Compositions and systems for measuring renal function

Abstract

To provide a system that measures a glomerular filtration rate (GFR).SOLUTION: This system includes a computer device, a power source, one or more sensors, and at least one tracer agent that emits fluorescence when being exposed to electromagnetic radiation. A sensor is used to detect the electromagnetic radiation, and a reduction speed of fluorescence in the body of a patient is used to calculate a glomerular filtration rate of the patient.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The field of the disclosure relates generally to methods and pharmaceutical compositions comprising pyrazine derivatives for assessing renal function in a patient. [Background technology]

[0002] Acute renal failure (ARF) is a common disease in hospitalized patients in general medical-surgical hospitals. Approximately half of patients who develop ARF die directly from ARF or from complications related to the underlying disease, and survivors face significantly increased mortality and prolonged hospitalization. Early diagnosis is generally considered important because renal failure is often asymptomatic and generally requires careful tracking of renal function markers in the blood. Dynamic monitoring of patients' renal function is desirable to minimize the risk of acute renal failure brought about by various clinical, physiological, and pathological conditions. Such dynamic monitoring tends to be particularly important in the case of critically ill patients, since the majority of critically ill patients tend to face the risk of multiple organ failure (MOF), which may lead to death. MOF is the sequential failure of the lungs, liver, and kidneys, which is triggered by one or more of acute lung injury (ALI), adult respiratory distress syndrome (ARDS), hypermetabolism, hypotension, persistent inflammatory focus, and septic syndrome. Common histological features of hypotension and shock leading to MOF generally include tissue necrosis, vascular congestion, interstitial or cellular edema, hemorrhage, and microthrombi. These changes generally affect, in order of decreasing frequency, the lungs, liver, kidneys, intestines, adrenal glands, brain, and pancreas. The transition from the early stages of trauma to clinical MOF generally corresponds to a certain degree of hepatic and renal failure, and an approximately 30% to 50% change in risk of death.

[0003] Traditionally, a patient's renal function has been determined using crude measurements of the patient's urine volume and plasma creatinine concentration. These values ​​are often misleading because they are influenced by age, hydration status, renal perfusion, muscle mass, dietary intake, and a variety of other clinical and anthropometric variables. In addition, a single value obtained several hours after sample collection is 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.).

[0004] Chronic kidney disease (CKD) is a disease characterized by the gradual loss of kidney function over time. CKD includes diseases that damage the kidneys and reduce their ability to properly remove waste products from the patient's bloodstream. Complications of CKD include high blood pressure, anemia (low red blood cell count), weak bones, malnutrition, nerve damage, and increased risk of heart disease. According to the National Kidney Foundation, approximately two-thirds of all cases of CKD are due to diabetes or high blood pressure. In addition to family history, other risk factors for kidney disease include age, ethnicity, high blood pressure, and diabetes. Glomerular filtration rate (GFR) is the best criterion for determining the level of kidney function and assessing the stage of CKD in patients.

[0005] GFR is an important endpoint for determining the level of renal function that determines the stage of CKD. The lower the GFR, the more severe the CKD, as shown in Table 1 and Figure 28. GFR can be estimated based on a blood test that measures the blood creatinine concentration in combination with other factors. A more accurate, and therefore more useful, method involves injecting the patient with an endogenous substance and then closely monitoring the urine output over a period of time. This is often done using a contrast agent (CA), which can cause kidney damage on its own. Radioisotopes or iodized aromatics are the two common categories of CAs used to measure GFR.

[0006] [Table 1]

[0007] Contrast-induced nephropathy (CIN) is a serious complication associated with the use of radioisotopes or iodized CAs. CIN is thought to result from renal vasoconstriction or tubular injury caused by CAs. The definition of CIN varies from study to study, but the most common definition based on patient symptoms includes an increase in serum creatinine of at least 25% from baseline 2–5 days after exposure to CAs in the absence of other causes of acute renal failure. CIN can be fatal in up to 20% of hospitalized patients with these complications. The more severe the CKD, the higher the risk of CIN. Thus, patients who most need accurate GFR data are at highest risk of developing complications, sometimes fatal, when obtaining that data.

[0008] With regard to conventional renal monitoring methods, an approximation of a patient's glomerular filtration rate (GFR) can be obtained using a 24-hour urine collection method, which (as the name suggests) typically requires approximately 24 hours of urine collection, several more hours of analysis, and meticulous bedside collection techniques. Unfortunately, the undesirably slow timing and significantly longer duration of this conventional method can reduce the chances of effectively treating the patient and / or saving the kidney. A further drawback of this type of method is that repeat data tends to be as laborious to obtain as the data obtained initially.

[0009] Also, sometimes it is necessary to adjust the change in a patient's serum creatinine based on measurements such as the patient's urine electrolytes and osmolality, and calculations such as the "renal insufficiency index" and / or "fractional excretion of sodium". Such adjustments of serum creatinine tend to disadvantageously require the simultaneous collection of additional serum and / or urine samples, and further calculations after some delay. Often, drug dosing is adjusted to renal function and is as inaccurate, time-consuming, and difficult to reassess as the measurements and calculations on which it is based. Finally, clinical decision-making in critically ill patient populations is often as much about timing as it is about precision.

[0010] It is known that hydrophilic anionic substances can generally be excreted by the kidney. Renal clearance generally occurs through two routes: glomerular filtration and tubular secretion. Tubular secretion can be characterized as an active transport process, and therefore substances that are eliminated through this route generally exhibit specific properties in terms of size, charge and lipophilicity.

[0011] Most of the substances passing through the kidney are filtered in the glomerulus, a group of tiny intertwining capillaries in the kidney's Malpighian corpuscles. Examples of exogenous substances that can be excreted from the kidney via glomerular filtration (hereafter referred to as "GFR substances") are shown in Figure 1. Examples of GFR substances include creatinine (1), o-iodohippuran (2), and 99mTc-DTPA (3). Examples of exogenous substances that can be cleared by the kidney through tubular secretion include 99mTc-MAG3 (4) and other substances known in the art. 99mTc-MAG3 (4) is also widely used to assess renal function by gamma scintigraphy or renal blood flow measurements. One drawback of the substances shown in Figure 1 is that o-iodohippuran (2), 99mTc-DTPA (3), and 99mTc-MAG3 (4) contain radioisotopes to allow for detection of those substances. Even if radioactive analogues (e.g., analogues of o-iodohippuran (2)) or other non-radioactive substances were used for renal function monitoring, such monitoring would generally require the use of undesirable ultraviolet radiation to excite these substances.

[0012] Pyrazine derivatives for use in renal monitoring are known in the art, including those disclosed in US Pat. Nos. 5,993,343; 5,993,635; 5,993,725; 5,993,645; and 6,103,663, the contents of which are incorporated herein by reference in their entireties. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 8,155,000 [Patent Document 2] U.S. Pat. No. 8,664,392 [Patent Document 3] U.S. Patent No. 8,697,033 [Patent Document 4] U.S. Pat. No. 8,722,685 [Patent Document 5] U.S. Pat. No. 8,778,309 [Patent Document 6] U.S. Patent No. 9,005,581 [Patent Document 7] U.S. Pat. No. 9,114,160 [Patent Document 8] U.S. Pat. No. 9,283,288 [Patent Document 9] U.S. Pat. No. 9,376,399 [Patent Document 10] U.S. Pat. No. 9,480,687 Summary of the Invention [Means for solving the problem]

[0014] In one aspect, disclosed herein is a compound represented by Formula I:

[0015] [ka]

[0016] During the ceremony, X 1 and X 2 are, independently of each other, -CO2R 1 , -CONR 1 R 2 , -CO(AA), and -CONH(PS); Y 1 and Y 2 are, independently of each other, NR 1 R 2 , and

[0017] [ka]

[0018] is selected from the group consisting of Z 1 is a single bond, -CR 1 R 2 -, -O-, -NR 1 -,-NCOR 1 selected from the group consisting of -, -S-, -SO-, and -SO2-; R 1 and R 2 are, independently of each other, H, -CH2(CHOH)aH, -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- is selected from the group consisting of AA is a polypeptide chain comprising one or more natural or non-natural amino acids linked together by peptide or amide bonds, each of the AAs may be identical or different from the others; PS is a sulfurized or non-sulfurized polysaccharide chain containing one or more monosaccharide units linked together by glycosidic bonds; a is a number ranging from 1 to 10; c is a number ranging from 1 to 100; m and n are each independently a number ranging from 1 to 3.

[0019] In another aspect, disclosed herein is a system for measuring a glomerular filtration rate (GFR) of a patient in need of such measurement. The system includes a computer device, a display device communicatively connected to the computer device, a power source operatively connected to the computer device to maintain electrical isolation of the system from an external power source, one or more sensor heads operatively connected to the computer device, and at least one tracer agent configured to emit spectral energy when exposed to electromagnetic radiation. The computer device is configured to operate and control the sensor heads, record one or more measurements received from the sensor heads, and calculate the GFR of the patient based on the measurements. The one or more sensor heads include at least one electromagnetic radiation source and are configured to generate and emit electromagnetic radiation, detect and measure the spectral energy emitted from the tracer agent, and transmit measurements of the spectral energy emitted from the tracer agent to the computer device. The tracer agent is administered to the patient and configured to emit spectral energy detectable by the sensor heads when exposed to electromagnetic radiation.

[0020] In yet another aspect, disclosed herein is a system for transcutaneously measuring a patient's glomerular filtration rate (GFR) normalized for body size, comprising a computer device, a display device communicatively connected to the computer device, a power source operatively connected to the computer device to maintain electrical isolation of the system from an external power source, one or more sensor heads operatively connected to the computer device, and at least one tracer agent 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 emit electromagnetic radiation, detect and measure the spectral energy emitted from the tracer agent, and transmit measurements of the spectral energy emitted from the tracer agent to the computer device. The tracer agent is administered to the patient and configured to emit spectral energy detectable by the sensor head when exposed to electromagnetic radiation. The computer device is configured to operate and control the sensor head, record one or more measurements received from the sensor head, determine an attenuation parameter from the spectral energy measured over the measurement time window, determine a quality metric associated with the spectral energy measured over the measurement time window, evaluate whether the attenuation parameter is sufficiently accurate using the quality metric, and if the attenuation parameter is assessed to be not sufficiently accurate, extend the length of the measurement time window until the quality metric is assessed to be sufficiently accurate, convert the attenuation parameter into a GFR measurement corrected for the patient's body size or a GFR measurement normalized for the volume of distribution of the tracer agent, and report the result on a display device.

[0021] In yet another aspect, disclosed herein is a method for measuring glomerular filtration rate (GFR) in a patient in need thereof, comprising administering to the patient a compound represented by formula I, or a pharma- ceutically acceptable salt thereof, measuring the concentration of the compound of formula I in the patient's body over a measurement time window, and determining the patient's GFR. [ka] During the ceremony, X 1 and X 2 are, independently of each other, -CO2R 1 , -CONR 1 R 2 , -CO(AA), and -CONH(PS); Y 1 and Y 2 are, independently of each other, NR 1 R 2 , and [ka] is selected from the group consisting of Z 1 is a single bond, -CR 1 R 2 -, -O-, -NR 1 -,-NCOR 1 selected from the group consisting of -, -S-, -SO-, and -SO2-; R 1 and R 2 are, independently of each other, H, -CH2(CHOH)aH, -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- is selected from the group consisting of AA is a polypeptide chain comprising one or more natural or non-natural amino acids linked together by peptide or amide bonds, each of the AAs may be identical to or different from the others; PS is a sulfurized or non-sulfurized polysaccharide chain containing one or more monosaccharide units linked together by glycosidic bonds; a is a number ranging from 1 to 10; c is a number ranging from 1 to 100; m and n are each independently a number ranging from 1 to 3.

[0022] In yet another aspect, disclosed herein is a method of assessing renal function in a patient, the method including administering to the patient a fluorescent compound or a pharma- ceutically acceptable salt thereof, exposing the patient to electromagnetic radiation causing emission of spectral energy from the fluorescent compound, detecting the emitted spectral energy from the fluorescent compound, and assessing the renal function of the patient based on the detected spectral energy. [Brief description of the drawings]

[0023] [Figure 1]FIG. 1 illustrates some known imaging agents for renal function monitoring. [Diagram 2] FIG. 1 is a diagram of a system for monitoring the GFR of a patient. [Figure 3A] 1 is a graph of MB-102 clearance showing a two-compartment pharmacokinetic model in a first patient with a GFR value of 120 mL / min. [Figure 3B] 1 is a graph of MB-102 clearance showing a two-compartment pharmacokinetic model in a second patient with a GFR value of 81 mL / min. [Figure 3C] 13 is a graph of MB-102 clearance showing a two-compartment pharmacokinetic model in a third patient with a GFR value of 28 mL / min. [Figure 3D] 13 is a graph of MB-102 clearance showing a two-compartment pharmacokinetic model in a fourth patient with a GFR value of 25 mL / min. [Figure 4] 1 is a graph comparing GFR measured using MB-102 with Omnipaque®. [Diagram 5] 1 is a bar graph of the recovery of MB-102 from the urine of human patients after 12 hours. [Figure 6] 1 is a bar graph of plasma half-life of MB-102 in human patients. [Figure 7] FIG. 1 is a graph showing the correlation between plasma concentrations of MB-102 and transcutaneous fluorescence intensity over time in a patient with a GFR of 117 mL / min / 1.73 m2. [Figure 8] FIG. 1 is a graph showing the correlation between plasma concentration of MB-102 and transcutaneous fluorescence intensity over time in a patient with a GFR of 61 mL / min / 1.73 m2. [Figure 9] FIG. 1 is a graph showing the correlation between plasma concentration of MB-102 and transcutaneous fluorescence intensity over time in a patient with a GFR of 23 mL / min / 1.73 m2. [Figure 10]Graph showing correlation between transcutaneously predicted GFR and plasma measured GFR measured using MB-102 and normalized to subject's body surface area (outliers removal method 1; hybrid offset method). [Figure 11] Graph showing correlation between transcutaneously predicted GFR and plasma measured GFR measured using MB-102 and normalized for the volume of distribution of the tracer agent in the subject's body (outlier removal method 1; hybrid offset method). [Figure 12] FIG. 1 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by iohexol, non-normalized (no outlier removal; fixed offset fitting method). [Figure 13] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR with iohexol and normalization with BSA (no outliers removal; fixed offset fitting method). [Figure 14] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by iohexol, normalized by Vd (method 1) (no outlier exclusion; fixed offset fitting method). [Figure 15] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by MB-102, non-normalized (no outlier removal; fixed offset fitting method). [Figure 16] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by MB-102, normalized by BSA (no outliers removal; fixed offset fitting method). [Figure 17] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by MB-102, normalized by Vd (method 1) (no outliers exclusion; fixed offset fitting method). [Figure 18]FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate, ie, GFR by MB-102, normalized by Vd (method 2) (no outliers exclusion; fixed offset fitting method). [Figure 19] Graph of plasma measured GFR correlated to transcutaneous fluorescence clearance rate: variable offset method (no outlier removal; MB-102 GFR measurements normalized by Vd (method 2)). [Figure 20] Graph of plasma measured GFR correlated to transcutaneous fluorescence clearance rate: Hybrid offset method (no outlier removal; MB-102 GFR measurements normalized by Vd (method 2)). [Figure 21] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate: outlier removal method 1 (hybrid offset method; MB-102 GFR measurements normalized to BSA). [Figure 22] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate: outlier removal method 1 (hybrid offset method; MB-102 GFR measurements normalized for Vd). [Figure 23] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate: outlier removal method 2 (hybrid offset method; MB-102 GFR measurements normalized to BSA). [Figure 24] FIG. 13 is a graph correlating plasma measured GFR with transcutaneous fluorescence clearance rate: outliers removal method 2 (hybrid offset method; MB-102 GFR measurements normalized by Vd (method 2)). [Diagram 25] 13 is a graph summarizing the optimization of RDTC transitions to determine the offset method used to fit the RDTC. [Figure 26] 1 is a graph summarizing the optimization of the outlier error threshold for fluorescence decay rate constants. [Figure 27] 1 is a graph summarizing the optimization of outlier error thresholds for plasma measured GFR. [Figure 28]A graph showing the five stages of chronic kidney disease based on GFR. [Figure 29A] Graph of eGFR versus plasma PK-measured GFR (nGFR) normalized for subject's body surface area. An error grid showing diagnostic accuracy is overlaid on the graph with CKD stage number. Measurements that fall within only the green side grid are correctly diagnosed by eGFR. Measurements that fall within both the green and yellow side grids are misdiagnosed by eGFR by one CKD stage. Measurements that fall within both the yellow and red side grids are misdiagnosed by eGFR by two CKD stages. [Figure 29B] Graph of transcutaneously measured GFR (tGFR) versus plasma PK measured GFR (nGFR) normalized for subject's body surface area. An error grid showing diagnostic accuracy is overlaid on the graph with CKD stage number. Measurements that fall within only the green side grid are correctly diagnosed by tGFR. Measurements that fall within both the green and yellow side grids are misdiagnosed by tGFR by one CKD stage. Measurements that fall within both the yellow and red side grids are misdiagnosed by tGFR by two CKD stages. [Figure 29C] Graph of transcutaneously measured GFR (tGFR) versus plasma PK measured GFR (nGFR) normalized for the volume of distribution of the tracer agent in the subject's body. An error grid showing diagnostic accuracy is overlaid on the graph with the CKD stage number. Measurements that fall within only the green side grid are correctly diagnosed by tGFR. Measurements that fall within both the green and yellow side grids are misdiagnosed by tGFR by one CKD stage. Measurements that fall within both the yellow and red side grids are misdiagnosed by tGFR by two CKD stages. [Diagram 30] This is a graph of transcutaneous GFR measured automatically at two body sites in real time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Any reference herein to "pyrazine", "pyrazine derivative", "pyrazine molecule", "pyrazine compound" or "pyrazine analog" applies to any compound of formula I. Additionally, any reference to pyrazine further includes any pharma- ceutically acceptable salt thereof, unless otherwise specified. The salt forms may be charged or uncharged, and may be protonated to form the appropriate cation or deprotonated to form the appropriate anion. All aspects and embodiments disclosed herein are applicable to compounds of formula I, with specific examples being merely illustrative and not limiting the scope of the disclosure.

[0025] In one aspect, disclosed herein is a compound represented by Formula I, or a pharma- ceutically acceptable salt thereof:

[0026] [ka]

[0027] During the ceremony, X 1 and X 2 are, independently of each other, -CO2R 1 , -CONR 1 R 2 , -CO(AA), and -CONH(PS); Y 1 and Y 2 are, independently of each other, NR 1 R 2 , and

[0028] [ka]

[0029] is selected from the group consisting of Z 1 is a single bond, -CR 1 R 2 -, -O-, -NR 1 -,-NCOR1 selected from the group consisting of -, -S-, -SO-, and -SO2-; R 1 and R 2 are, independently of each other, H, -CH2(CHOH)aH, -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- is selected from the group consisting of AA is a polypeptide chain comprising one or more natural or non-natural amino acids linked together by peptide or amide bonds, each of the AAs may be identical or different from the others; PS is a sulfurized or non-sulfurized polysaccharide chain containing one or more monosaccharide units linked together by glycosidic bonds; a is a number ranging from 0 to 10, c is a number ranging from 1 to 100; m and n are each independently a number ranging from 1 to 3.

[0030] In another embodiment, a is a number ranging from 1 to 10. In yet another embodiment, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0031] In some embodiments, at least one of X1 and X2 is -CO(PS) or -CO(AA). In yet other embodiments, both X1 and X2 are -CO(AA).

[0032] (AA) is a peptide chain comprising one or more natural or unnatural amino acids linked together by peptide or amide bonds. The peptide chain (AA) can be a single amino acid, a homopolypeptide chain, or a heteropolypeptide chain and can have any suitable length. In some embodiments, the natural or unnatural amino acid is an α-amino acid. In yet another aspect, the α-amino acid is a D-α-amino acid or an L-α-amino acid. The polypeptide chain comprises two or more amino acids, and in all aspects, each amino acid is selected independently from each other, including but not limited to, side chain structures and stereochemical structures. For example, in some embodiments, the polypeptide chain can comprise 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, or 1-10 amino acids. In some embodiments, the polypeptide chain can comprise 1-100 α-amino acids, 1-90 α-amino acids, 1-80 α-amino acids, 1-70 α-amino acids, 1-60 α-amino acids, 1-50 α-amino acids, 1-40 α-amino acids, 1-30 α-amino acids, 1-20 α-amino acids, or 1-10 α-amino acids. In some embodiments, the amino acids are selected from the group consisting of 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 acids of the polypeptide chain (AA) are selected from the group consisting of arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, homoserine, lysine, and serine. In some embodiments, the α-amino acids of the polypeptide chain (AA) are selected from the group consisting of aspartic acid, glutamic acid, homoserine, and serine.In some embodiments, a polypeptide chain (AA) refers to a single amino acid (eg, D-aspartic acid or D-serine).

[0033] In some embodiments, (AA) is a single amino acid selected from the group consisting of the 21 essential amino acids. In another aspect, (AA) is selected from the group consisting of 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.

[0034] 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-aminobutanoic 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-biphenylyl)butyric acid, cis-3-amino-cyclohexanecarboxylic acid, trans-3-amino-cyclohexanecarboxylic acid, ... Pentanecarboxylic 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 Ano-β-Phe-OH, 3,4-dimethoxy-β-Phe-OH, γ,γ-diphenyl-β-homo ala-OH, 4-fluoro-β-Phe-OH, β-Gln-OH, β-homo ala-OH, β-homo arg-OH, β-homo gln-OH, β-homo glu-OH, β-homo hyp-OH, β-homo leu-OH, β-homo lys-OH, β-homo met-OH, β2-homo phenylalanine, β-homo phe-OH, β3-homo pro-OH, β-homo ser-OH, β-homo thr-OH, β-homo trp-OH, β-homo trp-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, 3-(benzylamino)ethyl propionate, β-Ala-OH, 3-(amino)-5-hexenoic acid, 3-(amino)-2-methylpropionic acid, 3-(amino)-2-methyl 3-(amino)-4-(2-naphthyl)butyric acid, 3,4-difluoro-β-homophe-OH, γ,γ-diphenyl-β-homoala-OH, 4-fluoro-β-homophe-OH, β-Gln-OH, β-homoala-OH, β-homoarg-OH, β-homogln-OH, β-homoglu-OH, β-homohyp-OH, β-homoile-OH, β-homoleu-OH, β-homol ys-OH, β-homo met-OH, β-homo phe-OH, β3-homo proline, β-homo thr-OH, β-homo trp-OH, β-homo tyr-OH, β-Leu-OH, 2-methyl-β-homo phe-OH, 3-methyl-β-homo phe-OH, β-Phe-OH, 4-(3-pyridyl)-β-homo ala-OH, 3-(trifluoromethyl)-β-homo phe-OH, β-glutamic acid, β-homo alanine, β-homoglutamine, β-homoglutamine, β-homo hydroxyproline, β-homoisoleucine, β-homoleucine, β-homo methionine, β-homo phenylalanine, β-homo proline, β-homoserine, β-homo threonine, β-homo tryptophan, β-homo tyrosine, β-leucine, β-phenylalanine, pyrrolidine-3-carboxylic acid, and β-Dab-OH.

[0035] (PS) is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units linked together by glycosidic bonds. The polysaccharide chain (PS) may have any suitable length. For example, in some embodiments, the polysaccharide chain may comprise 1-100 monosaccharide units, 1-90 monosaccharide units, 1-80 monosaccharide units, 1-70 monosaccharide units, 1-60 monosaccharide units, 1-50 monosaccharide units, 1-40 monosaccharide units, 1-30 monosaccharide units, 1-20 monosaccharide units, or even 1-10 monosaccharide units. In some embodiments, the polysaccharide chain (PS) is a homopolysaccharide chain consisting of pentose or hexose monosaccharide units. In another embodiment, the polysaccharide chain (PS) is a heteropolysaccharide chain consisting of one or both of pentose and hexose monosaccharide units. In some embodiments, the monosaccharide unit (PS) of the polysaccharide chain is selected from the group consisting of 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 yet another aspect, the polysaccharide chain is an amino sugar in which one or more hydroxy groups on the sugar are replaced with amine groups. The bond to the carbonyl group can be made through the amine or hydroxy group.

[0036] In some embodiments, in the pyrazine derivatives of formula I, Y 1 and Y 2 At least one of

[0037] [ka]

[0038] It is. Z 1 is a single bond, -CR 1 R 2 -, -O-, -NR 1 -,-NCOR 1 selected from the group consisting of -, -S-, -SO-, and -SO2-; R 1 and R 2are, independently of each other, H, -CH2(CHOH)aH, -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- and a, c, m and n are as defined above.

[0039] In yet another embodiment, Y 1 and Y 2 At least one of, and R 1 and R 2 is as described above. In yet another embodiment, Y 1 and Y 2 Both, as well as R 1 and R 2 is as above. Alternatively, R 1 and R 2 are, independently of each other, H, -CH2(CHOH) a CH3, -(CH2) a SO3H, -(CH2 ) a In yet another embodiment, R is selected from the group consisting of NHSOH, and -(CH)POH. 1 and R 2 are both hydrogen.

[0040] In any embodiment of the pyrazine compounds, one or more atoms may be alternatively replaced with an isotopically labeled atom of the same element. For example, a hydrogen atom may be isotopically labeled with deuterium or tritium, a carbon atom may be isotopically labeled with 13 C or 14 Nitrogen atoms can be isotopically labeled with C, 14 N or 15 Pyrazine molecules can be isotopically labeled with N. Isotopic labels can be stable or unstable (i.e., radioactive). Pyrazine molecules can contain one or more isotopic labels. Isotopic labels can be partial or total. For example, pyrazine molecules can be labeled with 50% deuterium to provide them with a characteristic that can be easily monitored by mass spectrometry or other techniques. As another example, pyrazine molecules can be labeled with tritium to provide them with a radioactive signature that can be monitored both in vivo and ex vivo using techniques known in the art.

[0041] Pharmaceutically acceptable salts are known in the art. In any embodiment herein, the pyrazine may be in the form of a pharma-ceutically acceptable salt. Non-limiting examples of pharma-ceutically acceptable salts include those described in J.Pharm.Sci., 66(1), 1 (1977) by Berge et al., the teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts may be cationic or anionic.In some embodiments, the counterion of the pharma- ceutically acceptable salt is selected from the group consisting of acetate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, esterate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamate, salt, and the like. Acid salts, hydroxynaphthoate, iodide, isothioate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, acetate, succinate, sulfate, tannate, tartrate, theoclate, triethiodide, adipate salt, alginate, aminosalicylic acid, anhydromethylene citrate, arecoline, aspartic acid, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, jemisulfate, judrofluoride, judroiodide, methylene bis(salicylate), napadisilate, oxalate, pectinate, persulfate, phenylethyl barbiturate, picrate, propionate, thiocyanate, tosylate, undecanoate, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, benethamine, clemizole, diethylamine, piperazine, tromethamine, aluminum, calcium, lithium, magnesium, potassium, zinc sodium, barium, and bismuth. Functional groups in pyrazine derivatives capable of forming salts can optionally be formed using methods known in the art. As a non-limiting example, an amine hydrochloride salt can be formed by adding hydrochloric acid to pyrazine. A phosphate salt can be formed by adding a phosphate buffer to pyrazine.Acidic functional groups present, such as sulfonic, carboxylic, or phosphonic acids, can be deprotonated with a suitable base and the salt formed. Alternatively, amine groups can be protonated with a suitable acid to form an amine salt. The salt forms can be singly, doubly, or triply charged, and when more than one counterion is present, each counterion can be the same as or different from each of the other counterions.

[0042] In yet another aspect, disclosed herein is a method of measuring a glomerular filtration rate (GFR) in a patient in need of GFR measurement. The method includes administering a pyrazine compound or a pharma- ceutically acceptable salt thereof to the patient, measuring the transdermal fluorescence of the patient over a period of time, and determining (measuring) the GFR of the patient. The time used to determine a single measurement of GFR is referred to herein as the "Measurement Time Window." In many cases, it is clinically useful to assess GFR in real time over time. Thus, 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 agent. The total length of time after a single injection until a GFR measurement is provided is referred to herein as the "Single Injection Reporting Period." In some aspects, there is a temporal overlap between the Measurement Time Windows. In such cases, the time interval (reporting time) in which the GFR is reported is not necessarily the same as the Measurement Time Window. For example, in one embodiment, adjacent measurement time windows overlap each other by 50% and the reporting time is half the measurement time window. In some aspects, the measurement time windows can vary in length. In a preferred embodiment, when adjacent measurement time windows in time have different lengths, the overlap period is selected to be 50% of the length of the shorter of the two measurement time windows. In some aspects, the patient's GFR is measured using a system disclosed elsewhere herein.

[0043] In yet another embodiment, the measurement time window is automatically adjusted according to a metric related to signal quality (hereafter referred to as "Quality Metric"). The quality metric can be based on an estimate of the fluorescence signal-to-noise ratio (SNR), signal-to-background ratio (SBR), good-of-fit metrics, correlation coefficient, or any combination thereof. In one embodiment, a line is fitted to the logarithm of the fluorescence intensity versus time over the measurement time window (i.e., a single exponential is fitted to the fluorescence intensity versus time). The difference between the fitted line and the data ("fitting residuals") is used to estimate the "noise". In one embodiment, the noise is the root mean square (RMS) of the fitting residuals. In another embodiment, the noise is the median absolute deviation (MAD) of the fitting residuals. The "signal" is defined as the amplitude of the single exponential function derived by the fitting. In another embodiment, the signal is chosen as the difference between the fitted fluorescence at the beginning and end of the measurement time window. In another embodiment, the pre-injection fluorescence signal level is used to determine the "background" and the SBR is calculated by dividing the background into the signal level. When using SNR or SBR as the quality criterion, a minimum threshold is defined and the fit is considered valid for the purpose of measuring GFR only if the quality criterion exceeds the minimum threshold. In another embodiment, the estimated error of the time constant or rate constant determined by fitting to the fluorescence versus time is used as the quality criterion. In this case, the fit is considered valid only if the calculated quality criterion is below a predefined threshold. In some other embodiments, the time constant or rate constant that is fitted is defined as the signal, the estimated error determined by fitting is defined as the noise, and this version of the SNR is used as the quality criterion. In another embodiment, the correlation coefficient is used as the quality criterion. Various methods known in the art can be used to calculate the correlation coefficient, such as the Pearson correlation coefficient or the concordance correlation coefficient.In yet another embodiment, combinations of different quality criteria are combined into a single quality criterion or the fitting result is considered valid for the purpose of measuring GFR only if all selected quality criteria are passed.

[0044] In another aspect, a minimum measurement time window is defined and a quality criterion is used to determine whether to report the GFR or extend the length of the measurement time window. In one such embodiment, the length of the measurement time window is automatically extended until the quality criterion reaches a threshold, at which point the GFR is reported. In another aspect, a preliminary fit is used to the time or rate constant, or predicted GFR, and to set the measurement time window to a predefined length. In one embodiment, the minimum measurement time is set to 60 minutes, at which time a fit is performed and a preliminary estimate of the GFR is made. If the preliminary estimate of the GFR is 75 mL / min / 1.73 m 2 If so, the result is reported to the user and the measurement time window is maintained at 60 minutes. However, if the preliminary estimate of GFR is less than 75 mL / min / 1.73 m 2 If it is less than this, the results will not be reported to the user and the measurement time window will be extended to 120 minutes.

[0045] In another aspect, the remaining single injection report time is estimated and periodically provided to the user. The basis for estimating the remaining single injection report time can be SNR, SBR, or estimated fit error, as in the above-mentioned methods for determining the quality criterion. It should be noted that the quality criterion used to determine the measurement time window and the quality criterion used to determine the remaining single injection report time can be the same or different. In some aspects, in addition to the quality criterion, a fitted fluorescence decay time constant or rate constant is used to estimate the remaining single injection report time. In one embodiment, the fitted fluorescence decay time constant and the SNR are combined to predict the remaining single injection report time. The SNR is scaled to a minimum value of 0 to a maximum value of 1, and then multiplied by the fluorescence decay time constant. The product is then scaled to predict the single injection report time. The scaling factor is a calibration factor determined by analysis of previously collected data on human patients, animals, in vitro experimental systems, simulations, or any combination thereof.

[0046] In yet another embodiment, filtering and / or outlier rejection is applied to the fluorescence data within the measurement time window prior to fitting. Examples of suitable filtering include boxcar averaging, infinite response function filters, median filters, trimmed mean filters. Examples of outlier rejection methods include all of the quality assessment criteria described above, but applied to a subset of the measurement time window.

[0047] In some embodiments, a quality metric is calculated from the measured tracer agent emission energy as a function of time over a measurement time window. The quality metric can be used to determine whether to report the calculated GFR. In another embodiment, the quality metric is used to determine whether to extend the measurement time window. For example, the measurement time window is automatically extended until the quality metric exceeds a predefined threshold, at which point the GFR is reported.

[0048] The GFR normalized for the patient's body size is determined by fitting the measured tracer emission energy to a decay parameter as a function of time over a measurement time window. In some embodiments, the decay parameter is a rate constant (or its inverse is referred to as the time constant) determined by a monoexponential fit. In some embodiments, the offset of the fitted function is fixed to 0. In other embodiments, the offset is a variable term in the fit. In yet other embodiments, the offset is fixed or can be changed based on a preliminary evaluation of the decay parameter. The measurement time window is selected to begin after the tracer equilibrates in the body and spans a period of time during which the emission intensity decays due to renal clearance of the tracer. The fitted rate constant is multiplied by a calibration slope to determine the GFR normalized for the patient's body size. The calibration slope is determined by analysis of data previously collected on human patients, animals, in vitro experimental systems, simulations, or any combination thereof.

[0049] Since the patient's body size may affect the assessment of renal function, in some embodiments, a measure of body size is used to normalize the GFR calculation to further improve the measurement. In some embodiments, the measure of body size used to normalize the GFR is body surface area (BSA). In another embodiment, the measure of body size is the volume of distribution (Vd) of the tracer agent.

[0050] The methods and systems disclosed herein also allow for real-time monitoring of a patient's GFR. Additionally, multiple GFR measurements or determinations can be made with a single administration of tracer agent. In some embodiments, a single GFR measurement is determined after administration of the tracer agent. In other embodiments, multiple GFR measurements are determined after administration of the tracer agent to provide real-time GFR trends. In some such embodiments, an estimate is provided of the remaining time that the remaining concentration of tracer agent is sufficient to continue measuring the GFR.

[0051] In yet another aspect, disclosed herein is another method for measuring the glomerular filtration rate (GFR) of a patient in need of GFR measurement. The method includes administering a pyrazine compound or a pharma- ceutically acceptable salt thereof to the patient, measuring the transdermal fluorescence of the patient over a measurement time window, and determining (measuring) the GFR of the patient. In some aspects, the GFR of the patient is determined using a system disclosed elsewhere herein.

[0052] In yet another aspect, disclosed herein is yet another method of measuring GFR in a patient in need thereof, comprising administering to the patient a compound of formula I or a pharma- ceutically acceptable salt or formulation thereof, measuring the concentration of the compound of formula I in the patient's body over a measurement time window, and determining the patient's GFR.

[0053] In some aspects, and continuing with reference to the above method, measuring the concentration of pyrazine includes monitoring the patient's transdermal fluorescence. In yet another aspect, measuring the concentration of pyrazine includes 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 incorporates a quantifiable radioisotope. In yet another aspect, measuring the concentration of pyrazine includes collecting the patient's urine over a period of time to determine the rate at which the kidneys remove the compound from the patient's body.

[0054] In yet another embodiment, and continuing with reference to the method described above, the concentration of pyrazine in a patient is monitored by transdermal fluorescence. This may include contacting the patient's skin with a medical device configured to induce a fluorescent response from a compound of formula I, and detecting the fluorescent response. The medical device is contacted with the patient's skin at any suitable location. Particular locations known to be suitable are the sternum, substernal region, pectoralis major, occipital triangle, forehead, chin, upper buttocks, and lower buttocks. Other patient locations may be used as determined by convenience, medical device design, and / or medical need. In some embodiments, the method uses a system disclosed elsewhere herein.

[0055] In one embodiment of the above method, a display device is used to prompt the user to attach the sensor to one or more particular 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 to which the sensor is attached in order to proceed to the next step of the measurement set-up process. This has the advantage of preventing placement of the sensor on a body part that is inappropriate or not optimal for measuring GFR.

[0056] In another aspect, the next step is to set the light source output level and the detector gain level. In one such aspect, both the detector gain level and the light source output level are initially set to a low state, and then the light source output level is continuously increased until the target signal level is achieved. In one embodiment, the light source is an excitation source for a fluorescent GFR agent, and the light source drive current is increased until the target fluorescent signal is achieved or a predetermined maximum current is reached. If the maximum source current is reached without achieving the desired fluorescent signal level, the detector gain level is continuously increased until the target fluorescent signal is achieved or the maximum detector gain setting is reached.

[0057] In some embodiments, in addition to measuring the fluorescence of the skin and the GFR agent, measurements of the diffuse reflectance of the skin are made. In such embodiments, the diffuse reflectance signal can be used to determine optimal light source power levels and detector gain levels. In yet another embodiment, the diffuse reflectance measurements are made within the wavelength bands for excitation and emission of the fluorescent GFR agent. In such embodiments, the setting of the LED light source power levels and detector gain levels can be made using the diffuse reflectance instead of the fluorescence signal level to guide the settings. In one such embodiment, the target signal level or diffuse reflectance signal is in the range of 15%-35% of the signal level at which detector or amplifier saturation effects are observed. This provides headroom for signal variations that may be associated with patient movement or other physiological variations. The above-described techniques for optimizing the light source power and / or detector gain have the advantage of providing a means to compensate for physiological variations between different patients or between different body sites on the same patient. In one embodiment, the primary factor compensated for is the melanin content of the skin. Other physiological factors requiring compensation include blood volume, water content, and scattering within the tissue volume optically interrogated by the sensor. In another aspect, the user is not allowed to initiate a measurement if the desired signal target is not achieved, thus preventing inaccurate results from being reported.

[0058] Once the desired signal level is successfully achieved, in another embodiment, a baseline signal is recorded. In one such embodiment, the stability of the baseline is assessed, such as by fitting a slope to the signal over time, and the baseline is not considered valid unless the slope over time is below a predetermined threshold. In some embodiments, the display instructs the user not to begin administering the tracer agent until a stable baseline is achieved. In this manner, measurements are prevented if the sensor is not properly positioned or attached. Additionally, the user may be prevented from beginning measurements if the tracer agent injected during the previous measurement has not been cleared from the body to the desired extent.

[0059] Once a stable baseline has been obtained, in another embodiment of the above method, a tracer agent is injected into the patient's vascular space. The administration of the tracer agent is automatically detected as a rapid increase in the patient's transcutaneous fluorescence detected by one or more sensors. For this purpose, predetermined thresholds for rate of change, absolute signal change, or relative signal change can be used. The automatic detection of the tracer agent can be reported to a user on a display device such as a touch screen monitor. In another embodiment, once the tracer agent is detected, another threshold is used to determine whether there is sufficient tracer agent present to initiate a GFR measurement. In one such embodiment, the fluorescence (F meas The measurements of fluorescence (F) and diffuse reflectance (DR) are combined to reduce the effect of physiological variations on the combined result (referred to herein as intrinsic fluorescence or IF), thereby compensating for, for example, the effect of skin color on the measurements. The sufficiency of the tracer agent is then assessed by comparing the IF to a predefined threshold. In some embodiments, the IF is determined using the following equation (1):

[0060]

number

[0061] where the subscripts of each DR refer to measurements collected at the excitation (ex) and emission (em) wavelength bands of the tracer agent by both filtered and unfiltered detectors, and the superscripts of each DR are calibration factors determined by analysis of data previously collected on human patients, animals, in vitro experimental systems, simulations, or any combination thereof. In this way, the medical professional performing the measurement can be provided with the opportunity to administer additional tracer agent or to discontinue the measurement if not enough tracer agent has been administered for an accurate GFR assessment.

[0062] Once the tracer agent is administered, in another embodiment, equilibration of the tracer agent into the extracellular space is monitored. In one embodiment, the measurement time window does not begin until equilibration is determined to be sufficiently complete. Fitting to an exponential function can be used to assess the progress of equilibration. For example, the change in fluorescence intensity over time is fitted to a single exponential function, and equilibration is considered complete only when the fitted time constant stabilizes. In one such embodiment, a running estimate is provided to the user indicating the time until the first GFR measurement can be performed. In another embodiment, the user is prevented from starting the measurement until or unless sufficient equilibration is achieved. In one such embodiment, the equilibration time is compared to a predefined threshold, and if the equilibration time exceeds the threshold, the user is prevented from starting the GFR measurement. In this manner, evaluation of the GFR is prevented if the sensor is located at a site of insufficient exchange with the circulatory system.

[0063] In some embodiments, the reporting time, measurement time window, and / or single injection reporting time are set or modified based on the particular medical evaluation being performed. For example, in the case of a patient with chronic renal failure, a single measurement of GFR is sufficient. However, in the case of a patient with or at risk for acute renal failure, a real-time assessment or GFR trend offers a great potential benefit. In some embodiments, the reporting time may be about 15 minutes. In other embodiments, the reporting time may be about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 5 hours, about 8 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 embodiments, the reporting time may range from 15 minutes to 168 hours. In some embodiments, the single injection reporting time is based on the clearance half-life of the pyrazine compound. The clearance half-life may be predetermined for the patient, estimated based on the patient's medical condition, or determined transdermally using the methods described herein. In some embodiments, the single injection reporting time is the first clearance half-life, the second clearance half-life, the third clearance half-life, the fourth clearance half-life, the fifth clearance half-life, the sixth clearance half-life, the eighth clearance half-life, or the tenth clearance half-life. The maximum single injection reporting time is the time period during which pyrazine is no longer detectable in the patient's bloodstream. As used herein, "undetectable" means that the concentration of pyrazine is no longer detectable by the method used to measure. In some embodiments, if the device detection level is for a very long period of time (e.g., one week or more), "undetectable" means that the concentration level has fallen to less than 0.39% (i.e., the eighth half-life). In yet another embodiment, the reporting time is any range from about 1 hour to 168 hours in 1 hour increments.

[0064] Similarly, the measurement time window may be set or changed according to the patient's particular medical needs. In some embodiments, the measurement time window may be about 15 minutes. In another aspect, the measurement time window may be about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 5 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about hours, about hours, about hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 168 hours. In some embodiments, the measurement time window may range from 15 minutes to 168 hours. There may be one or more measurement time windows during each single injection reporting time. In some aspects, the single injection reporting time is divided into multiple measurement time windows, each measurement time window being identical to each other. In yet another aspect, the single injection reporting time is divided into multiple measurement time windows, each measurement time window being selected independently from the other measurement time windows, and may be the same or different from the other measurement time windows.

[0065] The methods and systems disclosed herein have the advantage of automatically adjusting for skin melanin content so that GFR measurements are accurate across a wide range of skin types and pigmentation levels. The Fitzpatrick scale is a numerical classification scheme for human skin color. The Fitzpatrick scale is widely recognized as a useful tool for dermatological studies of human skin pigmentation. Scores range from Type I (minimally pigmented fair skin) to Type VI (highly pigmented, dark brown). The systems and methods disclosed herein are suitable for use with all six categories of skin pigmentation on the Fitzpatrick scale. Specifically, the systems and methods disclosed herein are suitable for use with Type I, Type II, Type III, Type IV, Type V, and Type VI skin coloration.

[0066] In yet another embodiment, the pyrazine is combined with at least one pharma- ceutically acceptable excipient selected from the group consisting of a solvent, a pH adjusting agent, a buffering agent, an antioxidant, a tonicity adjusting agent, an osmolality adjusting agent, a preservative, an antibacterial agent, a stabilizer, a viscosity adjusting agent, a surfactant, and any combination thereof.

[0067] Pharmaceutically acceptable solvent can be aqueous or non-aqueous solution, suspension, emulsion or suitable combination thereof.Non-limiting examples of non-aqueous solvent are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate.Examples of aqueous carrier are water, alcoholic or aqueous solution, emulsion or suspension, including saline and buffered media.

[0068] As non-limiting examples, pharma- ceutically acceptable buffers include acetate, benzoate, carbonate, citrate, dihydrogen phosphate, gluconate, glutamate, glycinate, hydrogen phosphate, lactate, phosphate, tartrate, Tris-HCl, or any combination thereof, having a pH in the range of 4-9, preferably 5-8, more preferably 6-8, and most preferably 7.0-7.5. In yet another embodiment, the pH is in the range of 6.7-7.7. Other buffers known in the art may be selected based on the particular salt form of the pyrazine derivative produced or the particular medical application. A preferred buffer is phosphate buffered saline at physiological pH (about 7.2).

[0069] Examples of tonicity adjusting agents are glycerol, sorbitol, sucrose, or preferably sodium chloride and / or mannitol. Examples of viscosity adjusting agents include bentonite, calcium magnesium silicate, etc. Examples of diluents include ethanol, methanol, etc. Examples of antibacterial agents include benzalkonium chloride, benzethonium chloride, ethylparaben, methylparaben, etc. Examples of osmolality adjusting agents include aminoethanol, calcium chloride, choline, dextrose, diethanolamine, lactated Ringer's solution, meglumine, potassium chloride, Ringer's solution, sodium bicarbonate, sodium chloride, sodium lactate, TRIS, or any combination thereof. These examples are illustrative and are not intended to be a limiting list or a limitation.

[0070] Also disclosed herein is a method of assessing renal function in a patient in need thereof, comprising administering to the patient a compound of formula I, or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable formulation thereof; exposing the patient to electromagnetic radiation to cause emission of spectral energy from the compound of formula I; detecting the emitted spectral energy from the compound of formula I; and assessing the patient's renal function based on the detected spectral energy.

[0071] In some embodiments, the compound of formula I is not metabolized by the patient, but instead is completely eliminated by renal excretion and is not metabolized (e.g., not oxidized, glucuronidated, or otherwise conjugated). In some embodiments, at least 95% of the compound of formula I is not metabolized by the patient before renal excretion. In some embodiments, at least 96% of the compound of formula I is not metabolized by the patient before renal excretion. In some embodiments, at least 97% of the compound of formula I is not metabolized by the patient before renal excretion. In some embodiments, at least 98% of the compound of formula I is not metabolized by the patient before renal excretion. In some embodiments, at least 99% of the compound of formula I is not metabolized by the patient before renal excretion. In some embodiments, the compound of formula I is completely eliminated from the patient in less than a predetermined period of time. In some embodiments, the evaluation of the patient's renal function may further include measuring the patient's GFR.

[0072] The pyrazine can be administered by any suitable method. The method can be based on the medical needs of the patient and is selected by the medical professional administering the pyrazine, i.e., performing the treatment. Examples of administration methods include, but are not limited to, transdermal, oral, parenteral, subcutaneous, enteral, or intravenous administration. Preferably, the pyrazine compound is administered using an intravenous or transdermal administration method. In some embodiments, the pyrazine is administered by a single bolus intravenous injection. In yet another embodiment, the pyrazine is administered by multiple bolus intravenous injections. As used herein, both transcutaneous and transdermal refer to administration through the skin of a patient and are used interchangeably.

[0073] As used herein, "enteral administration" refers to any method of administration that uses the gastrointestinal tract to directly or indirectly deliver a drug to a patient. Examples of enteral administration include, but are not limited to, oral administration, sublingual administration, buccal administration, and rectal administration. As used herein, "parenteral administration" refers to any method of administration that directly or indirectly delivers a drug to a patient by injection or infusion. Examples of parenteral administration include, but are not limited to, intravenous administration, intraarterial administration, intradermal administration, transdermal administration, subcutaneous administration, and intramuscular administration.

[0074] Also disclosed herein is a stable parenteral composition comprising a pyrazine derivative of formula I and a pharma- ceutically acceptable buffer. The parenteral 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 adjuster as described elsewhere herein. The parenteral composition has a pH suitable for administration to a patient in need thereof, and can be adjusted using a buffer or other pH adjuster as described elsewhere herein. The parenteral composition has an osmolality suitable for administration to a patient in need thereof, and can be adjusted using an osmolality adjuster as described elsewhere herein. The parenteral composition is contained in a sealed container and terminally sterilized to reduce or eliminate the microbiological load of the composition. The parenteral composition is stable against degradation and other deleterious chemical reactions and has a pharma- ceutically acceptable shelf life.

[0075] As used herein, "stable" means maintained in a state suitable for administration to a patient. The compositions of the present disclosure have been found to be stable when kept at room temperature for at least 12 months. Additionally, the compositions of the present disclosure are generally stable when kept at room temperature for 12-24 months.

[0076] As used herein, a "sterile" composition refers to a composition that has not been exposed to microbiological contamination after being rendered sterile, i.e., the container that contains the sterile composition is intact. Sterile compositions are generally manufactured by pharmaceutical companies in accordance with the current Good Manufacturing Practice (cGMP) regulations of the U.S. Food and Drug Administration. In some embodiments, the sterile composition is contained in a container that has been heat sterilized. The container may be any container suitable for use in a medical setting, including, but not limited to, a vial, an ampoule, a bag, a bottle, and a syringe.

[0077] In some embodiments, the sterile composition can be in the form of a sterile, ready-to-use formulation for parenteral administration. This avoids the inconvenience of diluting a concentrated parenteral formulation in an infusion diluent prior to infusion or injection, and reduces the risk of microbiological contamination and any potential calculation or dilution errors during aseptic processing. Alternatively, the sterile composition can be a solid formulation that is diluted prior to administration to a patient.

[0078] The aqueous sterile pharmaceutical composition disclosed herein is suitable for parenteral administration to a patient in need thereof. For example, the aqueous sterile pharmaceutical composition may be administered in the form of a bolus injection or intravenous infusion. Suitable routes for 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 aspects, the composition is suitable for transdermal delivery to the epidermis or dermis of a patient. Transdermal delivery methods and devices are known in the art, and various methods are used to deliver pharmaceutical compositions to a patient.

[0079] The aqueous sterile pharmaceutical composition is formulated in combination with one or more pharma- ceutically acceptable excipients as described elsewhere herein. The aqueous sterile pharmaceutical composition is formulated to be suitable for administration to a patient in need thereof. Tonicity, osmolality, viscosity, and other parameters can be adjusted using agents and methods as described elsewhere herein.

[0080] In yet another aspect, disclosed herein is an aqueous sterile pharmaceutical composition for parenteral administration. The aqueous sterile pharmaceutical composition comprises about 0.1-50 mg / mL of a pyrazine compound of formula I. The aqueous sterile pharmaceutical composition also comprises about 0.01-2 M of a buffering agent, as disclosed elsewhere herein. The aqueous sterile pharmaceutical composition also comprises about 0-500 mg / mL of an osmolality adjusting agent and about 0-500 mg / mL of a tonicity adjusting agent. The aqueous sterile pharmaceutical composition may also optionally comprise one or more additional pharma- ceutically acceptable excipients. Examples of pharma-ceutically acceptable excipients may be selected from the group consisting of a solvent, a pH adjusting agent, a buffering agent, an antioxidant, a tonicity adjusting agent, an osmolality adjusting agent, a preservative, an antibacterial agent, a stabilizer, a viscosity adjusting agent, a surfactant, and any combination thereof. Specific examples of excipients are disclosed elsewhere herein.

[0081] The pyrazine compound used in the aqueous sterile pharmaceutical composition may be any compound disclosed herein. Specific examples include, but are not limited to, all of the compounds prepared in the Examples described below. A suitable example is the molecule illustrated in Example 12, (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid) (this molecule is also known as MB-102, or 3,6-diamino-N 2 ,N 5 -Bis(D-serine)-pyrazine-2,5-dicarboxamide.

[0082] The pH of the aqueous sterile pharmaceutical composition is suitable for administration to a patient. In some embodiments, the pH is in the range of 4 to 9, preferably 5 to 8, more preferably 6 to 8, and most preferably 7.0 to 7.5. In yet other embodiments, the pH is in the range of 6.7 to 7.7. Even more preferably, the pH is about 7.2 in phosphate buffered saline.

[0083] In some embodiments, the pyrazine is administered to a patient suspected or known to have at least one medical problem in the kidney, and the method disclosed herein is used to measure the level of renal dysfunction or failure in the patient. In some embodiments, the patient's estimated GFR (eGFR) or previously measured GFR is less than 110, less than 90, less than 60, less than 30, or less than 15. The patient's eGFR is measured using standard medical techniques known in the art. In some embodiments, the patient does not have or is not suspected of having a medical problem in the kidney. GFR monitoring is performed as part of the patient's general or periodic health evaluation, or as a preventive evaluation.

[0084] As known in the art, the rate at which a patient removes waste products from the bloodstream (i.e., clearance half-life) depends on the health and proper functioning of the patient's renal system. "Completely removed" as used in this context means that the level of pyrazine in the bloodstream has fallen to less than 0.39% (i.e., 8 half-lives). The clearance half-life depends on the patient's GFR and slows significantly as the function of the renal system decreases due to disease, age, or other physiological factors. For patients who do not have known risk factors associated with CKD and have a normal GFR and / or normal eGFR, the single injection reporting time is 24 hours. In some embodiments, for patients with a GFR or eGFR less than 110, the single injection reporting time is 24 hours. For patients with a GFR or eGFR less than 90, the single injection reporting time is 24 hours. For patients with a GFR or eGFR less than 60, the single injection reporting time is 48 hours. For patients with a GFR or eGFR less than 30, the single injection reporting time is 48 hours. In some embodiments, for patients with a GFR or eGFR less than 110, the single injection reporting time is equal to eight half-lives. For patients with a GFR or eGFR less than 90, the single injection reporting time is equal to eight half-lives. For patients with a GFR or eGFR less than 60, the single injection reporting time is equal to eight half-lives. For patients with a GFR or eGFR less than 30, the single injection reporting time is equal to eight half-lives.

[0085] The method disclosed herein is suitable for patients whose urine test results show increased protein concentration, because increased protein concentration in the urine of patients may indicate some renal dysfunction or failure.In some embodiments, the patient has high urine protein concentration as measured by standard medical tests (e.g., dipstick test).As a non-limiting example, the patient's urine test results may show high albumin concentration, high creatinine concentration, high blood urea nitrogen concentration (i.e., BUN test), or any combination thereof.

[0086] Continuing with 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 derivative to electromagnetic radiation may be performed at any suitable time, but is preferably performed while the pyrazine derivative is present in the patient's body. Due to this exposure of the pyrazine derivative to electromagnetic radiation, the pyrazine emits spectral energy (e.g., visible light and / or infrared light) that can be detected by a suitable detection device. The spectral energy emitted from the pyrazine derivative tends to exhibit a wavelength range that is larger than the wavelength range absorbed by the pyrazine derivative. By way of example, but not limited to, if an embodiment of the pyrazine derivative absorbs light at about 440 nm, the pyrazine derivative may emit light at about 560 nm.

[0087] Detection of the pyrazine (more specifically, the spectral energy emitted from the pyrazine) can be accomplished using optical fluorescence, absorbance, or light scattering techniques. In some aspects, the spectral energy is fluorescent light. In some embodiments, detection of the emitted spectral energy can be characterized as collection of the emitted spectral energy and generation of an electrical signal indicative of the collected spectral energy. The mechanism used to detect the emitted spectral energy from the pyrazine derivative present in the patient's body may be designed to detect only a selected wavelength (or range of wavelengths) and / or may include one or more appropriate spectral filters. A variety of catheters, endoscopes, earclips, handbands, headbands, surface coils, finger probes, and the like may be used to expose the pyrazine derivative to electromagnetic radiation and / or to detect the emitted spectral energy from the pyrazine derivative. The device for exposing the pyrazine to electromagnetic radiation and the device for detecting the emitted spectral energy from the pyrazine may be the same or different from each other. That is, one or two devices may be used. Detection of the spectral energy may be intermittent, one or more times, or may be substantially continuous.

[0088] The renal function of the patient can be determined based on the detected spectral energy. This is accomplished by using data indicative of the detected spectral energy to generate an intensity / time profile indicative of the clearance of the pyrazine derivative from the patient's body. The profile can be correlated to a physiological or pathological condition. For example, the renal function of the patient can be evaluated and the physiological condition of the patient can be diagnosed by comparing the clearance profile and / or clearance rate of the patient to a known clearance profile and / or clearance rate. When analyzing the presence of pyrazine derivatives in a body fluid, a concentration / time curve can be generated and analyzed (preferably in real time) to diagnose renal function. Alternatively, the clearance profile of the patient can be compared to one or more clearance profiles previously measured from the same patient to determine whether the renal function of the patient has changed over time. In some embodiments, the renal function assessment is performed using a system disclosed elsewhere herein.

[0089] Physiological function can be assessed by (1) comparing differences in how normal or damaged cells or organs clear pyrazine derivatives from the bloodstream, (2) measuring the excretion or accumulation rate of pyrazines in the patient's organs or tissues, and / or (3) obtaining tomographic images of the organs or tissues in which pyrazines are present. For example, blood pool clearance can be measured non-invasively from capillary surfaces present in earlobes or fingers, or invasively using appropriate instruments, such as intravascular catheters. Transdermal fluorescence can also be non-invasively monitored from the surface of the patient's body. Many locations on the patient's epidermis are suitable for non-invasive monitoring of transdermal fluorescence. The monitored sites are preferably those where equilibration from the vasculature to tissue occurs relatively quickly. Examples of suitable sites for monitoring on a patient include, but are not limited to, the sternum, substernal region, pectoralis major, occipital triangle, forehead, chin, upper buttocks, and lower buttocks. Accumulation of pyrazine derivatives in cells of interest can also be assessed using similar methods.

[0090] The modified pulmonary artery catheter may also be used to, among other things, perform the desired measurement of the spectral energy emitted from the pyrazine derivative. The ability of the modified pulmonary artery catheter to detect the spectral energy emitted from the pyrazine is significantly improved over current pulmonary artery catheters that only measure intravascular pressure, cardiac output, and other derivative measurements of blood flow. Traditionally, critically ill patients have been treated using only the above parameters, and such treatment has tended to rely on intermittent blood sampling and testing to assess renal function. These traditional parameters provide non-continuous data that is often misleading in many patient populations.

[0091] The modification of a standard pulmonary artery catheter only requires that the fiber optic sensor be wavelength specific. Catheters currently exist that incorporate fiber optic technology to measure mixed venous oxygen saturation. In one aspect, the modified pulmonary artery catheter incorporates a wavelength specific fiber optic sensor at the tip of a standard pulmonary artery catheter. This wavelength specific fiber optic sensor can be used to monitor renal function specific elimination of chemicals designed to be optically detectable, such as the pyrazine derivatives disclosed herein. Thus, renal function can be monitored in real time by the disappearance / clearance of the optically detected compounds.

[0092] In some embodiments, the pyrazine compounds are administered to a patient who has previously been diagnosed with at least stage 1 CKD. In other embodiments, the patient to whom the pyrazine compounds are administered has previously been diagnosed with stage 2 CKD, stage 3 CKD, stage 4 CKD, or stage 5 CKD. In yet other embodiments, the patient has not yet been diagnosed with CKD, but has one or more risk factors associated with CKD. In yet other embodiments, the patient has no known risk factors for CKD.

[0093] Administration of the pyrazine compound may be by any suitable method, based on the medical test being performed and the medical needs of the patient. Suitable methods are disclosed elsewhere herein. Preferably, the pyrazine is administered transdermally or intravenously.

[0094] Also disclosed herein is a system for measuring GFR or assessing renal function in a patient in need thereof, the system including a computer device, a display device communicatively connected to the computer device, a power source operatively connected to the computer device to maintain electrical isolation of the system from an external power source, one or more sensor heads operatively connected to the computer device, and at least one tracer agent configured to emit light when exposed to electromagnetic radiation. The computer device is configured to operate and control the sensor heads, record one or more light measurements transmitted from the sensor heads, and calculate the GFR of the patient based on the light measurements.

[0095] It should be noted that the term "connection" as used herein is not limited to direct, mechanical, electrical, and / or communication connections between components, but may include indirect, mechanical, electrical, and / or communication connections between multiple components. The display device and one or more sensor heads may communicate with the computing device using wired network connections (e.g., Ethernet or optical fiber), wireless communication means such as radio frequency (RF) (e.g., FM radio and / or digital audio broadcast), the Institute of Electrical and Electronics Engineers (IEEE) 802.11 standard (e.g., 802.11(g) or 802.11(n)), the Worldwide Interoperability for Microwave Access (WIMAX) standard, short-range wireless communication channels such as BLUETOOTH, cellular technology (e.g., Global System for Mobile Communications (GSM)), satellite communication links, and / or other suitable communication means. IEEE® is a registered trademark of the Institute of Electrical and Electronics Engineers, Inc., New York, NY, USA. WIMAX® is a registered trademark of the WiMax Forum, Beaverton, Oregon, USA. BLUETOOTH® is a registered trademark of Bluetooth SIG, Inc., Kirkland, Washington, USA.

[0096] In some embodiments, the 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 from the tracer agent, and transmit measurements of the electromagnetic radiation emitted from the tracer agent to a computer device. In a system having two or more sensor heads, each sensor head may be identical or different from one another, and the electromagnetic radiation emitted from each sensor head may be identical or different from one another. In some embodiments, the sensor heads are configured to be attached to the patient's skin. By way of example and not limitation, in a system having two sensor heads, one sensor head may emit and monitor electromagnetic radiation at a first wavelength, and the other sensor head may emit and monitor electromagnetic radiation at a second wavelength different from the first wavelength. This allows data to be compared, thereby increasing the accuracy of the information available to a medical professional performing GFR measurements and assessments. In yet another non-limiting example, in a system having two sensor heads, the two sensor heads may be used to separate local equilibrium dynamics from end-phase dynamics. This allows the medical professional to determine when equilibration is complete, thereby reducing artifacts due to local movement of the sensor.

[0097] In some embodiments, the tracer agent is administered to a patient by intravenous or transdermal administration, is cleared only by glomerular filtration in the patient's kidneys, and is configured to emit light detectable by the sensor head when exposed to electromagnetic radiation. In some embodiments, the tracer agent is a pyrazine compound of formula I as disclosed elsewhere herein. Preferably, the tracer agent is a compound prepared in one of the examples described below. Most preferably, the tracer agent is (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid) (also referred to as MB-102, or 3,6-diamino-N2,N5-bis(D-serine)-pyrazine-2,5-dicarboxamide). In some embodiments, the tracer agent is a pyrazine derivative in a formulation suitable for administration to a patient in need thereof. Such formulations are described elsewhere herein.

[0098] A system for measuring a patient's GFR or assessing renal function may be configured to perform the methods disclosed herein on a patient in need thereof. The computing device in the system may be any standard computer having all the capabilities implied therein, specifically including, but not limited to, a permanent memory, a processor capable of complex mathematical calculations, a user interface for interacting with the computer, and a display communicatively connected to the computing device. Thus, the permanent memory of the computing device may store any information, programs, and data required to perform the functions of the system to measure a patient's GFR or assess a patient's renal function. Such information, programs, and data may be baseline and / or control values ​​that may be used to compare transcutaneous fluorescence values ​​collected by one or more sensor heads to known values. In some embodiments, the computing device may store results from previous evaluations or GFR measurements of the patient for comparison with results obtained at a later date. This allows medical professionals to monitor the renal health of the patient over time. In some embodiments, the computing device is a laptop computer.

[0099] In various aspects, the computing device includes a processor and / or a memory device. In various other aspects, the processor is connected 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 a user by instructing the user via the display device and / or by receiving user input via a user interface. The term "processor" generally refers to any programmable system, including systems and systems that use microcontrollers, reduced instruction set circuits (RISC), application specific integrated circuits (ASIC), programmable logic circuits (PLC), and any other circuit or processor capable of performing the functions described herein. The above examples are merely examples and thus are not intended to limit in any way the definition and / or meaning of the term "processor."

[0100] In various aspects, a memory device includes one or more devices that allow for the storage and retrieval of information, such as executable instructions and / or other data. Additionally, 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), solid state disks, and / or hard disks. 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, validation results, and / or any other type of data.

[0101] The user interface is configured to receive at least one input from a user, such as a system operator, to measure the GFR or evaluate the patient's renal function. In one embodiment, the user interface includes a keyboard that allows the user to input relevant information. In various other embodiments, the user interface includes, but is not limited to, a pointing device, a mouse, a stylus, a touch panel (e.g., a touch pad, a touch screen), a gyroscope, an accelerometer, a position detector, and / or a voice input interface (e.g., a microphone, etc.).

[0102] The display device is configured to display information, such as input events and / or validation results, to a user. The display device may also include a display adapter connected to at least one display device. In one aspect, the display device may 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.

[0103] In various embodiments, the computing device generally includes a processor. The processor may be programmed by encoding operations with one or more executable instructions and providing the executable instructions to a memory device. In one embodiment, the processor is programmed to calculate a time constant of renal decay over a predetermined period of time. In one embodiment, transdermal fluorescence data of a patient is collected over a predetermined period of time and a graph of time (X-axis) versus fluorescence (Y-axis) is generated. The decay rate may be curvilinear or linear, and the time constant of the decay rate is calculated. In one embodiment, the decay rate is linear on a semi-log (y) plot. By comparing the time constant to a known value, the patient's GFR is determined. In some embodiments, the decay rate corresponds to standard first-order kinetics. In yet another embodiment, the decay rate may represent a multi-compartment pharmacokinetic model. Figures 3A-3D are diagrams illustrating two-compartment pharmacokinetics from which the time constant of renal decay can be determined by standard pharmacokinetic software.

[0104] GFR measurements were made using linear regression, outlier removal, calculation of correlation coefficient (R2), and standard error of calibration, as described in more detail in the Examples below.

[0105] This specification uses examples to disclose the invention including the best mode and to enable one of ordinary skill in the art to practice the invention, including making and using any device or system, and practicing any method incorporated herein. Any aspect or embodiment disclosed herein can be used in combination with any other aspect or embodiment understood by a person of ordinary skill in the art. Other embodiments are within the scope of the claims if they include elements that do not differ from the language of the claims, or if they include equivalent elements that do not differ substantially from the language of the claims.

[0106] Example 1

[0107] 3,6-Diamino-N 2 ,N 2 ,N 5,N 5 Preparation of -tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

[0108] [ka]

[0109] 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 h. The mixture was concentrated to dryness and the residue was partitioned with ethyl acetate and water. The layers were separated and the ethyl acetate 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 / 1CHCl3-methanol) afforded 228.7 mg (53% yield) of Example 1 as an orange foam: 1 H NMR (300 MHz, CDCl3), δ 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). 13 CNMR (75 MHz, CDCl3) 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 (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 3.14 min on a 30 mm column, (M+H). + = 429. UV / vis (100 μM in PBS) λ abs = 394 nm. Fluorescence (100 μm) λ ex =394nmλ em =550nm.

[0110] Example 2

[0111] 3,6-Diamino-N 2 ,N 5 -Bis(2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

[0112] [ka]

[0113] 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

[0114] [ka]

[0115] 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) at room temperature for 16 h. The mixture was concentrated to dryness and the residue was partitioned with ethyl acetate and water. The layers were separated and the ethyl acetate 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 bis-amide diastereomeric pair as a yellow solid: 1 NMR (300 MHz, CDCl3) 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 - diastereomeric mixture, J = 5.9 Hz, 4H), 1.35 (s, 6H), 1.26 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 165.7 (s), 146.8 (s), 126.8 (s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1 (t-diastereoisomeric pair), 27.6 (q), 26.1 (q).

[0116] Step 2. The product from step 1 was dissolved in THF (100 mL) and treated with 1.0 N HCl (2 mL). After hydrolysis was complete, the mixture was treated with K2CO3 (1 g), stirred for 1 h, and filtered through a plug of C18 with methanol. The filtrate was concentrated to dryness and the residue was triturated with methanol (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. This material was purified by radial flash chromatography to give 221 mg (36% yield) of Example 2 as an orange solid: 1 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, 6.66 Hz, 2H) 2.92 (dd, J=12.6, 3.3 Hz, 2H), 2.65 (dd, J=12.6, 8.4 Hz, 2H). LCMS (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 4.13 min on a 30 mm column, (M+H). + = 345. UV / vis (100 mM in H2O) λ abs = 432 nm. Fluorescence λ ex = 432 nm, λ em =558nm.

[0117] Example 3

[0118] (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid)

[0119] [ka]

[0120] Step 1. Synthesis of dimethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2'S)-bis(3-(benzyloxy)propanoate)

[0121] [ka]

[0122] A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (300 mg, 1.24 mmol), L-Ser(OBn)-OMe-HCl salt (647 mg, 2.64 mmol), HOBt-HO (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 h and concentrated. Workup as in Example 1 afforded 370 mg (51% yield) of the bisamide as a light yellow powder: 1 NMR (300MHz, CDCl3): δ8.47(d, J=8.74Hz, 2H), 7.25-7.37(m, 10H), 5.98(bs, 4H), 4.85(dt, J=8.7, 3.3Hz, 2H), 4.56 (ABq, J=12.6, Hz, Δv=11.9Hz, 4H), 3.99 (1 / 2 of ABq in d, J=8.7, 3.3Hz, 2H), 3.76-3.80 (1 / 2 of ABq unclear, 2H), 3.78 (s, 6H). 13 CNMR (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 (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 4.93 min on a 30 mm column, (M+H). + =581.

[0123] Step 2. Synthesis of (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)-bis(azanediyl))bis(3-(benzyloxy)propanoic acid)

[0124] [ka]

[0125] The product from 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 min, the reaction was judged complete by TLC. The pH was adjusted to approximately 2 by addition of 1.0 N HCl and the resulting solution was extracted with ethyl acetate (3 times). 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 (gradient 5-95% acetonitrile in 0.1% TFA over 10 min), retention time on 30 mm column = 4.41 min, (M+H). + =553.

[0126] Step 3. To the product from Step 2 (353 mg, 0.64 mmol) in methanol (20 mL) was added 5% palladium / C (300 mg) and ammonium formate (600 mg). The resulting reaction was heated under reflux for 2 h. The reaction was cooled to room temperature, filtered through a plug of celite, and concentrated. The residue was recrystallized from methanol-ether to give 191 mg (80% yield) of Example 3 as a yellow foam: 1 NMR (300 MHz, DMSO-d6) δ 8.48 (d, J = 6.9 Hz, 2H), 3.60 (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 3.48, J = 20.99, 6.3 Hz, 2H). 13CNMR (75 MHz, CDCl3) δ 172.9 (s), 164.9 (s), 147.0 (s), 127.0 (s), 62.9 (d), 55.7 (t). LCMS (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 1.45 min on a 30 mm column, (M+H). + = 373. UV / vis (100 μM in PBS) λ abs = 434 nm. Fluorescence λ ex = 449 nm, λ em =559nm.

[0127] Example 4

[0128] 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

[0129] [ka]

[0130] Step 1. Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic acid

[0131] [ka]

[0132] 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 solution. To this stirred mixture was added a solution of sodium nitrite (695 mg, 10.1 mmol) in water (10 mL) dropwise such that the temperature remained below 5 °C. The resulting mixture was stirred at 5-15 °C for 3 h, during which time the red mixture became a yellow solution. The yellow solution was poured into a solution of cupric bromide (2.23 g, 10.1 mmol) in water (100 mL) and the resulting mixture was stirred at room temperature. After an additional 3 h, the aqueous mixture was extracted with ethyl acetate (3 times). 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 pale yellow solid. 13 C NMR (75 MHz, CDCl3) δ 164.3(s), 148.8(s), 134.9(s). HPLC (gradient 5-95% acetonitrile in 0.1% TFA in 10 min), single peak retention time = 2.95 min on a 250 mm column.

[0133] 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

[0134] [ka]

[0135] The product obtained in 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 min. Bis(2-methoxyethyl)amine (620 mL, 559 mg, 4.20 mmol) was added and the resulting mixture was stirred at room temperature for 16 h and concentrated. The residue was partitioned with water and ethyl acetate. The ethyl acetate layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with 0.5 N HCl, saturated sodium bicarbonate, and brine. The organic layers were dried (Na2SO4), filtered, and concentrated to give 3-(bis(2-methoxyethyl)amino)-6-bromo-N 2 ,N 2 ,N 5 ,N 5 -Tetrakis(2-methoxyethyl) 2,5-dicarboxamide (26% yield) was obtained as a brown oil: LCMS (10 min gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 3.85 min on a 30 mm column, (M+H). + =608.

[0136] Step 3. To the product obtained in step 2 (116 mg, 0.19 mmol) was added bis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and one "spatula tip" of Pd(PPh3)4. The resulting mixture was heated at 140° C. for 2 h. The reaction was cooled and concentrated. The residue was purified by flash chromatography (SiO2, 10 / 1 CHCl3-methanol). The resulting material was purified again by medium pressure reverse phase chromatography (C18, manual gradient 10-50% acetonitrile in 0.1% TFA) to give 12 mg (10% yield) of Example 4 as an orange-brown film. LCMS (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 3.85 min on 250 mm column, (M+H) + =661.UV / vis (100μM in PBS) λ abs = 434 nm. Fluorescence λ ex = 449 nm, λ em =559nm.

[0137] Example 5

[0138] 3,6-Diamino-N 2 ,N 5 -Bis(2-aminoethyl)pyrazine-2,5-dicarboxamide bis(TFA) salt

[0139] [ka]

[0140] Step 1. 3,6-Diamino-N 2 ,N 5 Synthesis of -bis[2-(tert-butoxycarbonyl)-aminoethyl]pyrazine-2,5-dicarboxamide

[0141] [ka]

[0142] 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-HO (836 mg, 5.46 mmol) and EDC-HCl (1.05 g, 5.48 mmol) was stirred in DMF (25 mL) for 16 h and concentrated. Workup as in Example 1 afforded 770 mg (76% yield) of the bisamide as an orange foam: 1 NMR (300MHz, DMSO-d6) major conformers, δ8.44(t, J=5.7Hz, 2H), 6.90(t, J=5.7Hz, 2H), 6.48(bs, 4H), 2.93-3.16 (complex m, 8H), 1.37(s, 9H), 1.36(s, 9H). 13 C NMR (75MHz, DMSO-d6), conformers δ165.1(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).

[0143] Step 2. To the product from step 1 (770 mg, 1.60 mmol) in methylene chloride (100 mL) was added TFA (25 mL) and the reaction was stirred at room temperature for 2 h. The mixture was concentrated and the residue was dissolved in methanol (15 mL). Ether (200 mL) was added and the orange solid precipitate was isolated by filtration and dried under high vacuum to give 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). 13 C NMR (75 MHz, DMSO-d6) δ 166.4 (s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 3.62 min on a 30 mm column, (M+H). + = 283. UV / vis (100 μM in PBS) λ abs = 435 nm. Fluorescence (100 nM) λ ex = 449 nm, λ em =562nm.

[0144] Example 6

[0145] 3,6-Diamino-N 2 , N 5 -Bis(D-aspartic acid)-pyrazine-2,5-dicarboxamide

[0146] [ka]

[0147] Step 1. 3,6-Diamino-N 2 ,N 5 Synthesis of -Bis(benzyl DO-benzyl-aspartate)-pyrazine-2,5-dicarboxamide

[0148] [ka]

[0149] 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 ethyl acetate. The ethyl acetate layer was separated and washed successively with saturated sodium bicarbonate, water, and brine. The ethyl acetate solution was dried (Na2SO4), filtered, and concentrated. The residue was purified by flash chromatography (SiO2, 50 / 1 CHCl3-methanol to 10 / 1 CHCl3-methanol) to give 1.15 g (58% yield) of the bisamide as a yellow foam: 1 NMR (500MHz, CDCl3) δ8.61(d, J=8.4Hz, 2H), 7.29-7.39(m, 20H), 5.85(bs, 4H), 5.22( ABq, J=10.0Hz, Δv=17.3Hz, 4H), 5.10(ABq, J=12.2Hz, Δv=34.3Hz, 4H), 5.06-5.09(obs m, 2H), 3.11 (d ABq, J=17.0, 55.14Hz, Δv=77.9Hz, 4H). 13 C NMR (75 MHz, CDCl3) δ 170.7(s), 170.7(s), 165.4(s), 147.0(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 (10 min, gradient acetonitrile 50-95% in 0.1% TFA), single peak retention time = 5.97 min on a 250 mm column, (M+H). + =789.

[0150] Step 2. To the product obtained in step 1 (510 mg, 0.65 mmol) was added THF (20 mL) and water (10 mL). To the stirred mixture was added 10% palladium / C (500 mg) and ammonium formate (1 g). The resulting mixture was heated at 60° C. for 2 h and cooled to room temperature. The mixture was filtered through Celite and concentrated. The resulting material was purified again by medium pressure reverse phase chromatography (C18, 10-70% manual gradient acetonitrile in 0.18% TFA) to give 137.8 mg (54% yield) of Example 6 as an orange solid: 1 NMR (300MHz, DMSO-d6) δ8.62 (d, J=8.4Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J=8.4, 5.4Hz, 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 (gradient 5-95% acetonitrile in 0.1% TFA over 10 min), single peak retention time = 4.01 min on a 250 mm column, (M+H). + = 429. UV / vis (100 μM in PBS) λ abs = 433 nm. Fluorescence (100 nM) λ ex = 449 nm, λ em =558nm.

[0151] Example 7

[0152] 3,6-Diamino-N 2 ,N 5 -Bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl)pyrazine-2,5-dicarboxamide

[0153] [ka]

[0154] 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 (113 mg, 0.34 mmol) and the reaction was stirred at room temperature for 16 h. 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 / 10~80 / 20 0.1% TFA-ACN) to give 37.4 mg (35% yield) of Example 7 as an orange film: 1 H NMR (300MHz, DMSO-d6) δ8.47(t, J=5.7Hz, 2H), 7.96(t, J=5.4Hz, 2H), 3.20-3.60 (complex m, 36H), 3.47(s, 3H), 3.46(s, 3H), 2.30(t, J=6.3Hz, 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 (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 4.01 min on a 250 mm column, (M+H). + = 719, (M+Na) + = 741. UV / vis (100 μM in PBS). λ abs = 437 nm. Fluorescence (100 nM) λ ex = 437 nm, λ em =559nm.

[0155] Example 8

[0156] 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

[0157] [ka]

[0158] 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-octaoxahexacosane-26-oate (128 mg, 0.25 mmol) and the reaction was stirred at room temperature for 16 h. The reaction was concentrated and the residue was purified by medium pressure reverse phase chromatography (LiChroprep® RP-18 Lobar (B) 25×310 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 H NMR (300MHz, DMSO-d6) δ8.46 (t, J=5.7Hz, 2H), 7.96 (t, J=5.4Hz, 2H), 3.16-3.73 (complex m, 74H), 2.28-2.32 (m, 2H). 13 C NMR (75 MHz, DMSO-d6) - multiple conformations - 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 (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 5.90 min on a 250 mm column, (M+H). + =1071, (M+2H) 2+ = 536. UV / vis (100 μM in PBS) λ abs = 438 nm. Fluorescence (100 nM) λ ex = 438 nm, λ em =560nm.

[0159] Example 9

[0160] 3,6-Diamino-N 2 ,N 5-Bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxo-39-azahentetocalsurun41-yl)pyrazine-2,5-dicarboxamide

[0161] [ka]

[0162] To a solution of Example 5 (53.1 mg, 0.10 mmol) in DMF (5 mL) was added a solution of TEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodeca-oxaoctatriacontan-38-oate (144 mg, 0.21 mmol) in DMF (2.0 mL), and the resulting mixture was then stirred for 16 h. The reaction was concentrated and the residue was purified by medium pressure reverse phase chromatography (LiChroprep® RP-18 Lobar (B) 25×310 mm-EMD chemicals 40-63 μmm, ∼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 (300MHz, DMSO-d6) δ8.48 (t, J=5.7Hz, 2H), 7.96 (t, J=5.4Hz, 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) 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), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 5.90 min on a 250 mm column, (M+2H). 2+ = 712. UV / vis (100 μM in PBS) λ abs = 449 nm. Fluorescence (100 nM) λ ex = 449 nm, λem =559nm.

[0163] Example 10

[0164] Bis(2-(PEG-5000)ethyl)6-(2-(3,6-diamino-5-(2-aminoethylcarbamoyl)pyrazine-2-carboxamide)ethylamino)-6-oxohexane-1,5-diyldicarbamate

[0165] [ka]

[0166] 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 h. 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 medium pressure reverse phase chromatography (C18, manual gradient 10-70% acetonitrile in 0.18% TFA) to give 137.8 mg (54% yield) of Example 10 as a tan solid. Maldi MS m / z=11393.

[0167] Example 11

[0168] (R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamide)succinic acid

[0169] [ka]

[0170] Step 1. Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine

[0171] [ka]

[0172] A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in methanol (500 mL) was treated with NBS (34.3 g, 193.1 mmol) in portions over 1 h. The resulting mixture was then stirred for 16 h. TLC analysis at this point indicated 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 h. 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 h. 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 desiccator to give 45.4 g (97% yield) of 2-amino-5-bromo-3,6-dichloropyrazine as a white solid: 13 C NMR (75 MHz, CDCl3) δ 149.9(s), 145.6(s), 129.6(s), 121.5(s). LCMS (gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 4.51 min on a 30 mm column, (M+H). + =244, (M+H+ACN) + =285.

[0173] Step 2. Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile

[0174] [ka]

[0175] 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 at 130° C. for 2.5 h. To the resulting homogeneous mixture of sodium dicyanocuprate(I) was added a solution of the product of step 1 (15.6 g, 64.2 mmol) dissolved in DMF (150 mL) dropwise over 1 h. The temperature was gradually increased to 150° C. and the resulting mixture was stirred at this temperature for 10 h. The reaction was then cooled to room temperature and poured into water (1 L). The resulting mixture was extracted with ethyl acetate (3 times) and the combined extracts were filtered to remove fluffy dark solids, washed with brine, dried (Na2SO4), filtered again, and concentrated. Purification by flash column chromatography (SiO, 10 / 1 to 3 / 1 hexanes-ethyl acetate) 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 inlet: temperature = 280 °C, helium flow rate of 1.0 mL / min, temperature program: 100 °C (2 min hold), gradient to 300 °C, @10 °C / min (2 min hold), main peak retention time = 16.556 min, m / z (EI) = 188,190.

[0176] Step 3. Synthesis of 5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyrazine-2-carbonitrile

[0177] [ka]

[0178] To the product from step 2 (1.00 g, 5.29 mmol) in ACN (20 mL) was added bis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and the reaction mixture was heated to 70° C. for 16 h. The reaction was cooled and concentrated. The residue was partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic extracts were washed with brine, dried (Na2SO4), filtered and concentrated. Purification by flash column chromatography (SiO2, 10 / 1 to 1 / 1 hexane-ethyl acetate) afforded 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 (10 min, gradient 50-95% acetonyl in 0.1% TFA), single peak retention time = 4.91 min on a 250 mm column, (M+H). + = 286, (M+Na) + = 308, (M + Na + ACN) + =349.

[0179] Step 4. Synthesis of 3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile

[0180] [ka]

[0181] To the product obtained in step 3 (1.39 g, 4.88 mmol) in 48% hydrobromic acid (20 mL) at 0° C. (ice-salt bath) was added dropwise a solution of sodium nitrite (673 mg, 9.75 mmol) in water (10 mL) over 30 min. The resulting mixture was stirred at 0-5° C. for 1 h and poured into a stirred solution of CuBr2 (1.64 g, 7.34 mmol) in water (10 mL). The resulting mixture was then stirred at room temperature for 16 h. The mixture was extracted with ethyl acetate (3 times). The combined organic layers were dried (Na2SO4), filtered and concentrated. Purification by flash column chromatography (SiO2, 50 / 1CHCl3-methanol) afforded 1.00 g (58% yield) of the bromide as an orange-brown solid: 1 NMR (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 (10 min, gradient 50-95% acetonitrile in 0.1% TFA), single peak retention time = 4.55 min on a 250 mm column, (M+H). + =349, 351.

[0182] Step 5. Synthesis of 3-(bis(2-methoxyethyl)amino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile

[0183] [ka]

[0184] A mixture of the product from step 4 (1.0 g, 2.87 mmol), 2-furan boronic 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% palladium, 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 then heated to 75 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (100 mL), and filtered through a medium frit. Concentration and purification of the residue by flash chromatography (SiO2, 50 / 1CHCl3-methanol) afforded 757 mg of the furan adduct (78% yield) as a tan powder. LCMS (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 6.41 min on 250 mm column, (M+H) + =337.

[0185] Step 6. Synthesis of 6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxylic acid

[0186] [ka]

[0187] 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) sequentially. The resulting mixture was stirred vigorously at room temperature for 30 min and treated with sodium bicarbonate (2.10 g, 25.0 mmol) followed by water (5 mL). After stirring vigorously for an additional 15 min, a solution of the product of 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 h. The mixture was transferred to a separatory funnel and extracted with ethyl acetate. The aqueous layer was adjusted to pH ∼3.5 and extracted again with ethyl acetate (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 pale yellow solid: LCMS (10 min, gradient 5-95% acetonitrile in 0.1% TFA), single peak retention time = 5.05 min on 250 mm column, (M+H). + =315.

[0188] Step 7. Synthesis of (R)-dibenzyl 2-(6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamide)succinic acid

[0189] [ka]

[0190] A mixture of the product from step 1 (140 mg, 0.45 mmol), EDC-HCl (128 mg, 0.67 mmol), and HOBt-HO (102 mg, 0.67 mmol) in anhydrous DMF (25 mL) was stirred together at room temperature for 30 min. To this stirred mixture was added (R)-dibenzyl 2-aminosuccinic acid salt p-TsOH salt (213 mg, 0.44 mmol), followed by TEA (1 mL). The resulting mixture was then stirred for 16 h. The reaction mixture was concentrated and partitioned with ethyl acetate and saturated sodium bicarbonate solution. The ethyl acetate layer was separated, washed with saturated sodium bicarbonate and brine, dried (Na2SO4), filtered, and concentrated to give 240 mg of pyrazinamide (88% yield) as an orange foam. LCMS (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 8.76 min on 250 mm column, (M+H) + = 610, (M+Na) + =632.

[0191] Step 8. (R)-Dibenzyl 2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamide) succinate

[0192] [ka]

[0193] To the product from step 7 (240 mg, 0.39 mmol) was added morpholine (5 mL). The reaction mixture was heated to 70° C. for 2 h. The mixture was cooled and concentrated. The residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated and washed with saturated sodium bicarbonate and brine. The ethyl acetate layer was dried (Na2SO4), filtered, and concentrated. Purification by flash column chromatography (SiO2, 3:1 to 1:1 hexane-ethyl acetate) afforded 199 mg (75% yield) of the morpholine adduct as an orange foam. LCMS (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 8.76 min on 250 mM column, (M+H). += 661, (M+Na) + =683.

[0194] Step 9. Synthesis of Example 11

[0195] [ka]

[0196] To 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 h. The pH was adjusted to ∼2 with 1.0 N HCl and the solution was concentrated. Purification of the residue by medium pressure reverse phase chromatography (LiChroprep RP-18 Lobar (B) 25 × 310 mm - EMD chemicals 40-63 (μm, ∼70 g, 90 / 10 to 50 / 50 0.1% TFA-ACN)) gave 32 mg (27% yield) of Example 11 as an orange solid. LCMS (10 min, gradient 15-95% acetonitrile in 0.1% TFA), single peak retention time = 4.47 min on 250 mm column, (M+H) = 481. UV / vis (100 μM in PBS) λ abs = 438 nm. Fluorescence (100 nM) λ ex = 449 nm, λ em =570nm.

[0197] Example 12

[0198] (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid) ("D-serine isomer" or "MB-102")

[0199] [ka]

[0200] Step 1. Formation of dibenzyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)) (2R,2'R)-bis(3-hydroxypropanoic acid)

[0201] A round bottom flask (500 mL) equipped with a Claisen adapter and an addition funnel was charged with 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 atmosphere for 15 min. DIPEA (19.16 mL, 110.0 mmol) was added portionwise via addition funnel over 30 min, and after an additional 30 min, the cooling bath was removed and diacid (9.91 g, 50.0 mmol) was added in one portion. The brick red suspension was stirred for 30 min and HOBt-H2O (17.61 g, 115.0 mmol) was added in one portion. After 15 min, the reaction flask was cooled in an ice bath and EDC-HCl (22.05 g, 115.0 mmol) was added over 15 min. The resulting suspension was allowed to gradually warm to room temperature and stirred under N2 overnight (ca. 17 h).

[0202] The dark solution was concentrated under high vacuum (bath temperature 60° C.) to a syrupy residue and partitioned between ethyl acetate and Milli-Q water (400 mL each). The layers were separated and the aqueous layer was extracted with ethyl acetate (3 times, 200 mL). The combined ethyl acetate extracts were washed successively with 0.50 M KHSO4, saturated NaHCO3, water, 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 the bisamide (19.6 g, 71%) as an orange solid: Rf 0.45 [CHCl3:MeOH (9:1, v / v)]. 1H NMR (DMSO-d6) δ8.56 (d, J=8.0Hz, 2H, exchangeable with D2O), 7.40-7.33 (m, 10H), 6.76 (s, 4H, exchangeable with D2O), 5.37 (t , J=5.5Hz, 2H), 5.20(m, 4H), 4.66-4.63(dt, J=8.0, 4.0Hz, 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=4.44 min, 5~95% B / 6 min). C 26 H 28 Elemental analysis for N6O8: C, 56.52; H, 5.11; N, 15.21. Found: C, 56.39; H, 5.11; N, 14.99.

[0203] Step 2. Formation of (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid)

[0204] The bisamide (7.74 g, 14.0 mmol) was hydrogenated in ethanol:water (560 mL; 3:1, v / v) in the presence of 10% palladium / C (0.774 g). The reaction mixture was purged with argon and stirred under hydrogen atmosphere (using slow bubbling technique) at room temperature for 5.5 h. The reaction mixture was again purged with argon and the catalyst was removed by filtration through Celite. The Celite bed was washed with ethanol:water (400 mL; 1:1, v / v) and the combined filtrate was concentrated in vacuo. The product was dried under high vacuum. The residue was triturated with CH3CN to give the D-serine isomer (4.89 g, 94%) as an orange powder. 1H NMR (DMSO-d6) δ8.46 (d, J=8.3Hz, 2H, can be replaced with D2O), 6.78 (br s, 4H, interchangeable with D2O), 4.48-4.45 (dt, J=8.1, 3.9Hz, 2H), 3.88 (dd, J=11.1, 3.9Hz, 2H), 3.74 (dd, J=11.1, 3.7Hz, 2H). 13 C 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=2.86 minutes, 5~95% B / 6 minutes). C 12 H 16 Elemental analysis for N6O8: C, 38.71; H, 4.33; N, 22.57. Found: C, 38.44; H, 4.51; N, 22.33.

[0205] Example 13

[0206] (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("D-alanine isomer")

[0207] [ka]

[0208] Step 1. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2'R)-diethyl dipropionate

[0209] Under inert atmosphere, a flame-dried round-bottom flask (100 mL) equipped with a magnetic stir bar was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), D-alanine ethyl ester hydrochloride (1.86 g), EDC-HCl (2.7 g), HOBt-H2O (2.65 g), and Et3N (2.0 mL) and dissolved in DMF (anhydrous, 80 mL). Volatiles were removed under reduced pressure at 50 °C to yield a dark semi-solid. After cooling, acetonitrile (~100 mL) was added and the solution was allowed to stand for about 1 h. The red precipitate was isolated by centrifugation, washed with ethyl acetate, and dried. Total weight of the diester was 1.30 g (3.06 mmol, 60.6% isolated yield). This material (1.3 g) was carried on to the next step without further purification.

[0210] Step 2. Formation of (2R,2'R)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0211] The diester from step 1 (1.0 g) and LiOH (4 eq.) in THF / water were combined and stirred at room temperature for several hours. HPLC showed complete hydrolysis. TFA was added to make the pH acidic and the reaction mixture was left at room temperature overnight. The diacid was obtained by purifying the reaction mixture by preparative reverse phase HPLC. Program: 99:1 A:B for 5 min, then 5:95 A:B for 27 min @ 50 mL / min. Lambda UV 264 nm and fluorescence; λ x = 440 nm, 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.

[0212] Example 14

[0213] 3,3'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("β-alanine isomer")

[0214] [ka]

[0215] Step 1. Formation of dibenzyl 3,3'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionate

[0216] Under inert atmosphere, a flame-dried round-bottom flask (100 mL) equipped with a magnetic stir bar was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.3 g), benzyl 3-aminopropanoate p-toluenesulfonate (1.08 g), EDC-HCl (0.590 g), HOBt-H2O (0.582 g), and Et3N (1.50 g) and dissolved in DMF (anhydrous, 40 mL). The reaction mixture was stirred overnight at room temperature and concentrated in vacuo to approximately 10 mL. Residual DMF was removed with a toluene azeotrope. The reaction mixture was partitioned between ethyl acetate (3 x 125 mL) and saturated NaHCO3 (3 x 100 mL). The organic layers were combined and partitioned with citric acid (10% aqueous, 100 mL), and brine (100 mL). The organic layer was removed, dried (anhydrous Na2SO4) and concentrated in vacuo to give 0.58 g of a crystalline solid. TLC (silica on glass, 1:1 ethyl acetate:hexanes) Rf=0.22. The product was purified by flash chromatography on silica gel to give 0.49 g of product. Mass Spectrum (ES+) 521.36 (100%), 522.42 (30%), 523.34 (~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.

[0217] Step 2. Formation of 3,3'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0218] The dibenzyl ester from step 1 (0.92 g) was combined with ethanol (anhydrous, 75 mL) and transferred to a Fischer-Porter pressure bottle (approximately 170 g) equipped with inlet and outlet valves, a pressure gauge (0-approximately 689.48 kPa), and a Teflon-coated magnetic stir bar. Water (25 mL) and 10% palladium on carbon (0.2 g, Degussa / Aldrich wet) were added and the reaction vessel was sealed. After three vacuum / argon cycles, hydrogen was introduced to the vigorously stirred solution from a lecture bottle at approximately 68.95 kPa. After 3.5 hours, the reaction was filtered through a Celite pad and the resulting Celite / catalyst bed was rinsed with approximately 500 mL of 1:1 ethanol:H2O to give a solution that was concentrated in vacuo. 0.424 g of solid was isolated (70.5% isolated yield). Only a single peak was observed from HPLC / MS at 9.3 min. 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.

[0219] Example 15

[0220] 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic acid ("glycine isomer")

[0221] [ka]

[0222] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)) diacetate

[0223] A round bottom flask (300 mL) equipped with a magnetic stir bar was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (5.0 g), ethyl glycinate hydrochloride (5.04 g), EDC-HCl (8.1 g), HOBt-H2O (8.0 g), and DIPEA (5.9 g) and dissolved in DMF (anhydrous, 200 mL). A dry argon atmosphere was maintained throughout the reaction. The pyrazine was combined with the glycinate and DMF was added with stirring under an inert atmosphere. To this was added the base and HOBt. After about 15 minutes, EDC was added in portions over 45 minutes and the reaction was stirred overnight at room temperature under argon. The reaction mixture was concentrated in vacuo until a viscous semi-solid was obtained. The semi-solid was treated with toluene (about 30 mL) and the volatiles were removed in vacuo. After cooling, a solid formed. The crude product was dissolved in 500 mL of ethyl acetate 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 (2x150 mL ethyl acetate) and the organic layers were combined. The organic layers were washed with aqueous NaHSO4 and saturated brine, dried over Na2SO4 and concentrated to give a solid. Isolated yield: 5.46 g. HPLC analysis 96.9%. M / z 369.2. 1 H NMR and 13 C NMR was consistent with the proposed structure. This product was carried on to the next step without further purification.

[0224] Step 2. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic acid

[0225] The crude product from step 1 (700 mg) was dissolved in 40 mL of THF and 10 mL of water (DI). LiOH (4.2 eq.) was added and the mixture was stirred overnight at room temperature under an inert atmosphere. HPLC analysis showed complete conversion to the desired diacid (M / z=313.3). The reaction mixture was centrifuged (3000 rpm for 3 min) and the supernatant was checked by HPLC and discarded. The remaining solid was converted to the disodium salt by treatment with NaOH (6.25 N, 2 eq.) and the resulting solution was filtered (0.22 microns). The solution was lyophilized to give a solid with purity >95% by HPLC. The disodium salt was converted to the diacid by using a reverse phase preparative column after adding slightly more than 2 eq. of TFA. Proton and carbon NMR were consistent with the proposed structure.

[0226] Example 16

[0227] (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid ("L-alanine isomer")

[0228] [ka]

[0229] Step 1. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2'S)-diethyl dipropionate

[0230] 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 L-alaninate hydrochloride (1.86 g), EDC-HCL (2.70 g), and HOBT-H2O (2.65 g) and dissolved in DMF (anhydrous, 80 ML). Triethylamine was added (1.5 g). After 16 hours at room temperature, the reaction volatiles were removed in vacuo. A semi-solid was isolated. Water was added (70 ML) and the mixture was allowed to stand for approximately 1 hour. During this time a precipitate formed so the mixture was centrifuged and the solid was isolated and allowed to air dry overnight. This material was dissolved in ethyl acetate and washed with water, citric acid, and saturated sodium bicarbonate. The organic layer was dried (anhydrous sodium sulfate) and concentrated in vacuo to give the solid product (1.38 g). HPLC purity >95% pure. The crude product was used in the next step without further purification.

[0231] Step 2. Formation of (2S,2'S)-2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic acid

[0232] The crude product from step 1 (1.0 g) was dissolved in THF (30 mL) and LiOH-HO (4 equiv.) dissolved in water (10 mL) was added at room temperature. After 1 h, the volatiles were removed in vacuo. The product was purified by preparative reverse phase HPLC and lyophilized to give a solid that was the desired diacid product in >95% purity. Proton and carbon NMR were consistent with the proposed structure.

[0233] Example 17

[0234] 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoic acid) ("Dimethylglycine isomer")

[0235] Step 1. Formation of diethyl 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoate)

[0236] Under 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-aminopropane hydrochloride (1.86 g), EDC-HCl (2.7 g), HOBt-H2O (anhydrous, 80 mL) and dissolved in DMF (anhydrous, 80 ML). The reaction was initiated by the addition of triethylamine (1.50 g) and maintained at room temperature for 72 h. The volatiles were removed in vacuo. A dark viscous liquid separated. After cooling, it was dissolved in acetonitrile (~100 mL) and allowed to stand for ~1 h. The resulting precipitate was isolated by centrifugation and dried to give 1.30 g of the diethyl ester (61%) with a purity of >95% by reverse phase HPLC. This crude product was used directly in the next step.

[0237] Step 2. Formation of 2,2'-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoic acid)

[0238] The crude product from step 1 (1.0 g) was dissolved in THF:water (40 mL:5 mL). To this was added LiOH in water (2.5 eq. in 0.5 mL DI water). Another 2 eq. of LiOH was added to the reaction mixture and the reaction was allowed to proceed at room temperature overnight. Upon completion, the reaction mixture was acidified with TFA until a pH of about 4 was reached. The product was isolated by preparative RPHPLC. M / z 369.13. Proton and carbon NMR were consistent with the proposed structure.

[0239] Example 18

[0240] 3,6-Diamino-N 2 ,N 5 -Bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide

[0241] [ka]

[0242] 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, 5.4 mmol, 2.0 equiv.), and DMF (40 mL). To this was added triethylamine (0.546 g, 0.76 mL, 5.40 mmol, 2.0 equiv.), and PyBop® (3.1 g, 5.94 mmol, 2.2 equiv.). After 1 h, the reaction was complete by HPLC analysis and concentrated in vacuo while keeping the temperature below 40° C. The mixture was dissolved in water (10 mL) and passed through a Sephadex G-10 column, and fractions containing the fluorescent product were collected and lyophilized to give a cloudy solid. The target product is 3,6-diamino-N 2 ,N 5 -Bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide was obtained by preparative C-18 reverse phase HPLC: 160 mg, HRMS (theoretical) M+Na=547.1970; HRMS (observed) M+Na=547.1969.

[0243] Example 19

[0244] Protocol for assessing renal function

[0245] One embodiment of an in vivo renal monitoring assembly 10 is shown in FIG. 2 and includes a light source 12 and a data processing system 14. The light source 12 generally includes or is interconnected with a suitable device for exposing at least a portion of the patient's body to light. Examples of suitable devices that may be interconnected with or part of the light source 12 include, but are not limited to, catheters, endoscopes, fiber optics, ear clips, hand bands, head bands, forehead sensors, surface coils, and finger probes. Indeed, any of a number of devices capable of emitting visible and / or near infrared light of the light source may be used in the renal monitoring assembly 10. In one embodiment, the light source is an LED, one of which emits light near the maximum absorbance of the tracer agent, while the second LED emits light near the maximum fluorescence emission of the tracer agent. For example, one LED emits light at 450 nm, while the second LED emits light at 560 nm.

[0246] 2, the data processing system 14 of the renal monitoring assembly may 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., for directing and / or focusing the spectral energy), one or more filters (e.g., for removing undesirable wavelengths of the spectral energy), a photodiode or photomultiplier (e.g., for collecting and converting the spectral energy into an electrical signal indicative of the detected spectral energy), an amplifier (e.g., for amplifying the electrical signal from the photodiode or photomultiplier), and a processing unit (e.g., for processing the electrical signal from the photodiode or photomultiplier). The data processing system 14 is preferably configured to manipulate the collected spectral data and generate an intensity / time profile and / or a concentration / time curve indicative of the renal clearance of the pyrazine derivatives of the present disclosure from the patient 20. Indeed, the data processing system 14 may be configured to generate appropriate renal function data by comparing differences in how normal and damaged cells remove pyrazine derivatives from the bloodstream, measure the rate or accumulation of pyrazine derivatives in organs or tissues of the patient 20, and provide tomographic images of organs or tissues having the pyrazine derivatives.

[0247] As a non-limiting example, the system in one aspect includes two silicon photomultiplier tubes. The first photomultiplier tube includes a long pass filter, while the second photomultiplier tube does not have a filter. This configuration allows for measurements of both fluorescence emission and diffuse reflectance at the excitation and emission wavelengths. In one such embodiment, the fluorescence and diffuse reflectance measurements are combined into a unique fluorescence measurement that compensates for variations in the optical properties of tissue. An exemplary formula for combining measurements is shown below in Equation (1).

[0248] To measure renal function, in one embodiment, an effective amount of pyrazine derivative is administered to a patient in need thereof (e.g., in the form of a pharma- ceutically acceptable composition). At least a portion of the patient's 20 body is exposed to visible and / or near-infrared light from light source 12, as indicated by arrow 16. For example, light from light source 12 may be guided through an optical fiber attached to the ear of the patient 20. The patient may be exposed to light from light source 12 before or after administering the pyrazine derivative to the patient 20. In some cases, it may be beneficial to generate a background or baseline measurement of light emitted from the patient's 20 body (resulting from exposure to light from light source 12) before administering the pyrazine derivative to the patient 20. When the pyrazine derivative in the patient's 20 body is exposed to light from light source 12, the pyrazine derivative emits light (indicated by arrow 18), which is detected / collected by data processing system 14. First, administration of the pyrazine derivative to the patient 20 generally results in an initial spectral signal indicating the initial content of the pyrazine derivative in the patient 20. The spectral signal then tends to decay as a function of time as the pyrazine derivative is cleared from the patient 20. This decay in the spectral signal as a function of time is indicative of the patient's renal function. Furthermore, once the tracer agent is injected into the patient's vascular space, the initial kinetics reflect the equilibration of the tracer agent throughout the patient's extracellular space. In some embodiments, this equilibration is complete in less than two hours.

[0249] For example, in a first patient exhibiting healthy / normal renal function, the spectral signal may decay back to baseline at time T. However, a spectral signal indicative of a second patient exhibiting insufficient renal function may decay back to baseline at T+4 hours. The degree of renal impairment or failure affects the length of time required for the signal to decay back to baseline. Greater degrees of renal impairment require longer periods of time. Thus, patient 20 may be exposed to light from light source 12 for any time appropriate to provide the desired renal function data. Similarly, data processing system 14 may collect / detect spectral energy for any time appropriate to provide the desired renal function data.

[0250] Furthermore, the GFR measurement in a patient is not limited to a single measurement based on a single administration of a tracer agent. The time from administration of the tracer agent to when the tracer agent becomes undetectable in the patient can be subdivided into multiple smaller segments, and the patient's GFR is calculated for each smaller segment. In some embodiments, the time segments can overlap. As a non-limiting example, if the total period before the tracer agent becomes undetectable is 24 hours, the period can be divided into four equal segments of 6 hours; each new time segment begins at the end of the previous segment. In yet another embodiment, the time segments can overlap. For example, each individual time segment can be 4 hours long, but a new time segment can begin every two hours. This creates overlapping segments throughout the measurement. To more fully illustrate this non-limiting example, if a tracer agent is administered at time=0 and becomes undetectable at time=24 hours, the following time segments may be generated: 0-4 hours, 2-6 hours, 4-8 hours, 6-10 hours, 8-14 hours, 12-16 hours, 14-18 hours, 16-20 hours, 18-22 hours, and 20-24 hours. The patient's GFR may be calculated individually at each time segment. This data is used to more fully assess the patient's renal health. The time segments may be of any length that allows for GFR measurement, and may 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. Furthermore, the time segments need not be identical during the measurement. The length of each time segment is selected individually without regard to any other time segment.

[0251] Pharmacokinetic study results

[0252] In a clinical trial, 60 human patients were administered MB-102 ((2R,2'R)-2,2'-((3,6 diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid)) as prepared (in Example 12) intravenously. In addition to the methods and techniques disclosed herein, blood and urine were collected. Baseline pharmacokinetic data was collected and the methods and techniques disclosed herein were compared to Omnipaque® (iohexol), a known imaging agent used for GFR measurement.

[0253] Figures 3A-3D show data collected from 60 human patients studied with MB-102. Plasma pharmacokinetic data was collected, analyzed using methods known in the art, and compared to data measured using the methods and techniques disclosed herein.

[0254] Figures 3A-3D show a two compartment pharmacokinetic model for the elimination of MB-102. The model is consistent for patients regardless of GFR value. Figure 3A is a graph for a patient with normal renal function with a measured GFR (mGFR) of 120 mL / min. Figure 3B is a graph for a patient with an mGFR of 81 mL / min. Figure 3C is a graph for a patient with an mGFR of 28 mL / min. Figure 3D is a graph for a patient with an mGFR of 25 mL / min. The first compartment in the two compartment model is vascular to tissue equilibration, while the second compartment represents renal excretion only. The time to reach equilibrium averages about 1 hour and varies from subject to subject.

[0255] Shown in Figure 4 is comparative data comparing GFR measured using Omnipaque® using conventional methods to MB-102 using the methods disclosed herein for patients with eGFR ranging from about 20 to about 140. The data shows a correlation coefficient of 0.97, indicating that the method for measuring patient GFR used herein produces similar results compared to known methods.

[0256] As part of one clinical trial for MB-102, urine was collected from patients for a 12-hour period to measure the amount of recovery and the extent of secondary metabolism. As shown in FIG. 5, for patients with eGFR ≥ 60, over 99% of MB-102 was recovered without metabolism after 12 hours. For patients with mGFR values ​​< 60, a lower percentage of MB-102 was recovered, but was also unmetabolized. For patients with normal, stage 1, and stage 2 renal function, a 12-hour collection was sufficient for over 99% recovery of MB-102. In light of the pharmacokinetic data and plasma half-life measurements, it is easy to see that complete recovery is not possible for patients with more severe renal impairment.

[0257] FIG. 6 shows the plasma half-life of MB-102 for patients with either normal renal function, stage 1 renal impairment, stage 2 renal impairment, stage 3 renal impairment, or stage 4 renal impairment. Based on this data, it is clear that the urine recovery study described above requires more than 24 hours to remove all of MB-102 from the patient's bloodstream. The mean plasma half-life for the normal renal function group is 2 hours, so a collection time of 12 hours is about 6 half-lives, which is sufficient time to excrete most of the injected dose. The mean plasma half-life for the stage 2 and stage 3 groups is 2.5 hours, resulting in an excretion time of about 5 half-lives, which is also sufficient to recover most of the injected dose. However, since the half-life for stage 3 is 4 hours and the half-life for stage 4 is 8 hours, it is not possible to recover all of the injected dose within the 12-hour window of the clinical trial.

[0258] In clinical trials, plasma pharmacokinetics were correlated with transcutaneous fluorescence pharmacokinetics measured at the patient's sternum as a function of time (starting 2 hours after tracer injection and continuing until the end of the 12-hour study period). High correlations between plasma concentration and fluorescence intensity were observed for patients across a wide range of GFR values. Figures 7, 8, and 9 show that plasma concentrations of 23 mL / min / 1.73 m 2 (Stage 3b renal dysfunction) ~117mL / min / 1.73m 2(normal renal function) are shown. Thus, in patients across a wide range of GFRs, plasma pharmacokinetics correlates with transcutaneous fluorescence for MB-102.

[0259] Transcutaneous GFR measurements (fitting to the full data set)

[0260] Kinetic analysis was performed using LabView, Matlab, WinNonlin, and Microsoft Excel version 2010 as follows. Transdermal fluorescence data were fitted to a single exponential function from 2 hours (compared to the injection time to ensure that the tracer agent had equilibrated across the extracellular space) to the end of available data (usually around 12 hours). For each subject, two fits were performed: (1) with a fixed offset of 0 (2) with the offset varied. The exponential time constant determined from the fits is referred to as the renal decay time constant (RDTC), a decay parameter related to renal clearance.

[0261] In Microsoft Excel version 2010, I am trying to calculate linear regression, outlier exclusion, and correlation coefficient (R 2 ) and standard error of calibration (SEC) calculations were performed. The inverse of RDTC was correlated with GFR using four different GFR measurement methods.

[0262] Non-normalized - GFR determined from plasma PK analysis of iohexol and MB-102

[0263] BSA Normalization - Height and weight were used to estimate the body surface area (BSA) of each subject according to the method of Mosteller (N Engl J Med, 1987;317(17)). GFR, as determined by plasma PK analysis, was calculated as 1.73 m 2 Divided by the ratio of calculated BSA to (BSA for a "reference" size patient).

[0264] V dNormalization (Method 1) - GFR determined by plasma PK analysis was calculated using the volume of distribution (V d ) (also determined from PK analysis). V d The mean nGFR across all patients in group 1 was V d The nGFR was determined by equating the GFR normalization method and the BSA normalization method. As used herein, "nGFR" refers to the common method in which GFR is normalized for body size (BSA and V d (including both.)

[0265] V d Normalization (Method 2) - A single exponential function with offset fixed at 0 was fitted to the MB-102 plasma concentration versus time for 2-12 hours (relative to the time of tracer injection). The inverse of the fitted time constant was set to 14,760 mL (V for a "reference" size patient). d ; see above) to obtain GFR normalized for volume of distribution.

[0266] Correlation coefficients (R 2 ) and standard error of calibration (SEC) are summarized in Table 2 and Figures 12-18. Consistent with previous results by Rabito (J Nucl Med, 1993;34(2):199-207), normalization of GFR by BSA reduced R 2 The SEC increased and the EC decreased, confirming the assumption that the renal clearance rate of MB-102 provides a measure of renal efficiency that is independent of body size. Furthermore, these results suggest that the volume of distribution (V d ) has also been shown to be effective for normalizing GFR for standard body size. As shown in Table 2, BSA and the second V d The normalization method for body size, where σ i = σ i , was equally effective when no outlier rejection method was applied and the offset for the RDTC fitting was fixed to 0.

[0267] [Table 2]

[0268] RDTC fitting offset method

[0269] The transdermal fluorescence dynamics were fitted by two different offset methods: (1) the offset was fixed at 0, and (2) the offset was varied. Figures 18 and 19 show the correlation plots obtained for the fixed and variable offset methods, respectively. Note that when the offset was fixed at 0 (Figure 18), the data was more tightly clustered in the low GFR region of the correlation plot, whereas when the offset was variable (Figure 19), the data clustering was more tightly in the high GFR region.

[0270] This observation indicates instability in the baseline fluorescence. In healthy subjects with high rate constants for renal clearance, 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 perfectly match the initial pre-injection baseline fluorescence. By varying the offset in the fit for these healthy subjects, the fit was able to compensate for this baseline uncertainty, thereby improving the reliability of the extracted clearance rate constant. However, in many subjects with reduced renal function, the drug was not completely cleared within the 12 hours that measurements were collected. In these subjects, the inclusion of a variable offset in the fit was found to increase the uncertainty in the fitted clearance rate constant. Fixing the offset to 0 improved the reliability of the rate constant.

[0271] Based on these observations, a hybrid offset method was developed. In the hybrid method, the offset is allowed to vary if a fixed fitting of the offset indicates that the rate constant is high (i.e., healthy renal function), and the offset is fixed otherwise (i.e., impaired renal function). As shown in FIG. 25, R 2 The optimal transition point was selected to maximize R and minimize SEC. The transition points shown on the x-axis are expressed as time constants (in hours), which is the inverse of the clearance rate constant. As shown, the optimal time constants were in the range of 3.5 hours to 4.5 hours. A correlation plot employing the hybrid offset method (transition point: 3.5 hours) is shown in Figure 20. A comparison of the fixed, variable, and hybrid offset methods provided in Table 3 shows that the hybrid offset method has a significantly lower R compared to the other methods. 2 The results show that this results in a substantial increase in and a decrease in SEC.

[0272] [Table 3]

[0273] Data Exclusion

[0274] Some clinical subjects did not have the sensor remain fully attached to the skin for the entire 12 hours of the study. Reattachment of the sensor after injection often resulted in a significant shift in signal levels. This could be due to (1) heterogeneity in skin autofluorescence, (2) heterogeneity in the interstitial fluid fraction of the skin, or (3) changes in the coupling efficiency of light into and out of the skin. To address this in the future, the sensor has been redesigned to have a smaller footprint and to fit closer to the patient. In order to use the clinical data, some data exclusions were applied.

[0275] Five of the 60 clinical subjects were completely excluded from the fluorescence kinetics analysis: (1) subject 1 was excluded because the sensor repeatedly overheated (leading to a 60% reduction in the maximum allowable power level of the blue LED in all subsequent subjects); (2) subjects 8, 37, and 49 were excluded because the sensor was completely detached from the skin and the initial signal level could not be restored by reattachment; and (3) subject 46 was excluded because a large portion of the data was eliminated in the preprocessing step (presumably due to unintended changes in LED power and detector gain after tracer injection).

[0276] To further exclude data of interest, various criteria were tested, including: (1) correlation coefficient between IF and fit; (2) root mean square error (RMSE) of IF for the fit; (3) difference in RDTC determined from monoexponential and biexponential fits; (4) signal-to-noise ratio calculated by dividing the amplitude of the fitted exponential term by the RMSE; (5) coefficient of variation (CV) of the rate constant when fitted across multiple shorter time segments (e.g., 1-2 hours) within the full 12 hour data set; (6) estimation error of the rate constant fitted to the IF; and (7) estimation error of GFR determined from plasma data. None of these criteria included prior information on the correlation coefficient or SEC.

[0277] In one embodiment, the estimated error of the rate constants fitted to the IFs was divided by the rate constants (expressed as relative error) using the data exclusion method (6) described above, yielding the results shown in FIG. 26. By excluding subjects with relative errors in the rate constants greater than 1.4-1.8%, R 2 A significant improvement was observed in the CI and SEC. Choosing 1.75% as the cutoff criterion resulted in the exclusion of 10 of the 55 subjects. The resulting correlation plots are shown in Figures 21 and 22.

[0278] This method was further applied as an exclusion criterion to test whether errors in plasma-derived GFR measurements also contributed to data variance in the correlation plots. Figure 27 shows the optimization of this criterion. By choosing 5% as the cutoff for GFR acceptability, R 2 A slight improvement in CI and SEC was observed. However, this required the exclusion of an additional 10 subjects, reducing the remaining subjects to 35. The resulting correlation plots are shown in Figures 23 and 24. A numerical summary of the application of the different data exclusion criteria is shown in Table 4.

[0279] [Table 4]

[0280] The slope of the calibration determined from the above method can be applied to generate a transcutaneous measurement of GFR corrected for body size (referred to herein as "tGFR"). Figure 10 shows the correlation between predicted GFR and BSA-normalized plasma GFR values. The accuracy of the calibration can be represented by plotting tGFR against a "gold standard" determination of GFR corrected for body size derived from plasma PK analysis. Figure 10 shows the correlation obtained when plasma measured GFR values ​​are normalized for the patient's body surface area (BSA) (correlation coefficient (R 2 )=0.89). Volume of distribution (V d ) and correcting for body size instead of BSA, the correlation improved significantly to 0.96 (Figure 11).

[0281] Compared to eGFR, the method of estimating GFR most commonly used in clinical practice, these tGFR results show the potential for substantially improved accuracy. High accuracy is important to guide clinical decisions. Figure 28 and Table 1 show the five stages of chronic kidney disease (CKD). Misdiagnosis of CKD can affect the course of clinical treatment. As shown in Figures 29A-29C, an error grid plot was constructed to visualize CKD misdiagnosis of GFR measurements compared to the gold standard. Measurements that fall within the box with all green borders are correctly classified by CKD stage. Measurements that fall within the yellow and green borders are misdiagnosed by one CKD stage. Measurements that fall within the red and yellow borders are misdiagnosed by two CKD stages. Figure 29A shows the eGFR error grid for the same group of subjects used in the above analysis. In this case, 2 of 60 subjects were misdiagnosed by two CKD stages, 16 subjects were misdiagnosed by one stage, and 41 subjects were correctly diagnosed. Table 5 provides an overview of the percentage of CKD diagnostic errors among all measurements. Figures 29B and 29C show error grid plots for transcutaneous GFR (tGFR) measurements. Importantly, the tGFR error grid does not include misdiagnosis of the two CKD stages. Furthermore, V d The normalized tGFR shows a large reduction in one stage misdiagnosis when compared with the eGFR (see Table 5).

[0282] [Table 5]

[0283] Transcutaneous GFR measurement (window fitting)

[0284] In the above example, the GFR was determined using all available data sets (i.e., 10 hours of fluorescence decay after administration and equilibration of the tracer agent). This may be adequate for patients with stable renal function, but for patients with or at risk for acute kidney injury, a more rapid, real-time repeated assessment of GFR trends is required. Even for patients with stable renal function, waiting 12 hours for a GFR measurement may be inconvenient. Tables 6 and 7 show the results of varying the measurement time window and single injection reporting time for the same group of subjects, as described above.

[0285] In this example, the measurement time windows (first column in Tables 6 and 7) do not overlap, so the number of windows (second column in Tables 6 and 7) multiplied by the measurement time window gives the total number of hours after equilibration during which GFR estimation was performed. Best results were obtained when the measurement time window was long enough to allow substantial decay of the fluorescence intensity. The time required to achieve the same percentage of fluorescence intensity decay varies with renal health. Therefore, the offset was fixed to 0 except for the widest measurement time window in subjects with healthy kidneys. The standard error of calibration is summarized for all subjects (fifth column in Tables 6 and 7) and for the subset of subjects with nGFR less than or equal to 75 mL / min.

[0286] Using GFR normalized by BSA (Table 6) as the reference comparison, nGFR calibration accuracy of less than 15 mL / min was achieved using a measurement time window of at least about 1.5 hours for subjects with nGFR ≥ 75. For subjects with nGFR < 75, nGFR calibration accuracy of less than 10 mL / min was achieved using a measurement time window of at least about 3 hours. dUsing normalized GFR (Table 7), comparable nGFR calibration accuracy was achieved with measurement time windows of 0.5 and 2 hours, respectively. As shown in Tables 6 and 7, the above calibration accuracy goals were maintained over at least two non-overlapping measurement time windows. However, a significant increase in SEC was typically observed when extending the prediction to three or four measurement time windows. By increasing the measurement window time (e.g., by doubling or tripling) from that used for the first two GFR measurements, at least the third GFR measurement yielded comparable accuracy. These results demonstrate the utility of providing an automatically adjusted measurement time window, which not only accounts for the difference in SNR between different patients, but also for the decrease in SNR over time as the tracer agent is gradually cleared by the kidney.

[0287] [Table 6]

[0288] [Table 7]

[0289] Real-time transcutaneous GFR measurement

[0290] The method disclosed herein allows real-time transcutaneous GFR measurement in patients. After intravascular injection of the tracer agent into the subject, a waiting period of 2 hours allowed the tracer agent to equilibrate to the extravascular space. After accumulating 2 hours of mark data for an additional hour, an initial fit was performed on the RDTC. The initial fit was performed with a fixed offset term of 0. If the RDTC was less than 3.5 hours, the fit was repeated and the slope was varied, and if it was 3.5 hours or more, the initial RDTC (with fixed offset) was maintained. The estimated error in RDTC was then divided by RDTC. If the resulting relative error was less than 1.7%, the RDTC was converted to a BSA-normalized GFR by multiplying the inverse of the RDTC by the slope shown in FIG. 23. If the resulting relative error was 1.7% or more, no GFR was reported. After the initial RDTC fit, the fitting procedure was repeated at approximately 3 second intervals, and subsequent fits incorporated all data in the initial fit and all data accumulated thereafter (at approximately 1 / 2 time intervals). The same procedure was applied to data collected by two sensors: one placed on the manubrium of the sternum and the other on the pectoralis major muscle. The results generated in real time during the measurements are shown in Figure 30. Over the course of the study, the agreement between the tGFR obtained by the two sensors and the variation of tGFR obtained by each sensor was 2 mL / min / 1.73 m 2 It was within.

[0291] Stability study of MB-102

[0292] Samples of MB-102 were prepared and stored at 25°C and 60% relative humidity for 24 months. HPLC evaluation of each sample was performed at various time points to assess the stability of the samples. The results are shown in Table 8.

[0293] [Table 8]

[0294] When introducing elements of the disclosure or various variations, embodiments or aspects thereof, the articles "a," "an," "the," and "said" are intended to mean the presence of 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.

[0295] Although described in connection 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.

[0296] Embodiments of the invention may be described in the general context of computer-executable instructions, such as program modules, executed by one or more computers or other devices. Computer-executable instructions may be organized into one or more computer-executable components or modules. Generally, program modules include, but are not limited to, routines, programs, objects, components, and data structures that perform particular tasks or implement particular abstract data types. Aspects of the invention may be implemented with any number and arrangement of such components or modules. For example, aspects of the invention are not limited to the specific computer-executable instructions or the specific components or modules illustrated in the accompanying drawings and described herein. Other embodiments of the invention may include different computer-executable instructions or components having more or less functionality than those illustrated in the accompanying drawings and described herein. Aspects of the invention may also be implemented in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media, including memory storage devices.

[0297] In view of the above, it will be seen that the several advantages of the disclosure are achieved and other advantageous results obtained. Since various changes can be made in the method and system described above without departing from the scope of the disclosure, all matter contained in the above description and shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

1. 1. A computing device for measuring real-time glomerular filtration rate (GFR) in a patient in need of such measurement based on measurements of spectral energy received from a sensor, comprising: the sensor is placed on the patient's skin and is capable of detecting and measuring spectral energy, including light, emitted from a tracer agent administered to the patient, the tracer agent emitting spectral energy when exposed to electromagnetic radiation; the computing device measures the patient's GFR by correlating a decrease in light emitted from the tracer agent over a measurement time window with the patient's GFR and calculating an attenuation parameter related to renal clearance over the measurement time window; the decay parameter is a rate constant that is directly correlated to the GFR normalized for the patient's body size, the rate constant being determined by fitting an exponential function to the spectral energy as a function of time or a linear function to the logarithm of the spectral energy as a function of time, the fitting beginning after equilibration of the tracer agent between vascular and extravascular body spaces.

2. 2. The computer device of claim 1, The computing device further comprising a memory.

3. 3. The computer device of claim 2, The computing device further comprising a processor communicatively coupled to the memory.

4. 4. The computer device of claim 3, The sensor includes one or more sensor heads, the computing device configured to be operatively connected to the one or more sensor heads.

5. 5. A computer device according to claim 4, A computer device, wherein the processor is programmed to record one or more measurements of spectral energy transmitted from the one or more sensor heads over the measurement time window, before and / or after administration of the tracer agent to the patient.

6. 6. A computer device according to claim 5, The processor is further programmed to correlate a decrease in spectral energy emitted by the tracer agent over the measurement time window with the patient's GFR.

7. 5. A computer device according to claim 4, A computing device, wherein the operative connection between the computing device and the one or more sensor heads is a direct or indirect mechanical, electrical, and / or communicative connection.

8. 4. The computer device of claim 3, The computing device further comprising a display device communicatively connected to the processor.

9. 4. The computer device of claim 3, The computing device further comprising a user interface communicatively connected to the processor.

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