated. The residue was purified by medium pressure reverse phase chromatography (LiChroprep RP-18 Lobar (B) 25x310 mm-EMD chemicals 40-63 μm, ~70 g, 90 / 10 to 50 / 50 0.1% TFA-ACN) to give 32 mg (27% yield) of Example 11 as an orange solid: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 minutes), single peak retention time on a 250 mm column = 4.47 min, (M+H) + =481.UV / vis (100 μM PBS solution)λ abs =438nm.Fluorescence (100nM)λ ex =449nm,λ em =570nm.

[0234] Example 12

[0235] (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis-(azanediyl))bis(3-hydroxypropionic acid) ("D-serine isomer" or "MB-102")

[0236]

[0237] Step 1: Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2'R)-bis(3-hydroxypropionic acid)dibenzyl ester

[0238] To a 500 mL round-bottom flask equipped with a Claisen adapter and an addition funnel was added D-serine benzyl ester hydrochloride (24.33 g, 105.0 mmol), and anhydrous DMF (300 mL) was added via cannula. The solution was cooled in an ice bath and stirred under N₂ for 15 minutes. DIPEA (19.16 mL, 110.0 mmol) was added dropwise via the addition funnel over 30 minutes. After an additional 30 minutes, the cooling bath was removed, and the diacid (9.91 g, 50.0 mmol) was added in one portion. The brick-red suspension was stirred for 30 minutes, and HOBt·H₂O (17.61 g, 115.0 mmol) was added in one portion. After 15 minutes, the reaction flask was cooled in an ice bath, and EDC·HCl (22.05 g, 115.0 mmol) was added portionwise over 15 minutes. The resulting suspension was slowly warmed to room temperature and stirred under N₂ overnight (approximately 17 hours).

[0239] The dark solution was concentrated under high vacuum (bath temperature 60°C) to a syrupy residue, which was layered between EtOAc and ultrapure H2O (milli-Q H2O) (400 mL each). The layers were separated and the aqueous layer was extracted with EtOAc (3×200 mL). The combined EtOAc extracts were washed sequentially with 0.50 M KHSO4, saturated NaHCO3, H2O and brine (250 mL each). The solvent was removed under reduced pressure to give 23.7 g of an orange solid. The crude product was purified by flash chromatography on silica gel using a CHCl3:MeOH gradient to give bisamide (19.6 g, 71%) as an orange solid: R f 0.45[CHCl3:MeOH(9:1,v / v)]. 1H NMR (DMSO-d6) δ 8.56 (d, J = 8.0 Hz, 2H, exchangeable with D2O), 7.40-7.33 (m, 10H), 6.76 (s, 4H, exchangeable with D2O), 5.37 (t, J = 5.5 Hz, 2H), 5.20 (m, 4H), 4.66-4.63 (dt, J = 8.0, 4.0 Hz, 2H), 3.97-3.93 (m, 2H), 3.81-3.77 (m, 2H). 13 C NMR(DMSO-d6)δ170.1,164.9,146.4,135.8,128.4,128.0,127.6,125.9,66.2,61.1,54.4.RP-LC / MS(ESI)m / z 553.3(M+H)+(tR=4.44min,5-95% B / 6min).C 26 H 28 Calculated analysis of N6O8: C, 56.52; H, 5.11; N, 15.21. Found: C, 56.39; H, 5.11; N, 14.99.

[0240] Step 2. Formation of (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropionic acid)

[0241] The bisamide (7.74 g, 14.0 mmol) was hydrogenated in EtOH:HO (560 mL; 3:1 v / v) in the presence of 10% Pd / C (0.774 g). The reaction mixture was purged with argon and stirred at room temperature under a hydrogen atmosphere (slowly bubbling) for 5.5 hours. The reaction mixture was purged with Ar again, and the catalyst was removed by filtration through celite. The bed was washed with EtOH:HO (400 mL; 1:1, v / v), and the combined filtrates were concentrated under vacuum. The product was dried under high vacuum. The residue was triturated with CHCN to give the D-serine isomer (4.89 g, 94%) as an orange powder. 1 H NMR (DMSO-d6) δ 8.46 (d, J = 8.3 Hz, 2H, exchangeable with D2O), 6.78 (br s, 4H, exchangeable with D2O), 4.48-4.45 (dt, J = 8.1, 3.9 Hz, 2H), 3.88 (dd, J = 11.1, 3.9 Hz, 2H), 3.74 (dd, J = 11.1, 3.7 Hz, 2H). 13C NMR(DMSO-d6)δ171.6,164.7,146.4,125.9,61.2,54.3.RP-LC / MS(ESI)m / z 373.2(M+H)+(tR=2.86min,5_95% B / 6min).C 12 H 16 Anal. Calc. for N6O8: C, 38.71; H, 4.33; N, 22.57. Found: C, 38.44; H, 4.51; N, 22.33.

[0242] Example 13

[0243] (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("D-alanine isomer")

[0244]

[0245] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2'R)-dipropionate

[0246] Under an inert atmosphere, a flame-dried round-bottom flask (100 mL) equipped with a magnetic stir bar was charged with a solution of 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), D-alanine ethyl ester hydrochloride (1.86 g), EDC·HCl (2.70 g), HOBt·H₂O (2.65 g), and Et₃N (2.0 mL) in DMF (anhydrous, 80 mL). Volatiles were removed under reduced pressure at 50°C, yielding a dark semisolid. After cooling, acetonitrile (~100 mL) was added, and the solution was allowed to stand for approximately 1 hour. The red precipitate was isolated by centrifugation, washed with EtOAc, and dried. The total weight of the diester was 1.30 g (3.06 mmol, 60.6% isolated yield). This material (1.3 g) was carried forward to the next step without further purification.

[0247] Step 2. Formation of (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0248] The diester from step 1 (1.0 g) and LiOH (4 equivalents) were combined in THF / water and stirred at ambient temperature for several hours. HPLC indicated complete hydrolysis. The pH was made acidic by adding TFA and the reaction mixture was left at ambient temperature overnight. The reaction mixture was purified by preparative RPHPLC to give the diacid. Program: 99:1 A:B for 5 minutes, then 5:95 A:B for 27 minutes at 50 mL / min. λUV 264 nm and fluorescence; λ x=440nm,λ m = 565 nm. Fractions containing the desired product were combined and lyophilized (-85° C., 15 mtorr) to give an orange solid (0.821 g, 2.41 mmol, 95.6% isolated yield). M / z 341.13. Proton and carbon NMR were consistent with the proposed structure.

[0249] Example 14

[0250] 3,3'-((3,6-Diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("β-alanine isomer")

[0251]

[0252] Step 1. Formation of dibenzyl 3,3'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)dipropionate

[0253] Under an inert atmosphere, a flame-dried round-bottom flask (100 mL) was charged with a solution of 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.30 g), benzyl 3-aminopropionate p-toluenesulfonate (1.08 g), EDC·HCl (0.590 g), HOBt·H2O (0.582 g), and Et3N (1.50 g) in DMF (anhydrous, 40). The reaction mixture was stirred at ambient temperature overnight and concentrated in vacuo to approximately 10 mL. The remaining DMF was removed by azeotropy with toluene. The reaction mixture was partitioned between EtOAc (3 × 125 mL) and saturated NaHCO3 (3 × 100 mL). The organic layers were combined and washed with citric acid (10% aqueous solution, 100 mL) and brine (100 mL). The organic layer was removed, dried (anhydrous Na2SO4), and concentrated in vacuo to yield 0.58 g of a crystalline solid. TLC (silica on glass, 1:1 EtOAc:hexane) R f =0.22. The product was purified by silica gel flash chromatography to obtain 0.49 g of the product. Mass spectrum (ES+) 521.36 (100%), 522.42 (30%), 523.34 (about 6%). NMR, 1 H(DMSO-d6),400MHz: 2.55(4H,m),3.41(4H,m)5.01(4H,s),6.44(4H,s),7.21(10H,m),8.41(2H,m); 13 C(DMSO-d6):34.18,35.33,66.19,126.74,128.52,128.92,136.56,146.75,165.63,171.90.

[0254] Step 2. Formation of 3,3'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0255] The dibenzyl ester (0.92 g) from step 1 was combined with EtOH (anhydrous, 75 mL) and transferred to a Fischer-Porter pressure bottle (6 oz) equipped with inlet and outlet valves, a pressure gauge (0-100 psig), and a Teflon-coated magnetic stir bar. Water (25 mL) and 10% Pd / carbon (0.2 g, Degussa / Aldrich wet) were added, and the reaction vessel was sealed. After three vacuum / Ar cycles, H2 (g) was introduced from a lecture bottle at 10 psig to the vigorously stirred solution. After 3.5 hours, the reaction was filtered through a pad of Celite, and the resulting Celite / catalyst bed was rinsed with approximately 500 mL of 1:1 EtOH:HO to give a solution that was concentrated in vacuo. 0.424 g of a solid was isolated (70.5% isolated yield). HPLC / MS yielded a single peak at 9.3 minutes. Mass spectrum (ES+) 341.32 (100%), 342.37 (30%), 344.29 (18%), 270.30 (62%). NMR, 1 H(DMSO-d6),400MHz: 2.54(2H,m),3.42(2H,m),6.52(2H,s),7.21(4H,m),8.38(2H,m),11.9(2H,bs); 13 C(DMSO-d6):34.20,35.33,126.77,146.75,165.55,173.57.

[0256] Example 15

[0257] 2,2'-((3,6-Diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic acid ("glycine isomer")

[0258]

[0259] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetate

[0260] A round-bottom flask (300 mL) equipped with a magnetic stir bar was charged with a solution of 3,6-diaminopyrazine-2,5-dicarboxylic acid (5.0 g), glycine ethyl ester hydrochloride (5.04 g), EDC·HCl (8.1 g), HOBt·H2O (8.0 g), and DIPEA (5.9 g) in DMF (anhydrous, 200 mL). A dry argon atmosphere was maintained throughout the reaction. The pyrazine and glycine salts were combined, and DMF was added with stirring under an inert atmosphere. Base and HOBt were added to this. After approximately 15 minutes, EDC was added portionwise over 45 minutes, and the reaction was stirred at ambient temperature under Ar overnight. The reaction mixture was concentrated in vacuo until a viscous semisolid was obtained. The semisolid was treated with toluene (approximately 30 mL), and the volatiles were removed in vacuo. A solid formed upon cooling. The crude product was dissolved in 500 mL of EtOAc and mixed until two layers formed. The solution was washed with brine and saturated NaHCO₃, and the aqueous layer was removed. The aqueous layer was washed (2×EtOAc, 150 mL), and the organic layers were combined. The organic layer was washed with aqueous NaHSO₄ and saturated brine, dried over Na₂SO₄, and concentrated to obtain a solid. Isolated yield: 5.46 g. HPLC analysis: 96.9%. M / z: 369.2. 1 H and 13 C NMR was consistent with the proposed structure. The product was carried forward to the next step without further purification.

[0261] Step 2. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic acid

[0262] The crude product (700mg) of step 1 was dissolved in 40mL of THF and 10mL of water (DI). LiOH (4.2 equivalents) was added, and the mixture was stirred overnight at ambient temperature under an inert atmosphere. HPLC analysis showed complete conversion to the desired diacid (M / z=313.3). The reaction mixture was centrifuged (3000rpm for 3 minutes), the supernatant was checked by HPLC and discarded. The remaining solid was converted into the disodium salt by treating with NaOH (6.25N, 2 equivalents), and the resulting solution was filtered (0.22 microns). The solution was freeze-dried to obtain a solid with an HPLC purity of >95%. By adding a little more than 2 equivalents of TFA, the disodium salt was then converted into the diacid by reverse phase preparative column. Proton and carbon NMR were consistent with the proposed structure.

[0263] Example 16

[0264] (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("L-alanine isomer")

[0265]

[0266] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2'S)-dipropionate

[0267] Under an inert atmosphere, a flame-dried round-bottom flask (100 mL) was charged with a solution of 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), L-alanine ethyl ester hydrochloride (1.86 g), EDC·HCl (2.70 g), and HOBt·H2O (2.65 g) in DMF (anhydrous, 80 mL). Triethylamine (1.50 g) was added. After 16 hours at ambient temperature, the reaction volatiles were removed in vacuo. A semisolid separated. Water (70 mL) was added and the mixture was allowed to stand for approximately 1 hour. During this time, a precipitate formed, and the mixture was centrifuged, and the isolated solid was air-dried overnight. This material was dissolved in EtOAc and washed with water, citric acid, and saturated sodium bicarbonate. The organic layer was dried (anhydrous sodium sulfate) and concentrated in vacuo to yield a solid product (1.38 g). HPLC purity was >95%. The crude product was used in the next step without further purification.

[0268] Step 2. Formation of (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0269] The crude product from step 1 (1.0 g) was dissolved in THF (30 mL) and LiOH·H O (4 equivalents) dissolved in water (10 mL) was added at ambient temperature. After one hour, the volatiles were removed in vacuo. The product was purified by preparative reverse phase HPLC and lyophilized to give a solid of the desired diacid product with a purity of >95%. Proton and carbon NMR were consistent with the proposed structure.

[0270] Example 17

[0271] 2,2'-((3,6-Diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropionic acid) ("dimethylglycine isomer")

[0272]

[0273] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropionate)

[0274] Under an inert atmosphere, a flame-dried round-bottom flask (100 mL) was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), ethyl gem-dimethyl-3-aminopropionic acid hydrochloride (1.86 g), EDC·HCl (2.70 g), and HOBt·H2O (2.65 g) in DMF (anhydrous, 80 mL). The reaction was initiated by the addition of triethylamine (1.50 g) and maintained at ambient temperature for 72 hours. Volatiles were removed in vacuo. A dark, viscous liquid separated. After cooling, it was dissolved in acetonitrile (approximately 100 mL) and allowed to stand for approximately 1 hour. A precipitate formed, which was isolated by centrifugation and dried to yield 1.30 g of the diethyl ester (61%) with a RPHPLC purity of >95%. The crude product was used directly in the next step.

[0275] Step 2. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoic acid)

[0276] The crude product from step 1 (1.0 g) was dissolved in THF:water (40 mL:5 mL). An aqueous solution of LiOH (2.5 equivalents in 0.5 mL of deionized water) was added. An additional 2 equivalents of LiOH were added to the reaction mixture, and the reaction was allowed to proceed overnight at ambient temperature. Upon completion, the reaction mixture was acidified with TFA until a pH of approximately 4 was reached. The product was isolated by preparative RPHPLC. M / z 369.13. Proton and carbon NMR were consistent with the proposed structure.

[0277] Example 18

[0278] 3,6-Diamino-N 2 ,N 5 -Bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide

[0279]

[0280] A round-bottom flask (100 mL) equipped with a magnetic stir bar was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.535 g, 2.70 mmol), (1R,2S,3R,4R)-1-aminopentane-1,2,3,4,5-pentanol (0.978 g, 5.40 mmol, 2.0 equiv), and DMF (40 mL). Triethylamine (0.546 g, 0.76 mL, 5.40 mmol, 2.0 equiv) and PyBop (3.1 g, 5.94 mmol, 2.2 equiv) were added. After one hour, the reaction was complete by HPLC analysis and concentrated in vacuo, maintaining the temperature below 40°C. The mixture was dissolved in water (10 mL) and passed through a Sephadex G-10 column. Fractions containing the fluorescent product were collected and lyophilized to yield an impure solid. The target product 3,6-diamino-N- 2 ,N 5 -Bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide: 160 mg, HRMS (theoretical value) M+Na=547.1970; HRMS (observed value) M+Na=547.1969.

[0281] Example 19

[0282] Protocol for assessing renal function

[0283] Figure 2 An example of an in vivo renal monitoring assembly 10 is shown in FIG, which includes a light source 12 and a data processing system 14. The light source 12 typically includes or is interconnected with a suitable device to expose at least a portion of the patient's body to the light therein. Examples of suitable devices that can be interconnected with the light source 12 or be part of the light source 12 include, but are not limited to, catheters, endoscopes, optical fibers, ear clips, handbands, headbands, forehead sensors, surface coils, and finger probes. In fact, any of a number of devices capable of emitting visible light and / or near-infrared light from a light source can be used in the renal monitoring assembly 10. In one aspect, the light source is an LED, wherein one LED emits light near the maximum absorbance of the tracer and a second LED emits light near the fluorescence emission maximum of the tracer. For example, one LED emits light at 450 nm and a second LED emits light at 560 nm.

[0284] Still refer to Figure 2The data processing system 14 of the renal monitoring assembly can be any suitable system capable of detecting spectral energy and processing data indicative of the spectral energy. For example, the data processing system 14 may include one or more lenses (e.g., to direct and / or focus the spectral energy), one or more filters (e.g., to filter out undesirable wavelengths of spectral energy), a photodiode or photomultiplier tube (e.g., to collect the spectral energy and convert it into an electrical signal indicative of the detected spectral energy), an amplifier (e.g., to amplify the electrical signal from the photodiode or photomultiplier tube), and a processing unit (e.g., to process the electrical signal from the photodiode or photomultiplier tube). The data processing system 14 is preferably configured to manipulate the collected spectral data and generate an intensity / time curve (profile) and / or a concentration / time curve indicative of the renal clearance of the pyrazine derivatives of the present disclosure from the patient 20. In practice, the data processing system 14 can be configured to generate appropriate renal function data by comparing the differences in how normal cells and damaged cells remove the pyrazine derivatives from the bloodstream, to determine the rate or accumulation of the pyrazine derivatives in an organ or tissue of the patient 20, and / or to provide a tomographic image of an organ or tissue having pyrazine derivatives associated therewith.

[0285] By way of example and not limitation, in one aspect, the system includes two silicon photomultiplier tubes. The first photomultiplier tube includes a longpass filter, while the second photomultiplier tube is unfiltered. This arrangement allows for measurement of both fluorescence emission and diffuse reflectance at both excitation and emission wavelengths. In one such embodiment, the fluorescence and diffuse reflectance measurements are combined into an intrinsic fluorescence measurement that compensates for variations in tissue optical properties. An exemplary formula for the combined measurement is provided in Equation 1.

[0286] In one aspect of determining renal function, an effective amount of a pyrazine derivative is administered to a patient in need thereof (e.g., in the form of a pharmaceutically acceptable composition). As indicated by arrow 16, at least a portion of the body of patient 20 is exposed to visible light and / or near-infrared light from light source 12. For example, light from light source 12 can be delivered via an optical fiber secured to the ear of patient 20. The patient can be exposed to light from light source 12 before or after the pyrazine derivative is administered to patient 20. In some cases, it may be beneficial to generate a background or baseline reading of light emitted from the body of patient 20 (due to exposure to light from light source 12) before the pyrazine derivative is administered to patient 20. When the pyrazine derivative in patient 20 is exposed to light from light source 12, the pyrazine derivative emits light (indicated by arrow 18) that is detected / collected by data processing system 14. Initially, administering the pyrazine derivative to patient 20 typically results in an initial spectral signal indicating the initial content of the pyrazine derivative in patient 20. The spectral signal then tends to decay as a function of time as the pyrazine derivative is cleared from patient 20. The decay of the spectral signal as a function of time indicates the patient's renal function. Additionally, if the tracer is injected into the patient's vascular space, the initial kinetics reflect the equilibrium of the tracer throughout the patient's extracellular space. In some aspects, this equilibrium is completed in less than 2 hours.

[0287] For example, in a first patient exhibiting healthy / normal renal function, the spectral signal may decay back to baseline within a time period of T. However, the spectral signal indicating a second patient exhibiting renal impairment may decay back to baseline within a time period of T+4 hours. The extent of the renal impairment or defect will affect the length of time required for the signal to decay back to baseline. More severe renal impairment requires a longer time period. In this way, the patient 20 can be exposed to light from the light source 12 for any amount of time suitable to provide the desired renal function data. Similarly, the data processing system 14 can be allowed to collect / detect spectral energy for any amount of time suitable to provide the desired renal function data.

[0288] In addition, the determination of GFR in a patient is not limited to a single determination based on a single administration of a tracer. The time between the administration of the tracer and its becoming undetectable in the patient can be subdivided into multiple smaller segments, and the patient's GFR is calculated for each smaller segment. In some aspects, the time periods can overlap. As an example and not a limitation, if the entire time period before the tracer becomes undetectable is 24 hours, then the time period can be divided into four equal six-hour segments; each new time period begins at the end of the previous segment. In yet another aspect, the time periods can overlap. For example, each individual time period may be as long as four hours, but a new time period may start every two hours. This will generate overlapping segments throughout the measurement process. To more fully illustrate this non-limiting example, if the tracer is administered at time 0 and becomes undetectable at time 24 hours, the following time periods can be generated: 0-4 hours, 2-6 hours, 4-8 hours, 6-10 hours, 8-12 hours, 10-14 hours, 12-16 hours, 14-18 hours, 16-20 hours, 18-22 hours, and 20-24 hours. The patient's GFR can be calculated separately in each time period. This data will be used to more fully assess the health of the patient's kidneys. The time period can be any length that allows for GFR determination and can be 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or 12 hours. In addition, the time periods do not have to be the same during the measurement period. The length of each time period is selected independently without regard to any other time period.

[0289] Pharmacokinetic study results

[0290] In a clinical study, MB-102 ((2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropionic acid) prepared in Example 12) was administered intravenously to 60 human patients. In addition to the methods and techniques disclosed herein, blood and urine were collected. Standard pharmacokinetic data were collected and compared with known contrast agents used for GFR determination. (iohexol) for comparison.

[0291] Figures 3A to 3D Shown in Figure 2 are data collected from 60 human patients tested with MB-102. Plasma pharmacokinetic data were collected and analyzed using methods known in the art and compared to data measured using the methods and techniques disclosed herein.

[0292] Figures 3A to 3D A two-compartment pharmacokinetic model for the elimination of MB-102 is shown. This model is consistent across patients regardless of their GFR value. Figure 3AFor use in patients with normal renal function, whose measured GFR (mGFR) is 120 mL / min. Figure 3B For patients with an mGFR of 81 mL / min. Figure 3C For patients with an mGFR of 28 mL / min. Figure 3D For patients with an mGFR of 25 mL / min. The first compartment of the two-compartment model represents the equilibrium between blood vessels and tissues, while the second compartment represents renal excretion only. On average, equilibrium time is approximately one hour and varies depending on the subject.

[0293] Figure 4 The use of Comparative data comparing GFR measured using conventional methods with MB-102 using the methods disclosed herein for patients with an eGFR range of about 20 to about 140. The data showed a correlation coefficient of 0.97. This indicates that the methods used herein for determining a patient's GFR provide similar results compared to known methods.

[0294] As part of the MB-102 clinical 1 study, urine was collected from patients for 12 hours to determine the amount recovered and the extent of secondary metabolism. Figure 5 As shown, for patients with eGFR greater than 60, greater than 99% of MB-102 was recovered and unmetabolized after 12 hours. For patients with mGFR values ​​less than 60, a lower percentage of MB-102 was recovered, but it was also unmetabolized. For patients with normal, stage 1, and stage 2 renal function, a 12-hour collection period was sufficient to achieve greater than 99% recovery of MB-102. Incomplete collection is understandable in patients with more severe renal impairment, given the pharmacokinetic data and plasma half-life measurements.

[0295] Figure 6 The plasma concentration half-lives of MB-102 for patients with normal renal function, stage 1 renal impairment, stage 2 renal impairment, stage 3 renal impairment, or stage 4 renal impairment are shown in Table 1. Based on these data, it is clear that in the urine collection studies described above, more than 24 hours were required to clear all of the MB-102 from the patient's bloodstream. The mean plasma half-life in the normal renal function group was 2 hours, so a 12-hour collection time was approximately 6 half-lives and was sufficient time to excrete the majority of the injected dose. The mean plasma half-life in the stage 2 and stage 3 groups was 2.5 hours, resulting in an excretion time of approximately 5 half-lives, which was also sufficient to collect the majority of the injected dose. However, the 4-hour half-life in stage 3 and the 8-hour half-life in stage 4 did not allow the collection of all injected doses within the 12-hour window for clinical studies.

[0296] In clinical studies, plasma pharmacokinetics as a function of time (beginning 2 hours after tracer injection and continuing until the end of the 12-hour study period) correlated with transdermal fluorescence pharmacokinetics measured suprasternally in patients. A high correlation between plasma concentrations and fluorescence intensity was observed for patients across a wide range of GFR values. Figure 7 、 8 Figures 9 and 9 show three individual patients with GFR values ​​of 23 mL / min / 1.73 m 2 (Stage 3b renal injury) to 117 mL / min / 1.73 m 2 (normal renal function). Thus, plasma pharmacokinetics correlated with transdermal fluorescence of MB-102 in patients across a wide GFR range.

[0297] Transdermal GFR measurements (fit to full data set).

[0298] Kinetic analysis was performed using LabView, Matlab, WinNonlin, and Microsoft Excel, version 2010, as described below. Transdermal fluorescence data were fitted to a monoexponential function between 2 hours (relative to the time of injection, to ensure that the tracer had equilibrated across the extracellular space) and the end of the available data (typically approximately 12 hours). For each subject, two fits were performed: (1) with the offset fixed to zero and (2) with the offset allowed to vary. The exponential time constant determined by the fit is called the renal decay time constant (RDTC).

[0299] Linear regression, outlier removal, and correlation coefficient (R) were performed in Microsoft Excel, version 2010. 2 ) and standard error of calibration (SEC) were calculated. The reciprocal of RDTC was correlated with GFR using four different GFR determination methods.

[0300] Unnormalized-GFR as determined from plasma PK analysis of iohexol and MB-102.

[0301] BSA-normalized - Body surface area (BSA) was estimated for each subject using height and weight according to the method of Mosteller (N Engl J Med, 1987; 317(17)). GFR determined by plasma PK analysis was divided by the calculated BSA with 1.73 m 2 ratio (BSA of a “standard” sized patient).

[0302] V d - Normalized (Method 1) - GFR determined by plasma PK analysis divided by volume of distribution (V d ) (also determined by PK analysis) compared to 14,760 mL, a V for a “standard” sized patientd By forcing the mean nGFR of all patients in group 1 to be d and BSA normalization methods are equivalent to determine the V of standard-sized patients. d “nGFR” is used here to refer to the common method (including BSA and V d ), where GFR is normalized to body size.

[0303] V d - Normalization (Method 2) - Monoexponential, with offset fixed at zero, fitted to MB-102 plasma concentration versus time, 2 to 12 hours (relative to the time of tracer injection). The inverse of the fitted time constant was multiplied by 14,760 mL (V for a "standard" sized patient). d ; see above), yielding GFR normalized by volume of distribution.

[0304] Table 2 and Figures 12 to 18 The correlation coefficient (R 2 ) and standard calibration error (SEC). In agreement with the previous study of Rabito (J Nucl Med, 1993; 34 (2): 199-207), the increase in R by normalizing GFR with BSA 2 and reduced SEC, confirming the hypothesis that renal clearance of MB-102 provides a measure of renal efficiency that is independent of body size. In addition, these results suggest that the volume of distribution (V d ) is also effective in normalizing GFR to standard body size. As can be seen from Table 2, BSA and the second V d The body size normalization method was also effective when no outlier rejection method was applied, and the offset of the RDTC fit was fixed to zero.

[0305] Table 2

[0306]

[0307] RDTC fitting offset method

[0308] Transdermal fluorescence dynamics were fitted by two different offset methods: (1) the offset was fixed to zero, and (2) the offset was allowed to vary. Figure 18 and 19 The resulting correlation plots for the fixed and variable offset methods are shown. Note that when the offset is fixed to zero ( Figure 18 ), the data are more tightly clustered in the low GFR region of the correlation plot, whereas when the offset is allowed to vary ( Figure 19 ), the data clusters are tighter in high GFR areas.

[0309] This observation indicates that the baseline fluorescence is unstable. In healthy subjects with high renal clearance constants, the fluorescent agent was cleared before the end of the 12-hour data collection period. In these subjects, it was observed that the final plateau level of fluorescence did not always fully match the baseline fluorescence before the initial injection. Allowing the offset to vary in the fitting of these healthy subjects allows the fitting to compensate for this baseline uncertainty, thereby improving the reliability of the extracted clearance rate constant. However, in many subjects with impaired renal function, the agent was not completely cleared within the 12-hour window in which the measurements were collected. In these subjects, it was found that including a variable offset in the fitting increased the uncertainty of the clearance rate constant of the fitting. By fixing the offset to zero, the reliability of the rate constant was improved.

[0310] Based on these observations, a hybrid offset method was developed. In the hybrid method, if the fit with a fixed offset indicates a high rate constant (i.e., healthy renal function), the offset is allowed to vary; otherwise (i.e., impaired renal function), the offset is fixed. The optimal transition point is chosen so that R 2 Maximize and minimize SEC, such as Figure 25 The transition point shown on the X-axis is expressed as a time constant (in hours), which is the inverse of the clearance rate constant. As can be seen from the figure, the optimal time constant is in the range of 3.5 to 4.5 hours. The correlation diagram using the hybrid offset method (transition point: 3.5 hours) is shown in Figure 20 The comparison of fixed, variable and hybrid offset methods provided in Table 3 shows that the hybrid offset method results in R 2 There was a significant increase in and a decrease in SEC.

[0311] Table 3

[0312]

[0313] Data Exclusion

[0314] For some clinical subjects, the sensor was not fully attached to the skin for the entire 12 hours of the study. Reattachment of the sensor after injection often resulted in significant changes in signal levels. This may be due to: (1) heterogeneity of skin auto-fluorescence, (2) heterogeneity of the interstitial fluid fraction in the skin, or (3) changes in the coupling efficiency of light into and out of the skin. To address this issue in the future, the sensor was redesigned to have a smaller footprint and be closer to the patient. In order to utilize the clinical data, some data exclusions were applied.

[0315] Five of the 60 clinical subjects were completely excluded from the fluorescence kinetic analysis: (1) Subject 1 was excluded because the sensor repeatedly overheated (the maximum allowed blue LED power level was reduced by 60% in all subsequent subjects), (2) Subjects 8, 37, and 49 were excluded because the sensor completely detached from the skin and the original signal level could not be restored by reattachment, and (3) Subject 46 was excluded because a large portion of the data was excluded during the preprocessing step (possibly due to unexpected changes in LED power or detector gain after tracer injection).

[0316] Different metrics that further excluded subject data were tested, including: (1) the correlation coefficient between the IF and the fit; (2) the root mean square error (RMSE) of the IF relative to the fit; (3) the difference between the RDTC determined from one and two exponential fits; (4) the signal-to-noise ratio, calculated as the magnitude of the fitted exponential term divided by the RMSE; (5) the coefficient of variation (CV) of the rate constant when fitted over multiple shorter time periods (e.g., 1-2 hours) within the full 12-hour dataset; (6) the estimated error of the rate constant fitted to the IF; and (7) the estimated error of the GFR determined from the plasma data. None of these metrics included a priori information about the correlation coefficient or SEC.

[0317] In one aspect, using the data exclusion method (6) above, the estimated error in the rate constant fitted to the IF is divided by the rate constant (expressed as relative error) to yield Figure 26 By excluding subjects with relative errors of rate constants greater than 1.4–1.8%, R 2 and SEC. 1.75% was selected as the cutoff metric, excluding 10 of the 55 subjects. The resulting correlation plot is shown in Figure 21 and 22 middle.

[0318] This approach was further used as an exclusion metric to test whether errors in the plasma-derived GFR determination also contribute to the data scatter in the correlation plots. Figure 27 An optimization of this metric is shown. By choosing 5% as the cutoff for GFR acceptability, it was observed that R 2 and SEC were slightly improved. However, this required the exclusion of an additional 10 subjects, reducing the remaining subjects to 35. The resulting correlation plot is shown in Figure 23 and 24 A numerical summary of the application of different data exclusion metrics is provided in Table 4.

[0319] Table 4

[0320]

[0321] The calibration slope determined by the method described above can be used to generate a transcutaneous measurement of body size corrected GFR (referred to herein as "tGFR"). Figure 10 The correlation between predicted GFR values ​​and plasma GFR values ​​with BSA normalization is shown. Calibration accuracy can be depicted by plotting tGFR against a "gold standard" determination of body size corrected GFR obtained from plasma PK analysis. Figure 10 The correlation coefficient (R) is shown when the plasma GFR values ​​are normalized to the patient's body surface area (BSA). 2 ) is 0.89. Using the distribution volume (V d ) to correct for body size rather than BSA resulted in a significantly improved correlation of 0.96 ( Figure 11 ).

[0322] These tGFR results show the potential for significantly improved accuracy when compared with eGFR, the most commonly used method for estimating GFR in clinical practice. High accuracy is important for guiding clinical decision-making. Figure 28 Table 1 illustrates the five stages of chronic kidney disease (CKD). Misdiagnosis of CKD may affect the clinical treatment process. An error grid plot was constructed to provide a visualization of CKD misdiagnosis relative to the gold standard GFR measurement, as shown in Figure 1. Figures 29A-29C As shown. Measurements falling within the box with all green boundaries are correctly classified by CKD stage. Measurements contained within the yellow and green boundaries are misclassified as one CKD stage. Measurements within the red and yellow boundaries are misclassified as two CKD stages. Figure 29A The eGFR error grid for the same group of subjects used in the above analysis is shown. In this case, 2 of the 60 subjects were misdiagnosed as CKD stage 2, 16 subjects were misdiagnosed as stage 1, and 41 subjects were correctly diagnosed. Table 5 provides a summary of CKD diagnostic errors expressed as a percentage of the total measurements. Figure 29B and 29C The error grid for transdermal GFR (tGFR) measurements is provided. Importantly, the tGFR error grid does not include misdiagnosis of 2 CKD stages. In addition, when compared with eGFR, the V d The tGFR normalized method showed a significant reduction in stage 1 misdiagnosis (see Table 5).

[0323] Table 5

[0324]

[0325] Transdermal GFR measurement (window fitting)

[0326] In the above examples, GFR was determined using the complete available data set (i.e., 10 hours of fluorescence decay after tracer administration and equilibrium). This may be suitable for patients with stable renal function, but for patients with acute kidney injury or at risk of acute kidney injury, a more rapid and real-time repeated assessment of GFR trends is required. Even for patients with stable renal function, waiting 12 hours for GFR determination may be inconvenient. Tables 6 and 7 show the results of varying the measurement time window and single injection reporting period for the same group of subjects as described above.

[0327] The measurement time windows (column 1 in Tables 6 and 7) did not overlap in this example, so the number of windows (column 2 in Tables 6 and 7) multiplied by the measurement time window represents the total number of hours after equilibrium during which GFR was estimated. The best results were obtained when the measurement time window was long enough to allow the fluorescence intensity to decay significantly. The time required to achieve the same fractional fluorescence intensity decay will vary depending on the health of the kidneys. Therefore, the offset was fixed to zero except for the widest measurement time window for subjects with healthy kidneys. The standard calibration error is summarized for all subjects (column 5 in Tables 6 and 7), as well as for the subgroups of subjects with nGFR below or above 75 mL / min.

[0328] Using BSA normalized GFR as a reference comparison (Table 6), for subjects with nGFR above 75, a measurement time window of at least about 1.5 hours was used to achieve an nGFR calibration accuracy of less than 15 mL / min. For subjects with nGFR below 75, a measurement time window of at least about 3 hours was used to achieve an nGFR calibration accuracy of less than 10 mL / min. d Normalized GFR was used as a reference for comparison (Table 7), and equivalent nGFR calibration accuracy was achieved using measurement time windows of 0.5 hours and 2 hours, respectively. As can be seen in Tables 6 and 7, the above calibration accuracy targets remained unchanged for at least 2 non-overlapping measurement time windows. However, a significant increase in SEC was generally observed when the prediction was extended across 3 or 4 measurement time windows. By increasing the measurement window time from that used for the first two GFR measurements (e.g., doubling or tripling), at least the third GFR measurement provided equivalent accuracy. These results show the utility of providing an automatically adjusted measurement time window to account not only for different SNRs in different patients, but also for the decreasing SNR over time as the tracer is gradually cleared by the kidneys.

[0329] Table 6.

[0330]

[0331]

[0332] Table 7.

[0333]

[0334] Real-time transdermal GFR measurement

[0335] The method disclosed herein enables real-time transdermal GFR determination in patients. After the tracer is injected intravascularly into the subject, a two-hour waiting time is used to allow the tracer to equilibrate to the extravascular space. After two hours, the labeled data are accumulated for a further hour before a first fit of the RDTC is performed. The first RDTC fit is performed with the offset term fixed to zero. If the RDTC is less than 3.5 hours, the fit is repeated, allowing the slope to vary, otherwise the original RDTC (with fixed offset) is retained. The estimated error in the RDTC is then divided by the RDTC. If the relative error obtained is less than 1.7%, the error is corrected by inverting the RDTC and multiplying by Figure 23 The slope shown in is used to convert RDTC to BSA-normalized GFR; otherwise, GFR is not reported. After the first RDTC fit, the fitting procedure is repeated at intervals of approximately 3 seconds, with subsequent fits incorporating all data from the first fit as well as all data accumulated subsequently (at intervals of approximately one second). The same procedure is applied to data collected by two sensors: one placed on the manubrium of the malleus of the sternum and the second placed on the pectoralis major muscle. Results generated in real time during the measurement are shown in Figure 2. Figure 30 Throughout the study, the agreement between the tGFR reported by the two sensors and the change in tGFR reported by each sensor was within 2 mL / min / 1.73 m 2 Inside.

[0336] Stability test of MB-102

[0337] MB-102 samples were prepared and stored at 25°C and 60% relative humidity for 24 months. HPLC analysis of each sample was performed at various time points to assess the stability of the sample. The results are shown in Table 8.

[0338] Table 8.

[0339]

[0340] When introducing elements of the present disclosure or embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0341] Although described in conjunction with an exemplary computing system environment, embodiments of the invention are operational with numerous other general purpose or special purpose computing system environments or configurations. The computing system environment is not intended to suggest any limitation as to the scope of use or functionality of any aspect of the invention.

[0342] Embodiments of the present invention can be described in the general context of computer-executable instructions (e.g., program modules) executed by one or more computers or other devices. Computer-executable instructions can be organized into one or more computer-executable components or modules. Typically, program modules include, but are not limited to, routines, programs, objects, components, and data structures that perform specific tasks or implement specific abstract data types. Any number and organization of such components or modules can be used to implement various aspects of the present invention. For example, various aspects of the present invention are not limited to the specific computer-executable instructions or specific components or modules shown in the accompanying drawings and described herein. Other embodiments of the present invention may include different computer-executable instructions or components with more or less functionality than shown and described herein. Various aspects of the present invention can also be implemented in a distributed computing environment, where tasks are performed by remote processing devices linked by a communication network. In a distributed computing environment, program modules can be located in local and remote computer storage media including memory storage devices.

[0343] In view of the foregoing, it will be seen that the several advantages of the present disclosure are achieved and other advantageous results obtained. As various changes could be made in the above-described processes and composite materials without departing from the scope of the present disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

1. A system for determining real-time glomerular filtration rate (GFR) in a patient in need thereof, the system comprising: computing devices, a display device communicatively coupled to the computing device, a power supply operatively coupled to the computing device to maintain electrical isolation of the system from an external power source, one or more sensor heads operably coupled to the patient's skin and operably coupled to the computing device, and at least one tracer configured to be administered to the patient and to emit spectral energy when exposed to electromagnetic radiation, wherein the spectral energy is detectable by the one or more sensor heads; Wherein, the computing device is configured as follows: operating and controlling the sensor head, recording one or more measurements sent from the sensor head, and calculating the patient's GFR based on the measured values; wherein the one or more sensor heads include at least one electromagnetic radiation source and are configured to generate and deliver electromagnetic radiation, detecting and measuring the spectral energy emitted by the tracer, and sending the measurements emitted by the tracer to the computing device; and wherein the computing device determines the patient's GFR by correlating a decrease in light emitted by the tracer over a measurement time window to the patient's GFR and by calculating a decay parameter correlated to renal clearance over the measurement time window; wherein the decay parameter is a rate constant directly related to GFR normalized to the patient's body size; wherein the rate constant is determined by fitting an exponential function to the spectral energy as a function of time or fitting a linear function to the logarithm of the spectral energy as a function of time; wherein fitting of the data is initiated after the tracer is equilibrated between the vascular and extravascular space; wherein a quality metric is used to evaluate whether the attenuation parameter is sufficiently accurate, and if the attenuation parameter is not sufficiently accurate, increasing the measurement time window until the quality metric evaluation indicates sufficient accuracy; and The body mass index used to normalize GFR is either body surface area or the volume of distribution of the tracer.

2. The system of claim 1, wherein the tracer is a compound of formula I in, X 1 and X 2 Each independently is -CO2R 1 、-CONR 1 R 2 , -CO(AA) or -CONH(PS); Y 1 and Y 2 Each independently selected from -NR 1 R 2 and Z 1 For single bond, -CR 1 R 2 -、-O-、-NR 1 -、-NCOR 1 -, -S-, -SO- or -SO2-; R 1 to R 2 Each independently selected from H, -CH2(CHOH) a H, -CH2(CHOH) a CH3, -CH2(CHOH) a CO2H, -(CHCO2H) a CO2H, -(CH2CH2O) c H, -(CH2CH2O) c CH3, -(CH2) a SO3H, -(CH2) a SO3 - 、-(CH2) a SO2H, -(CH2) a SO2 - 、-(CH2) a NHSO3H, -(CH2) a NHSO3 - 、-(CH2) a NHSO2H, -(CH2) a NHSO2 - ,-(CH2) a PO4H3, -(CH2) a PO4H2 - 、-(CH2) a PO4H 2- 、-(CH2) a PO4 3- 、-(CH2) a PO3H2, -(CH2) a PO3H - and -(CH2) a PO3 2- ; AA is a peptide chain comprising one or more amino acids selected from natural amino acids and unnatural amino acids, said one or more amino acids being linked together by peptide or amide bonds, and each instance of AA may be the same as or different from every other instance; PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units linked by glycosidic bonds; and "a" is a number from 1 to 10, "c" is a number from 1 to 100, and each of "m" and "n" is independently a number from 1 to 3.

3. The system of claim 1, wherein the tracer is or a pharmaceutically acceptable salt thereof.

4. A system as described in claim 1, wherein the electromagnetic radiation source and / or the amplification of the detected spectral energy is adjusted to account for inter-patient variations in skin melanin content or other tissue optical properties.

5. The system of claim 1, wherein the tracer is eliminated solely by glomerular filtration in the patient's kidneys.

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