Compositions and methods to treat alzheimer's disease

Abstract

Aspects of the invention are drawn to methods for identifying or treating Alzheimer's Disease and / or Alzheimer's Disease and Related Dementias (AD / ADRD) in a subject. Further aspects of the invention are drawn to methods for screening the presence of an Alzheimer's Disease and / or Alzheimer's Disease and Related Dementias (AD / ADRD) signature.

Description

COMPOSITIONS AND METHODS TO TREAT ALZHEIMER’S DISEASE

[0001] This application is an International Application which claims priority from U.S. provisional patent application no. 63 / 547,282. filed on November 3, 2023, and U.S. provisional patent application no. 63 / 625,488, filed on January 26, 2024, the entire contents of each of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.GOVERNMENT SUPPORT

[0004] This invention was made with government support under P20 GM121307 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION|0005| Aspects of the invention are drawn to methods of identifying and treating Alzheimer’s Disease and / or Alzheimer’s Disease and Related Dementias (AD / AD RD). Further aspects of the invention are draw n to methods for screening the presence of an AD / ADRD signature.BACKGROUND OF THE INVENTION

[0006] There is growing evidence that vascular stress is an important contributor to the pathogenesis of Alzheimer’s Disease and / or Alzheimer’s Disease and Related Dementias (AD / ADRD). It was previously reported that individuals with AD / ADRD have elevated levels of total, acid-labile, and bound plasma sulfides, which were associated with measures of cognitive dysfunction, brain atrophy and cerebral microvascular disease. While the increased levels of these sulfide species have now been described in human AD / ADRD, the tissue andenzymatic sources remain unclear. Identifying these sources can provide important insights into the pathomechanisms linking plasma sulfides with disease activity and progression in AD / ADRD.SUMMARY OF THE INVENTION

[0007] An aspect of the invention is directed to method of identifying and / or treating a subject afflicted with Alzheimer's Disease or Alzheimer's Disease and Related Dementias (AD / ADRD). In embodiments, the method comprises determining the protein level of one or more H2S-generating enzymes in a biological sample obtained from a subject. In embodiments, the protein level of one or more H2S-generating enzymes is indicative of a subject afflicted with AD / ADRD. In embodiments, treating the subject afflicted with AD / ADRD comprises administering an H2S-generating enzyme inhibitor, an H2S-generating enzyme substrate. H2S- generating enzyme genetic augmentation, or a combination thereof.

[0008] In embodiments, the method further comprises obtaining the biological sample from the subj ect.

[0009] In embodiments, the protein level of the one or more H2S-generating enzymes in the biological sample is at least 20% higher or lower than the protein level of the one or more H2S- generating enzymes in an age-matched control sample is indicative of a subject afflicted with AD / ADRD.

[0010] In embodiments, the one or more HzS-generating enzymes comprises Cystathionine beta synthase (CBS) and / or Cystathionine gamma-lyase (CSE). Optionally, the one or more H2S -generating enzymes further comprises 3-mercaptosulfo transferase (3-MST).

[0011] In embodiments, the protein level of CBS is indicative of a subject afflicted with AD / ADRD. For example, a protein level of CBS about two times higher than the protein level of CBS in an age-matched control sample is indicative of AD / ADRD.

[0012] In embodiments, the protein level of CSE is indicative of a subject afflicted with AD / ADRD. For example, a protein level of CSE about 20% lower than the protein level of CSE in an age-matched control sample is indicative of AD / ADRD.

[0013] In embodiments, H2S generating inhibitors comprise hydroxylamine (HA) and aminooxyacetic acid (AOAA).

[0014] Aspects of the invention are further directed to a method for screening the presence of an Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) signature. In embodiments, the method comprises obtaining a biological sample from a subj ectand detecting the presence of at least one AD / ADRD biomarker in a biological sample from the subject. In embodiments, the level of the AD / ADRD biomarker in the biological sample is higher or lower than the level of AD / ADRD biomarker in an age-matched control sample. In embodiments, the AD / ADRD biomarker is indicative of a subject afflicted with AD / ADRD. In embodiments, the AD / ADRD biomarker comprises one or more H2S-generating enzymes. In embodiments, the method comprises treating the subject afflicted with AD / ADRD. In embodiments, treating comprises administering a pharmaceutically effective amount of a therapeutic. In embodiments, the therapeutic comprises a PkS-generating enzyme inhibitor, an additional H2S-generating enzyme substrate, FhS-generatmg enzy me genetic augmentation, or a combination thereof.

[0015] In embodiments, the one or more FhS-generating enzymes comprise Cystathionine beta synthase (CBS) and / or Cystathionine gamma-lyase (CSE). Optionally, the I-bS-generating enzyme comprises 3 -mercaptosulfo transferase (3-MST).

[0016] In embodiments, the one or more FkS-generating enzymes comprises Cy stathionine beta synthase (CBS) and / or Cystathionine gamma-lyase (CSE). Optionally, the one or more H2S -generating enzymes further comprises 3-mercaptosulfo transferase (3-MST).

[0017] In embodiments, the prtotein level of CBS is indicative of a subject afflicted with AD / ADRD. For example, a protein level of CBS about two times higher than the protein level of CBS in an age-matched control sample is indicative of AD / ADRD.|0018| In embodiments, the protein level of CSE is indicative of a subject afflicted with AD / ADRD. For example, a protein level of CSE about 20% lower than the protein level of CSE in an age-matched control sample is indicative of AD / ADRD.

[0019] In embodiments, H2S generating inhibitors comprise hydroxylamine (HA) and aminooxyacetic acid (AOAA).

[0020] In embodiments, detecting comprises performing an assay to determine the protein level of one or more H2S-generating enzymes in the biological sample. For example, an assay comprises an immunoassay, a colorimetric assay, a fluorometric assay, or a combination thereof. For example, the immunoassay comprises a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or a combination thereof.

[0021] In embodiments, the method further comprises incubating the biological sample with an agent that binds to the one or more H2S-generating enzymes. For example, the agent comprises an antibody. For example, the antibody comprises an anti-CBS antibody or an anti- CSE antibody.

[0022] In embodiments, the biological sample comprises plasma, blood, urine, or saliva.

[0023] In embodiments, the method further comprises isolating extracellular vesicles and / or microparticles from the biological sample to determine the level of the one or more H2S- generating enzymes.

[0024] In embodiments, the therapeutic comprises a CBS inhibitor if the one or more H2S- generating enzy mes is CBS.

[0025] In embodiments, the therapeutic comprises a CSE substrate if the one or more H2S- generating enzymes is CSE.

[0026] Aspects of the invention are further directed to a method of treating a subject afflicted with Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD). In embodiments, the method comprises administering an EES-generating enzyme inhibitor, an H2S -generating enzyme substrate, and H2S -generating enzyme genetic augmentation, or any combination thereof.

[0027] In embodiments, the one or more H2S -generating enzymes includes CSE, and the therapeutic includes a CSE inhibitor.

[0028] In embodiments, the one or more H2S -generating enzymes includes CBS, and the therapeutic includes one of a CBS substrate, a CBS genetic augmentation, or both.

[0029] In embodiments, the one or more EES-generating enzymes includes CSE and CBS, and the therapeutic includes (i) a CSE inhibitor and (ii) a CBS substrate, a CBS genetic augmentation, or both.|0030| In embodiments, the method further comprises administration of a further therapeutic. For example, the further therapeutic includes one of a biologic and / or an immunotherapy.

[0031] Disclosed herein are methods of identifying a subject responsive to one or more CBS inhibitors, the subject afflicted with Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD), the method comprising determining H2S levels in a first biological sample obtained from the subject; administering to the subject a therapeutically effective dose of the one or more CBS inhibitors; and determining H2S levels in a second biological sample obtained from the subject; wherein the subject is responsive to the one or more CBS inhibitors if H2S levels in the second biological sample are lower than the H2S levels in the first biological sample. In some embodiments, the H2S levels in the second biological sample are at least and / or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, 100% lower than the H2S levels in the first biological sample. In some embodiments, the method includes further determining CBS levels in the first biological sample. In some embodiments, a CBS level in the first biologicalsample is at least and / or about two times higher than the level of CBS in an age-matched control sample is indicative of AD / ADRD. In some embodiments, the one or more CBS inhibitors comprise hydroxylamine (HA), aminooxyacetic acid (AOAA), or both. In some embodiments, the second biological sample is obtained within about 1-60 minutes, 1 to 24 hours, 1 to 30 days after administering to the subject the CBS inhibitor.

[0032] Disclosed herein are methods of delaying in a subject the onset of Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) comprising administering to the subject a therapeutically effective dose of one or more CBS inhibitors. In some embodiments, the one or more CBS inhibitors comprises hydroxylamine (HA), aminooxy acetic acid (AOAA), or both. In some embodiments, the subject has not been diagnosed with Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) before administering the one or more CBS inhibitors. In some embodiments, CBS levels in a biological sample obtained from the subject before the administering the therapeutic dose of the one or more CBS inhibitor are higher than about 0. 1, 0.2, 0.3, 0.4, 0.5, one or two times the level of CBS in an age-matched control sample.

[0033] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0034] Certain illustrations, charts, or flow charts are provided to allow for a better understanding for the present invention. It is to be noted, however, that the drawings illustrate only selected embodiments of the inventions and are therefore not to be considered limiting of scope. Additional and equally effective embodiments and applications of the present invention exist.

[0035] FIG. 1 provides dot blots showing plasma cystathionine beta synthase (CBS) and plasma cystathionine gamma-lyase (CSE) in Alzheimer's Disease and Alzheimer's Disease and Related Dementias (AD / ADRD) samples.

[0036] FIG. 2 provides a bar graph showing the comparison of sulfide producing enzymes across groups. CBS = cystathionine beta synthase; CSE = cystathionine gamma-lyase; 3-MST = 3-mercaptosulfo transferase. *p<0.05.

[0037] FIG. 3 provides scatterplots for Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) score versus plasma enzyme levels for each of the groups.

[0038] FIG. 4 provides scaterplots for plasma enzyme levels versus age at assessment for each of the groups. CBS and CSE levels were significantly correlated with age.

[0039] FIG. 5 provides scaterplots showing beta-amyloid and plasma sulfide enzyme levels. MST was found to be strongly correlated with beta-amyloid level.

[0040] FIG. 6 provides scaterplots showing beta-amyloid and plasma sulfide enzyme levels. Acid-labile sulfide was correlated with beta-amyloid level.

[0041] FIG. 7 provides a violin plot showing plasma beta-amyloid was significantly elevated in Alzheimer’s Disease, with plasma beta-amyloid 19% higher in Alzheimer’s samples than in controls, p-value = 0.0121, (Control, n=32; AD, n= 38)

[0042] FIG. 8 provides a violin plot showing plasma CBS was significantly elevated in Alzheimer’s Disease, with plasma CBS 2.38 times higher in Alzheimer’s samples than in controls, p-value = 0.0001, (Control, n=32; AD, n= 38)

[0043] FIG. 9 provides a violin plot showing plasma CSE was significantly lower in Alzheimer's Disease, with plasma CSE 0.21 times lower in Alzheimer’s samples than in controls, p-value = 0.034, (Control, n=32; AD, n= 38)

[0044] FIG. 10 provides a violin plot showing ATIR.Rb was significantly elevated in Alzheimer’s Disease, with ATlR.Rb 2.263 times higher in Alzheimer’s samples than in controls, p-value = 0.0001, (Control, n=32; AD, n= 38)

[0045] FIG. 11 provides a violin plot showing ACE.Ms was significantly elevated in Alzheimer’s Disease, with ACE.Ms 1.61 times higher in Alzheimer’s samples than in controls, p-value = 0.0024, (Control, n=32; AD, n= 38)

[0046] FIG. 12 provides a violin plot showing CSE.Rb was significantly lower in Alzheimer's Disease, with CSE.Rb 21% lower in Alzheimer’s samples than in controls, p- value = 0.0364, (Control, n=32; AD, n= 38)

[0047] FIG. 13 provides a violin plot showing MST3.Ms was not significantly different in Alzheimer’s Disease samples compared to control samples. (Control, n=32; AD, n= 38)

[0048] FIG. 14 provides a violin plot showing ACE2.Ms was not significantly different in Alzheimer's Disease samples compared to control samples. (Control, n=32; AD, n= 38)

[0049] FIG. 15 provides a violin plot showing TGFBIP.Rb was not significantly different in Alzheimer’s Disease samples compared to control samples. (Control, n=32; AD, n= 38)

[0050] FIG. 16 provides a violin plot showing Chymase.Ms was not significantly different in Alzheimer’s Disease samples compared to control samples. (Control, n=32; AD, n= 38)

[0051] FIG. 17 provides a violin plot showing APP.Rb was not significantly different in Alzheimer’s Disease samples compared to control samples. (Control, n=32; AD, n= 38)

[0052] FIG. 18 depicts a graph showing that plasma CBS was significantly higher in AD than in control.DETAILED DESCRIPTION OF THE INVENTION

[0053] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0054] The singular forms ’‘a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one.” and “one or more than one.”

[0055] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0056] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0057] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0058] As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0059] Aspects of the invention are drawn to methods of identifying a subject afflicted with Alzheimer’s Disease and / or Alzheimer’s Disease and Related Dementias (AD / ADRD). For example, embodiments comprise determining a protein level of one or more FbS-generating enzymes in a biological sample obtained from a subject, wherein an elevated or lowered protein level of one or more bbS-generating enzymes is indicative of a subject afflicted with AD / ADRD. For example, embodiments described herein comprise treating the subject afflicted with AD / ADRD by administering an FhS-generating enzyme inhibitor, an FhS-generating enzyme substrate, FhS-generating enzyme genetic augmentation, or a combination thereof.

[0060] Aspects of the invention can pertain to neurological disease or a neurodegenerative disorder, such as methods of identifying a neurological disease or a neurodegenerative disease, and / or methods of treating subjects afflicted with the same. ‘"Neurological diseases” can refer to diseases of the central and peripheral nervous system, such as the brain, spinal cord, cranial nerves, peripheral nen es, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. Non-limiting examples of neurological diseases include, but are not limited to, Alzheimer's disease (AD). Alzheimer’s Disease and Related Dementias (ADRD), dementia, age-related dementia. Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy. In some embodiments, the neurological disease or disorder is AD or ADRD.

[0061] In embodiments, the terms "Alzheimer's disease" and "AD" can refer to a progressive neurodegenerative disorder of the brain and the most common cause of dementia after the age of 65 years. The pathological criteria of AD include intraneuronal neurofibrillary tangles (NFT) composed of paired helical filaments of hyperphosphorylated tau protein, deposits of the proteolytic fragments Ab40 and Ab42 of the amyloid precursor protein (APP) in form of extracellular neuritic (senile) plaques and congophilic angiopathy, and loss of neurons. The lesions develop in the hippocampal region and spread to other brain regions in a characteristic spatio-temporal pattern. The degree of dementia may correlate with the number of NFT lesions rather than with the burden of neuritic plaques, and definite neuropathological diagnosis of AD can be established only in combination with the clinical diagnosis.

[0062] The rate of progression is different for each person. If Alzheimer's disease develops rapidly, it is likely to continue to progress rapidly. If it has been slow to progress, it will likely continue on a slow course. Two types of Alzheimer's disease include early onset Alzheimer’s disease and late onset Alzheimer’s disease. In early onset Alzheimer's disease, symptoms firstappear before age 60. Early onset Alzheimer's disease is much less common, accounting for only 5- 10% of cases. However, it tends to progress rapidly. Early onset disease can run in families and involves autosomal dominant, inherited mutations that may be the cause of the disease. So far, three early onset genes have been identified. Late onset Alzheimer's disease, the most common form of the disease, develops in people 60 and older and is thought to be less likely to occur in families. Late onset Alzheimer's disease may run in some families, but the role of genes is less direct and definitive. These genes may not cause the disease itself, but simply increase the likelihood of formation of plaques and tangles or other Alzheimer's disease- related pathologies in the brain.

[0063] The etiology of AD is multifactorial, but in certain rare families AD segregates as an autosomal dominant disorder with age of onset in the 40s and 50s. Disease-causing mutations have been identified in the amyloid precursor protein gene (APP), and in the presenilin 1 and presenilin 2 genes (PSEN1 and PSEN2). At the biochemical level these mutations are associated with a change in the proteolytic cleavage of APP increasing the production of either total Abeta or selectively the highly amyloidogenic fragment Ab42. This change in Ab production and the extent to which Ab initiate the pathogenic process leading to neuritic plaques and, most importantly, to formation of intraneuronal NFT and neuron loss has been studied intensively.

[0064] The cause of Alzheimer's disease is not entirely known but is thought to include both genetic and environmental factors. A diagnosis of Alzheimer's disease is made based on characteristic symptoms and by excluding other causes of dementia. The only way to validate a case of Alzheimer's disease is by microscopic examination of a sample of brain tissue after death.

[0065] The brain tissue shows "neurofibrillary tangles", "neuritic plaques" (abnormal clusters of dead and dying nerve cells, other brain cells, and protein), and "senile plaques" (areas where products of dying nerve cells have accumulated around protein). Although these changes occur to some extent in all brains with age, there are many more of them in the brains of people with Alzheimer's disease. The destruction of nerve cells (neurons) leads to a decrease in neurotransmitters (substances secreted by a neuron to send a message to another neuron). The correct balance of neurotransmitters is critical to the brain. By causing both structural and chemical problems in the brain, Alzheimer's disease appears to disconnect areas of the brain that normally work together.

[0066] Other types of Alzheimer’s disease encompass familial Alzheimer's disease and sporadic Alzheimer's disease. The term "familial Alzheimer's disease" refers to Alzheimer'sdisease associated with genetic factors (ie, demonstrates inheritance), while "sporadic Alzheimer's disease" refers to Alzheimer's disease that is not associated with a family history of the illness.

[0067] The main pathologies within brain tissue include extracellular neuritic 1 -amyloid plaques, neurofibrillar tangles, neurofibrillar degeneration, granulovascular neuronal degeneration, synaptic loss and extensive neuronal cell death.

[0068] “Alzheimer’s Disease and Related Dementias7’ and “(AD / ADRD)” can refer to debilitating conditions that impair memory, thought processes, and functioning, primarily among older adults. ADRDs share many cognitive and pathological features with and can be difficult to distinguish from AD. As used herein, AD and ADRD can be used interchangeably.

[0069] “Dementia” can refer to a condition characterized by a decline in a person’s cognitive functioning — thinking, remembering, reasoning — and behavioral abilities to such an extent that it interferes with daily life and activities, such as memory, language skills, visual perception, problem solving, self-management, and the ability to focus and pay attention. Some dementias are reversible if caught early enough and current research is showing that some dementias may be preventable.

[0070] The terms “identifying” and / or “diagnosing” are known to the skilled artisan, and can refer to becoming aware of a particular medical condition, disease, complication, or risk. For example, the particular medical condition can be AD / ADRD. In embodiments, for example, a subject can be identified or diagnosed as having AD / ADRD by determining a level of one or more H2S -generating enzymes in a biological subject obtained from a subject. For example, a subject can be identified or diagnosed as having AD / ADRD by having a significantly increased level of CBS as compared to an age-matched control.

[0071] The term “staging” or “disease staging” can refer to a measure of the severity of a disease or disorder, such as AD / ADRD, that uses objective medical criteria to assess the stage of disease progression of a particular medical condition, disease, complication, or risk. For example, a subject can be staged with a particular medical condition, such as AD / ADRD by determining a level of one or more H2S-generating enzymes in a biological subject obtained from a subject. The stages of Alzheimer’s disease, for example, can be separated into three categories: mild Alzheimer's disease, moderate Alzheimer's disease and severe Alzheimer's disease.

[0072] In the early stage of Alzheimer's, a person may function independently. He or she may still drive, work and be part of social activities. Despite this, the person may feel as if he or she is having memoiy lapses, such as forgetting familiar words or the location of everydayobjects. Symptoms may not be widely apparent at this stage, but family and close friends may take notice and a doctor would be able to identify symptoms using certain diagnostic tools. Common difficulties include coming up with the right word or name, remembering names when introduced to new people, having difficulty performing tasks in social or work settings, forgetting material that was just read, losing or misplacing a valuable object, experiencing increased trouble with planning or organizing.

[0073] Middle-stage Alzheimer's is typically the longest stage and can last for many years. As the disease progresses, the person with Alzheimer's will require a greater level of care. During the middle stage of Alzheimer’s, the dementia symptoms are more pronounced, the person may confuse words, get frustrated or angry, and act in unexpected ways, such as refusing to bathe. Damage to nerve cells in the brain can also make it difficult for the person to express thoughts and perform routine tasks without assistance. Symptoms, which vary from person to person, may include being forgetful of events or personal history', feeling moody or withdrawn, especially in socially or mentally challenging situations, being unable to recall information about themselves like their address or telephone number, and the high school or college they attended, experiencing confusion about where they are or what day it is, requiring help choosing proper clothing for the season or the occasion, having trouble controlling their bladder and bowels, experiencing changes in sleep patterns, such as sleeping during the day and becoming restless at night, showing an increased tendency to wander and become lost, demonstrating personality and behavioral changes, including suspiciousness and delusions or compulsive, repetitive behavior like hand-wringing or tissue shredding.

[0074] In the final stage of the disease (e.g., late-stage Alzheimer’s disease), dementia symptoms are severe. Individuals lose the ability to respond to their environment, to carry on a conversation and, eventually, to control movement. They may still say words or phrases, but communicating pain becomes difficult. As memory and cognitive skills continue to worsen, significant personality changes may take place and individuals need extensive care. At this stage, individuals may require around-the-clock assistance with daily personal care, lose awareness of recent experiences as well as of their surroundings, experience changes in physical abilities, including walking, sitting and. eventually, swallowing, have difficulty communicating, become vulnerable to infections, especially pneumonia.

[0075] Embodiments as described herein comprise determining the protein level of one or more FhS-generating enzy mes in a biological sample obtained from a subject. “Hydrogen sulfide” and “HzS” can refer to a colorless gasotransmitter that is known to alleviate a variety of illnesses such as cancer, heart disease, and neurological conditions. Hydrogen sulfide isproduced through [ HS-generating enzymes, non-limiting examples of which include cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), and 3- mercaptopyruvate sulfurtransferase (3-MST). The term “HiS-generating enzymes” can refer to enzymes that can naturally synthesize hydrogen sulfide (H2S). For example, CBS, CSE, and 3-MST enzymatically regulate the production of H2S through direct or indirect use of sulfur- containing amino acids as their substrates.

[0076] As shown in Example 2 and Example 3. protein levels of plasma CBS was significantly increased in AD / ADRD Example 2 further shows that elevated CBS was associated with poorer cognitive performance and that MST was positively correlated with beta-amyloid levels. These results indicate that the pathologically elevated bound and acid labile sulfides in AD / ADRD are associated with increased levels of circulating CBS. As used herein the term “circulating” or “circulating levels” can refer to the amount of an analyte, such as CBS, CSE, and / or 3-MST, in the blood of a mammal, such as in the liquid portion of the blood. In embodiments, a protein level of the one or more H2S -generating enzymes in a biological sample that is at least 20% higher or at least 20% lower than in an age-matched control sample is indicative of a subject afflicted with AD / ADRD. For example, the protein level of the one or more FhS-generating enzymes (e.g., CBS) in a biological sample can be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% higher than in an age-matched control sample. For example, the protein level of the one or more H2S -generating enzymes (e.g.. CSE) in a biological sample can be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1 ,000%, 5,000% lower than in an age-matched control sample.

[0077] Referring to FIG. 18, for example, protein levels of plasma CBS is significantly increased in AD / ADRD relative to control. For example, protein levels of plasma CBS can be significantly increased in AD / ADRD relative to control by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%. For example, protein levels of plasma CBS can be significantly increased in AD / ADRD relative to control by at least 40%.

[0078] In embodiments, the protein level of one or more H2S -generating enzymes in a biological sample obtained from a subject can be changed when compared to a control sample. As used herein, “changed as compared to a control” sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator (e.g., marker) to be detected at a level that is statistically different than a sample from a normal, untreated, or abnormal state control sample. Determination of statistical significance is within the ability' of those skilled inthe art, e.g., the number of standard deviations from the mean that constitute a positive or negative result.

[0079] In embodiments, the H2S -generating enzyme can comprise cystathionine beta synthase. “Cystathionine beta synthase” and “CBS” can refer to the first (and rate-limiting) H2S -generating enzyme in the transsulfuration pathway. CBS is an important mammalian enzyme in health and disease, and its biochemical functions under physiological conditions include the generation of hydrogen sulfide (H2S). a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system, and the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor). In embodiments, a protein level of CBS that is about two times higher than the protein level of CBS in an age-matched control sample is indicative of AD / ADRD.

[0080] In embodiments, the FES-generating enzyme can comprise cystathionine gammalyase. “Cystathionine gamma-lyase” and “CSE” can refer to a H2S-generating enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. CSE can use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2S). In embodiments, a protein level of CSE that is about 20% lower than the protein level of CSE in an age-matched control sample is indicative of AD / ADRD.

[0081] In embodiments, the FES-gencrating enzyme can comprise 3-mercaptosulfo transferase. “3-mercaptosulfo transferase” and “3-MST” can refer to a EES-generating enzyme that mediates the reaction of 3 -mercaptopyruvate with dihydrolipoic acid and thioredoxin to produce hydrogen sulfide.

[0082] The methods described herein typically involve obtaining a biological sample from the subject, such as a subject with a AD / ADRD. As used herein, the phrase “obtaining a biological sample” can refer to any process for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample may be obtained (e.g., at a point-of- care facility, e.g., a physician's office, a hospital, laboratory facility) by procuring a tissue or fluid sample (e.g., blood draw, marrow sample, spinal tap) from a subject. Alternatively, a biological sample may be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject.

[0083] The biological sample can be a biological fluid, i.e., a bodily fluid. The bodily fluid comprises peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid,pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some embodiments, the biological sample comprises blood or a blood derivative, such as peripheral blood, sera, or plasma.

[0084] In embodiments, a ‘"control sample” can refer to a biological sample that serves as a reference, usually a known reference, for comparison to a test sample. In embodiments, a test sample can be taken from a subject suspected of having a given disease (e.g. a neurological disease, such as AD / ADRD) and compared to a sample taken from a known normal (nondiseased) individual (e.g. a control sample). A control sample can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population). For example, control subjects can be healthy individuals with a similar medical background and similar age. A control sample can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, the value of a control sample can be devised to compare therapeutic benefit based on pharmacological data (e.g.. half-life) or therapeutic measures (e.g., comparison of side effects). Control samples are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in control samples, variation in test samples will not be considered as significant.|0085| In embodiments, the control sample can be an age-matched normal, unimpaired subject.

[0086] Aspects of the invention are further drawn tow ards methods of screening for the presence of an AD / ADRD signature. The term “screening” can refer to checking whether a subject with characteristics indicative of AD / ADRD (for example, positive symptoms or other tests) or with risk factors for AD / ADRD (for example, increasing age, familial inheritance, exposure to aluminium, traumatic brain injury (TBI), and associated co-morbidities such as vascular disease and infection), have AD / ADRD.

[0087] For example, embodiments comprise detecting the presence of at least one AD / ADRD biomarker in a biological sample from the subject. The term “biomarker” can refer to a measurable substance in an organism whose presence is indicative of some phenomenon such as disease, infection, or environmental exposure. In embodiments, the biomarker can comprise one or more H2S-generating enzy mes. For example, the H2S -generating enzymes can comprise CBS and CSE. Optionally, the H2S-generating enzyme can comprise 3-MST.

[0088] In embodiments, one or more molecular biomarkers, such as those provided in Example 2, can provide a “molecular signature’7that can be used in a variety of diagnostic, prognostic, and therapeutic methods. For example, the molecular signature can be used to diagnose and / or prognose a neurological disease, such as AD / ADRD. For example, the molecular signature can be used to track the efficacy of a treatment.

[0089] In one embodiment, the comparison of the herein described biomarker level relative to a reference or control level allows the person skilled in the art to identify a subject afflicted with AD / ADRD and / or treat the subject afflicted with AD / ADRD. As used herein, a “low” level of a biomarker in a sample can be a level that is less than the level of the biomarker from the reference and / or control sample(s). A “low” level of a biomarker in a sample can also refer to a level that is decreased in comparison to the level of the biomarker reached upon treatment, for example with an anti-AD / ADRD treatment. A “low” level of a biomarker can also refer to a level that is present in comparison to an individual that does not have AD / ADRD. In certain cases, the low level of a biomarker in a sample can be an indication of AD / ADRD, or of AD / ADRD status, or of progression of AD / ADRD.

[0090] A “high” level of a biomarker in a sample can be a level that is elevated in comparison to the level of a biomarker from the reference and / or control sample(s). A “high” level of a biomarker in a sample can also refer to a level that is elevated in comparison to the level of the biomarker reached upon treatment, for example with an anti-AD / ADRD treatment. A “high” level of a biomarker can also refer to a level that is present in comparison to an individual that does not have AD / ADRD. In certain cases, the high level of a biomarker in a sample can be an indication of AD / ADRD, or of AD / ADRD status, or of progression of AD / ADRD.

[0091] “Determining a level” or “detecting a level” of an AD / ADRD biomarker can refer to an assessment performed on a biological sample. For example, the level can be the “protein level” of an AD / ADRD biomarker in a biological sample.

[0092] Embodiments described herein can comprise determining a protein level of one or more FES-generating enzymes in a biological sample or detecting the presence of at least one AD / ADRD biomarker in a biological sample. A “biological sample” can refer to a material containing, for example, a nucleic acid, protein or other biological or chemical material of interest. In embodiments, the biological sample can be obtained from a subject by methods known to the skilled artisan. In an embodiment, the biological sample is selected from, but without being limited thereto, blood and fractions thereof, blood serum, blood plasma, urine, excreta, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper'sfluid), pleural effusion, tears, saliva, sputum, sweat, biopsy, ascites, amniotic fluid, lymph, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions, and breast secretions. In embodiments, extracellular vesicles and / or microparticles can be isolated from the biological sample.

[0093] Extracellular vesicles” can refer to lipid bound vesicles secreted by cells into the extracellular space. The three main subtypes of extracellular vesicles are microvesicles, exosomes, and apoptotic bodies, which are differentiated based upon their biogenesis, release pathways, size, content, and function. CBS is highly expressed in the brain, and endothelial cells release CBS into extracellular vesicles in response to inflammatory' stimuli (Veerareddy et al., 2021). Human brain endothelial cells exposed to inflammatory stimuli increase the shedding of CBS within apical extracellular vesicles, which matches the appearance of plasma CBS in the vascular compartment in AD / ADRD (Veerareddy et al., 2021). While the exact likelihood of CBS shedding by endothelial cells is unclear, it is possible that removal of CBS from the endothelial cells by the process of extracellular vesicle shedding could have negative vascular consequences. In embodiments, extracellular vesicles and / or microparticles can be isolated from the biological sample to determine the level of one or more H2S-generating enzymes. For example, extracellular vesicles and / or microparticles can be isolated from the biological sample to determine the level of CBS in the biological sample.

[0094] In embodiments, the detecting and / or the determining can comprise performing an assay to determine the presence of and / or the protein level of one or more H2S-generating enzymes in a biological sample. For example, the H2S-generating enzymes can comprise CBS and / or CSE. Optionally, the H2S-generating enzymne can comprise 3-MST. Non-limiting examples of such assays comprise an immunoassay (e g., comprise a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation), a colorimetric assay, a fluorometric assay, or others known in the art.

[0095] Aspects of the invention are also drawn to methods of treating a subject afflicted with AD / ADRD or preventing the development of or progression of AD / ADRD. The terms “treat,” “treatment.” and “treating” can refer to the management and care of a subject for the purpose of combating a condition, disease or disorder, such as a AD / ADRD, in any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. The term can include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of an active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease or disorder; curing or eliminating the condition, disease or disorder; and / orpreventing the condition, disease or disorder. "Preventing" or "prevention" can refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, such as AD / ADRD, and includes the administration of an active compound to prevent or reduce the risk of the onset of symptoms or complications.

[0096] Aspects of the invention are also draw n tow ards methods of alleviating one or more symptoms of AD / ADRD in a subject. The term “alleviating a symptom of' can refer to relieving a symptom or making it less intense or severe. Non-limiting examples of such symptoms include, among others, mild or severe dementia, progressive memory impairment (ranging from slight forgetfulness to disorientation and severe memory' loss), poor visual- spatial abilities, changes in personality, poor impulse control, poor judgment, distrust of others, increased arrogance, restlessness, poor planning capacity, poor decision-making and withdrawal from social relationships. In severe cases, patients lose the ability to use language and communicate, and require help for personal hygiene, eating and dressing, and ultimately remain confined to bed.

[0097] In embodiments, a subject afflicted with AD / ADRD can be treated by administering an H2S-generating enzyme substrate. The term “substrate” can refer to a molecule that an enzyme reacts with, such as molecule that an H2S-generating enzyme reacts with. In embodiments, the FbS-generating enzyme substrate can be a CSE substrate. In embodiments, the FhS-generaling enzyme substrate can be a CBS substrate. For example, the CBS substrate can comprise homocysteine and / or serine. CBS catalyzes the conversion of these substrates into cystathionine, releasing H2S in the process. In embodiments, the FBS-generating enzyme substrate can be a 3-MST substrate.

[0098] In embodiments, a subject afflicted with AD / ADRD can be treated by administering a therapeutic that binds to one or more FbS-generating enzymes. The term “therapeutic” or “therapeutic agent” can refer to a compound or an agent having a beneficial, therapeutic effect in vivo. Therapeutics can be used in patients with active disease (e.g., to treat the disease itself or its signs and symptoms), in preventive medicine (e.g., to prevent the disease or delay the onset or progression of its signs and symptoms), or as palliative care (e.g., to lessen the severity of signs and symptoms of the disease).

[0099] In embodiments, the therapeutic can be an antibody. The term “antibody” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, or any antigen binding fragment or a single chain thereof. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity ofbinding to the antigen. Non-limiting examples include a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the antigen and does not react with other polypeptides. In embodiments, the antibody can be an anti-H2S- generatying enzyme antibody. In embodiments, the antibody can be a monoclonal antibody, such as an anti-H2S-generating enzyme monoclonal antibody’. For example, the antibody can comprise an anti -CBS antibody, an anti-CSE antibody, and / or an anti-3-MST antibody.

[0100] In embodiments, the therapeutic can be a small molecule. The term “small molecule” can refer to a chemical compound that is small enough in size so that it can readily pass through a cellular membrane unassisted. In general, a small molecule can refer to a chemical compound that is not a polymer, such as a nucleic acid, polypeptide, or polysaccharide, although the term can encompass small polymers that can readily crossing the cellular membrane. Non-limiting examples of such small molecules comprise amino acids, lipids, sugars, fatty acids, alkaloids, and others.|00101| In embodiments, the therapeutic can be a peptide or peptidemimetic. The term "peptide" can refer to a macromolecule which comprises a multiplicity of amino or imino acids (or their equivalents) in peptide linkage. In the polypeptide or peptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyterminal direction, in accordance with standard usage and convention. Peptides can include moieties other than amino acids (e.g, can be glycoproteins, proteoglycans, etc.) and / or can be otherwise processed or modified. Non-limiting examples of such peptides and / or peptidemimetics comprise D-amino acid substitutions, peptoids, urea peptidomimetics, peptide sulfonamides, oligocarbamates, partial or full retro-inverso peptides, azapeptides, (3-peptides and N-modified peptides.

[0102] Peptides can contain L-amino acids, D-amino acids, or both and can contain any of a variety7of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acety lation, amidation, glycosylation, biotinylation, substitution with D- amino acid or unnatural amino acid, and / or cyclization of the peptide. In some embodiments, peptides can comprise natural amino acids, non-natural amino acids, synthetic amino acids,and combinations thereof. A "short peptide" can refer to any peptide containing up to 25 amino acids (e.g., up to 20. 15. 12. 10. 9, 8, 7. 6, 5, 4, or 3). In some embodiments, a short peptide contains 5-25 amino acids. Peptides also can include peptidomimetics unless indicated otherw ise. Herein the terminologies "mimic," "mimetic," “peptidomimetic" and the like can be used herein interchangeably.

[0103] In embodiments, the therapeutic can be in a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent. In embodiments, a pharmaceutically acceptable excipient, carrier, or diluent can comprise any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

[0104] In embodiments, the therapeutic can comprise an H2S-generating enzyme inhibitor, an H2S-generating enzyme substrate, and H2S-generating enzyme genetic therapy, or any combination thereof.

[0105] The term ‘’inhibitor” can refer to any agent which reduces the level and / or activity of a protein or protein complex. The term “inhibiting” can refer to decreasing, limiting, and / or blocking a certain action, function, or interaction. Non-limiting examples of inhibitors include small molecule inhibitors, degraders, antibodies, enzymes, or polynucleotides (e.g.. siRNA). In embodiments, a neurological disease, such as AD / ADRD, can be “inhibited” if at least one symptom of the disease is alleviated, terminated, slowed, or prevented. In embodiments, the H2S -generating enzy me inhibitor can be a CBS inhibitor. For example, the CBS inhibitor can comprise aminooxy acetic acid (AOAA) and hydroxylamine. AOAA interferes with the activity of CBS and can be used to study the physiological roles of H2S produced by CBS. In embodiments, the H2S -generating enzyme inhibitor can be a CSE inhibitor. For example, the CSE inhibitor can comprise dl-propargy Iglycine (PAG). PAG is an analog of the substrate L- cysteine and can selectively inhibit CSE activity, reducing H2S production. In embodiments, the H2S-generating enzyme inhibitor can be a 3-MST inhibitor. For example, the 3-MST inhibitor can comprise P-cyano-L-Alanine (BCA). BCA can be used to block the activity of 3- MST and modulate the functions of H2S produced by this enzyme.

[0106] In embodiments, the H2S-generating enzyme inhibitor can be an antagonist. The term “antagonist” can refer to a compound or composition that can decrease, block, inhibit, abrogate, or interfere with a biological response by binding to or blocking a cellular constituent.

[0107] In embodiments, “genetic therapy" or “genetic augmentation” can refer to the transfer of the genetic material of interest (e.g., DNA or RNA) into a host, to treat or prevent a genetic or acquired disease or disorder. The genetic material of interest encodes a product (e.g., a polypeptide of a protein, a functional peptide or RNA) that is intended to be produced in vivo. For example, the genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide of therapeutic value.

[0108] Pharmaceutical compositions can be in a form adapted to any route of administration, such as oral, subcutaneous, parenteral (intravenous, intraperitoneal, intradermal), intramuscular, rectal, epidural, intratracheal, inhalation, intranasal, transdermal (i.e., topical), transmucosal, vaginal, buccal, ocularly, or pulmonary administration, for example, in a form adapted for administration by a peripheral route or is suitable for oral administration or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions can be prepared in a manner well-known to the person skilled in the art, e.g., as described in “Remington's Pharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton. Pa., U.S.A., 1985 and more recent editions and in the monographs in the “Drugs and the Pharmaceutical Sciences” series, Marcel Dekker. The compositions can appear in conventional forms, for example, solutions and suspensions for injection, capsules and tablets, such as in the form of enteric formulations for oral administration. The composition can also be in a form suited for local or systemic injection or infusion and can, as such, be formulated with sterile water or an isotonic saline or glucose solution. The compositions can be in a form adapted for peripheral administration only, with the exception of centrally administrable forms. The compositions can be in a form adapted for central administration.

[0109] For example, pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition can be sterile and can be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0110] In embodiments, sterile injectable solutions can be prepared by incorporating the therapeutic in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the therapeutic into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-dry ing which yields a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.

[0111] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the therapeutic can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Oral formula of the drug can be administered once a day, twice a day, three times a day, or four times a day, for example, depending on the half-life of the drug.

[0112] In embodiments, the terms ‘ administration ’, “administer’, or “administering’’ can refer to the physical introduction to the subject a composition containing a therapeutic using any of a variety of methods and delivery systems known to those of skill in the art. In embodiments, the therapeutic can comprise an H2S-generating enzyme inhibitor, an H2S- generating enzyme substrate, and H2S -generating enzyme genetic augmentation, or any combination thereof. For example, the therapeutic can be administered intranasally. intramuscularly, orally, intravenously, pulmonary, subcutaneously, or intraperitoneally.

[0113] In embodiments, the therapeutic can be administered by bolus injection or by infusion. A bolus inj ection can refer to a route of administration in which a syrine is connected to the IV access device and the medication is injected directly into the subject. The term “infusion” can refer to an intravascular injection.

[0114] The therapeutic can be administered to a subject one time (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0115] In embodiments, the therapeutic can be administered to a subj ect in a therapeutically effective amount. A ‘Therapeutically effective amount” or “therapeutically effective dose” can refer to that amount of the therapeutic agent sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the neurological disease, such as AD / ADRD, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose can refer to that ingredient alone. When applied to a combination, a therapeutically effective dose can refer to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0116] In embodiments, a therapeutically effective amount, such as a therapeutically effective amount of the therapeutic, can comprise a dose of less than about 0. 1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg. about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, about 300 mg / kg, about 325 mg / kg, about 350 mg / kg, about 375 mg / kg, about 400 mg / kg, about 425 mg / kg, about 450 mg / kg. about 475 mg / kg, about 500 mg / kg, about 525 mg / kg, about 550 mg / kg, about 575 mg / kg, about 600 mg / kg, about 625 mg / kg, about 650 mg / kg, about 675 mg / kg, about 700 mg / kg, about 725 mg / kg, about 750 mg / kg, about 775 mg / kg, about 800 mg / kg, about 825 mg / kg, about 850 mg / kg, about 875 mg / kg, about 900 mg / kg, about 1.0 g / kg, about 1.5 g / kg, about 2.0 g / kg, about 2.5 g / kg. about 5 g / kg, about 10 g / kg, about 25 g / kg. about 50 g / kg, or more than 50 g / kg of compound per body weight of a subject.

[0117] In embodiments, the therapeutically effective amount comprises less than about 0.1 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about100 mg, about 120 mg, about 135 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg. about 275 mg, about 300 mg, about 325 mg, about 350 mg. about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 1.0 g, about 1.5 g, about 2.0 g. about 2.5 g, about 5 g, about 10 g. about 25 g. about 50 g. or more than 50 g.

[0118] A therapeutically effective amount of the therapeutic will depend on the age and weight of the subject and the concentration and / or formulation of the therapeutic.

[0119] As describe herein, aspects of the invention are drawn to methods of identifying a subject afflicted with AD / AD RD. Typical subjects can include mammals, for example primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals, for example, pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g.. mice. rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted herein or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g.. a liver or other organ) from the living subject. The terms “subject”, “individual”, and “patient” can be used interchangeably.

[0120] Embodiments as described herein further comprise administering one or more additional active agents to a subject with the therapeutic. Non-limiting examples of such additional active agents can comprise a vaccine, an anti-inflammatory agent, a pain reliever, a steroid, or any combination thereof.

[0121] Embodiments as described herein further comprise administering one or more additional further therapeutics to a subject with the therapeutic. Non-limiting examples of such therapeutics can comprise a biologic, an immunotherapy, or both. The terms “biologic” or “biological drugs” can refer to drugs that are produced using a living system, such as a microorganism, plant cell, or animal cell. Non-limting examples of biologies can comprise gene therapies, transplant tissue, recombinant proteins, stem cell therapies, and monoclonal antibodies. The term “immunotherapy” can refer to treatments that use the body’s own immune system to combat diseases. Non-limiting examples of immunotherapies used to treat AD / ADRD can comprise AN1792. Amilomotide. UB-311, Aducanumad, Donanemab, Lecanemab, Gantenerumab, Crenezumab, and Solanezumab. (Song et al., 2022).

[0122] Aspects of the invention are also directed towards kits.

[0123] In embodiments, the kit can comprise reagents for detecting AD / ADRD in a subject with at least 80% sensitivity, comprising a reagent for detecting a plurality of biomarkers for AD / ADRD in a biological sample from the subject. For example, the kit may comprise a negative control containing a biomarker at a concentration of about the concentration of the biomarker which is present in a biological sample of an individual who does not have AD / ADRD. The kit also may include a positive control containing the biomarker at a concentration of about the concentration of the biomarker which is present in a biological sample of an individual who has AD / ADRD or has increased risk for AD / ADRD.

[0124] In embodiments, the kit can comprise one or more anti -AD / ADRD therapeutics, such as those described herein. For example, the kit can include (a) a therapeutic agent, such as FbS -generating enzyme inhibitor, an additional H2S -generating enzyme substrate, H2S- generating enzyme genetic augmentation, or a combination thereof, as described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that is drawn to the methods described herein and / or the use of the therapeutic agents for therapeutic benefit.

[0125] The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the reagents therein, including molecular weight, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material comprises methods of using the reagents therein. The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.

[0126] The composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The reagents can be provided in any form, e.g., liquid, dried or lyophilized form, or for example, substantially pure and / or sterile. When the reagents are provided in a liquid solution, the liquid solution is an aqueous solution. When the reagents are provided as a dried form, reconstitution can be by the addition of a suitable solvent. The solvent, e.g.. sterile water or buffer, can optionally be provided in the kit.

[0127] The kit can include one or more containers for the reagent(s). In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undividedcontainer. For example, the reagents can be contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., in a given ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (e.g.. impermeable to changes in moisture or evaporation), and / or light-tight. The kit optionally includes a device suitable for administration of anti-AD / ADRD therapies, e.g., a syringe or other suitable delivery' device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.EQUIVALENTS

[0128] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.EXAMPLES

[0129] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0130] Alzheimer 's disease diagnosis using analysis of cystathionine beta synthase ( CBS) and cystathionine gamma lyase (CSE) in human plasma

[0131] Description of the Invention

[0132] We have previously described changes in the levels of plasma sulfide species in Alzheimer's disease and related dementias (ADRD). These chemical species are found in plasma and appear to reflect the enzymatic activity of CBS and CSE enzymes (Disbrow et al., 2022, Disbrow E, Stokes KY, Ledbetter C, Patterson J, Kelley R, Pardue S, Reekes T, Larmeu L, Batra V, Yuan S, Cvek U, Trutschl M, Kilgore P, Alexander JS, Kevil CG. Plasma hydrogen sulfide: A biomarker of Alzheimer's disease and related dementias. Alzheimers Dement. 2021 Aug; 17(8): 1391-1402. doi: 10.1002 / alz. 12305. Epub 2021 Mar 12. PMID: 33710769; PMCID: PMC8451930.). We have previously described the use of plasma sulfide generating enzyme measurement to diagnose multiple sclerosis (Veerareddy P, Dao N, Yun JW, Stokes KY, Disbrow E. Kevil CG, Cvek U, Trutschl M, Kilgore P, Ramanathan M, Zivadinov R, Alexander JS. Dysregulated Sulfide Metabolism in Multiple Sclerosis: Serum and Vascular Endothelial Inflammatory Responses. Pathophysiology. 2022 Sep 17;29(3):570-582. doi: 10.3390 / pathophysiology29030044. PMID: 36136071; PMCID: PMC9502521.) This approach to measure enzymatic sources of sulfides as opposed to the sulfides themselves is much simpler, inexpensive and less subject to sample instability.

[0133] This invention describes a new method for diagnosing and staging Alzheimer's disease (AD) or Alzheimer's disease and related dementia (ADRD) using circulating levels of CBS and / or CSE and / or 3 -mercaptosulfo transferase (3MST) in plasma specimens. We have found that levels of CBS were highly significantly elevated (p=0.0000000046) compared to age matched controls. Similarly, CSE was significantly decreased in AD / ADRD (p=0.036). By measuring the relative increase and decrease of these enzymes, without wishing to be bound by theory, we will diagnose, stage and evaluate the response to therapy in AD / ADRD.

[0134] The testing methodology relies on either a comparison of a cohort of AD / ADRD specimens with a cohort of controls and then to an unknown specimen. Without wishing to bebound by theory, we will employ enzyme linked immunosorbent analysis (ELISA) of these enzymes. Determination of the ranges of these enzymes in control and AD / ADRD also provides another and simpler method for validating whether a specimen is within the 'range' of a described disease state. FIG. 1 provides dot blots showing plasma CBS and CSE in AD / ADRD samples. The data show a significant increase in CBS as well as a significant decrease in CSE. (Control. n=32, AD n= 38)

[0135] By using both CBS and CSE. we can create a principal component analysis of both enzymes which may reveal disease with even greater selectivity and specificity. Because these enzymes are far more stable to isolation, storage and evaluation than their sulfide products, this approach constitutes a novel and powerful means of diagnosing and staging disease. This testing platform uses dot blotting using a grid containing control and AD / ADRD specimens against which individual specimens can be compared. Alternatively, enzyme linked immunosorbent analysis of CBS and CSE can provide even more sensitive analysis of these enzymes which can be used to diagnose, stage and gauge the response towards AD / ADRD therapeutic approaches.EXAMPLE 2

[0136] Increased Plasma Cystathionine Beta-Synthase (CBS) in Human Alzheimer’s Disease and Related Dementias (ADRD): Correlation with Cognitive Performance100137] Abstract

[0138] There is growing evidence that vascular stress is an important contributor to the pathogenesis of Alzheimer’s disease (AD). We have previously reported that individuals with Alzheimer's disease (ADRD) had elevated levels of total, acid-labile, and bound plasma sulfides, which were associated with measures of cognitive dysfunction, brain atrophy and cerebral microvascular disease. While the increased levels of these sulfide species have now been described in human ADRD, the tissue and enzymatic sources remain unclear, but could provide important insights into the pathomechanisms linking plasma sulfides with disease activity and progression in ADRD. We conducted a comparison of the levels of 3 different enzymatic sources of plasma sulfides, cystathionine beta synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptosulfo transferase (3-MST) in healthy controls (n=29) and individuals who met the criteria for ADRD (based on cognitive scores (Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-cog) >=17; n=35). We found that plasma CBS was significantly increased in ADRD. Plasma beta-amyloid was also significantly increased in ADRD. We also found that CBS was associated with poorer cognitive performance(r(63)= 534, p<0.0001) and that MST was positively correlated with beta-amyloid levels. Finally, acid labile sulfides were significantly elevated in ADRD compared to control groups, and acid labile sulfide was significantly positively correlated with beta-amyloid level. These results indicate that the pathologically elevated bound and acid labile sulfides in ADRD are associated with increased levels of circulating CBS. A better understanding of the underlying vascular pathomechanisms and consequences that confer greater risk due to excess sulfide burden may lead to improved detection and treatment of ADRD.

[0139] Introduction

[0140] Plasma Sulfides, ADRD and Vascular Abnormalities

[0141] Recent evidence indicates that vascular stress is an important contributor to Alzheimer’s disease (AD) pathogenesis. There is a well-documented relationship between AD and cardiovascular disease in human aging (Stakos et al, 2020), and vascular disturbance is a risk factor for AD (Jellinger et al. , 2005). The ‘ Vascular Cognitive Impairment and Dementia" hypothesis (VCID; Hachinski et al., 1993) proposes that peripheral cardiovascular factors such as heart failure and arterial fibrillation result in cerebral damage due to cerebral hypoperfusion and stroke, and imbalances between blood flow-based substrate delivery and brain energy requirements intensify common cardiovascular risks for AD such as hypertension6’8, cerebrovascular disease9’11, and sedentary' lifestyle12, 13. Prior to beta-amyloid deposition and phospho-Tau proteinopathy, AD is preceded by brain microvascular abnormalities15and cerebral hypoperfusion16, 17. In addition, arterial stiffening, another peripheral cardiovascular factor, results in endothelial dysfunction, BBB breakdown, decreased beta amyloid clearance and neurovascular decoupling leading to disrupted neuronal activity, apoptosis and dementia (see Eisenmenger et al., 2023, for review). Cerebrovascular dysfunction may occur early in AD15, setting off a cascade of events including metabolic stresses18and excitotoxic calcium signaling19’21which progressively derange brain structure / function culminating in amyloid plaques and phospho-Tau tangles.

[0142] Currently, the mechanisms and mediators underlying VCID are unclear but may involve ’gaso transmitters' e.g., nitric oxide, carbon monoxide and hydrogen sulfide and their metabolic products. We have recently described how the cerebral microvasculature may be dysregulated in Alzheimer's disease and related dementias (ADRD) which contributes to cognitive dysfunction in this condition (Disbrow et al., 2021, Reekes et al., 2023). Specifically, hydrogen sulfide and its metabolites are known to be important both in neuromodulation and in regulation of vascular homeostasis. Our recent work indicates links exist between redox- related disturbances in sulfide metabolism and measures of cerebrovascular disease andcognitive disturbance in human ADRD. For example, we reported that people with ADRD had increased total plasma sulfides driven by elevated acid-labile and bound sulfide species. Total sulfides were used to discriminate between ADRD and control subjects with an accuracy of 0.93, a specificity of 0.98 and sensitivity of 0.80 (Disbrow et al., 2021). Moreover, we linked sulfide dysregulation to brain atrophy, microvascular disease and cognitive dysfunction (Disbrow et al., 2021; Reekes et al., 2023). While hydrogen sulfide is generally considered to be a weak vasodilator at low concentrations but a vasoconstrictor at high concentration (Kubo et al., 2007, Capmada et al., 2017), without wishing to be bound by theory, bound and acid- labile sulfides may therefore reflect high rates of sulfide generation explaining pathological vasoconstriction in ADRD, yet the exact pathways that yield these sulfides are not yet well understood.

[0143] Three enzymes, namely cystathionine y-lyase (CSE), cystathionine [3-synthase (CBS), and 3 -mercaptopyruvate sulfur transferase (MST), are responsible for producing sulfides. CBS is highly expressed in the brain, and we showed that endothelial cells release CBS into extracellular vesicles in response to inflammatory stimuli (Veerareddy et al.. 2021). In Multiple Sclerosis, we found that CBS protein appeared to be reduced in serum in active disease, here we also find an increase in the amount of CBS protein which tracks with disease. Alterations in sulfide generation which reflect increased total formation with an increased proportion of acid labile and bound sulfides may help explain relationships between sulfides and ADRD progression. An improved understanding of the relationships between plasma sulfides, vascular function and cognition could reveal novel diagnostic, prognostic, and therapeutic approaches for ADRD and perhaps other mechanistically related forms of neurodegeneration.

[0144] Methods

[0145] 2, 1, Data selection

[0146] In order to evaluate the relationships among plasma sulfides and related enzymes and cognitive performance in an older population, participants had to meet the following studyenrollment criteria: Greater than 60 years of age, fluent English speaker, no known or diagnosed neurological or psychiatric disorders associated with cognitive function (not including ADRD), no reported severe head trauma, no history of substance or alcohol use disorder within the 10 years preceding enrollment, not pregnant, and able to give meaningful, informed consent and understand the study procedures (or same ability from a legally authorized representative). Consenting participants who met these criteria completed a neuropsychological assessment including the Alzheimer's Disease Assessment Scale -Cognitive (ADAS-Cog), with a cutoff score for identification of ADRD of 17 or more on the ADAS-Cog. The blood samples were analyzed using a blood-based hydrogen sulfide analysis and a blood plasma-based enzyme assay. Sixty-four participants completed the cognitive testing and blood draw.

[0147] 2,2, Cognitive assessments

[0148] Cognitive function was assessed using Alzheimer's Disease Assessment Scale- Cognitive subscale (ADAS-Cog). which consists of 11 participant or observer-based tasks to assess cognitive domains of memory, language and praxis.

[0149] 2,3 Blood Sample Analysis

[0150] 2.3.1. Blotting Analysis

[0151] Frozen plasma samples were thawed, and 0.5 pL of each of the samples was loaded onto nitrocellulose membranes and left to dry overnight. Ponceau S staining was used for protein loading standardization. A measure of 5% milk and TBST were used to block (2 h) and rinse the blots (3X) after staining. Membranes were immunoblotted for cystathionine y-lyase (CSE), cystathionine [3-synthase (CBS), and 3-mercaptopyruvate sulfur transferase (MST) and incubated in Clarity Western Peroxide Reagent and visualized using Clarity Western Luminol / Enhancer Reagent (Biorad, Hercules, CA, USA). All primary antibodies were used at 1:500 dilution and incubated overnight. Secondary antibodies were used at 1:2000 dilution and incubated for 1 h at room temperature before washing and ECL reactions.|00152| ChemiDoc imaging system (Biorad. Hercules, CA, USA) was used to develop images of membranes. Densitometry was performed using ImageJ analysis software, v. 153 (NIH, Bethesda, MD, USA). Data were normalized to total protein using Ponceau S Staining (Sigma biochemicals, St. Louis, MO, USA).

[0153] 2.3.2. Antibodies

[0154] Rabbit anti-Human Cystathionine-gamma-lyase (CSE) Polyclonal Antibody (cat. No. MBS2014844, MyBioSource, San Diego, CA, USA), Cystathionine-beta-synthase (CBS) (N-Term) Antibody (cat. No. ABIN629598, Antibodies Online, Limerick, PA, USA), Recombinant Anti-MST3 Antibody [EP1468Y] (cat. No. Ab51137, Abeam, Cambridge. MA, USA), and Anti-Rabbit IgG (whole molecule)-Peroxidase antibody produced in goat (cat. No. 028M4755V, Sigma- Aldrich, St. Louis, MO, USA) were used in blotting analysis procedures.

[0155] 2.3.2. Sulfide Analysis

[0156] Lithium-heparin vacutainer tubes were used to collect whole blood, which was processed within 15 minutes of collection. The blood samples were then centrifuged at 1400 RCF for 4 minutes, and the resulting plasma was mixed with a 5: 1 ratio of plasma tostabilization buffer (a degassed 100 mM Tris-HCl buffer with a pH of 9.5 and 0. 1 mM DTP A). The mixture was frozen and stored in liquid nitrogen until analysis.

[0157] To calculate the levels of the individual sulfide pools, three measurements were performed. First the free sulfide alone, which is present in all measurements, was measured by derivatization of hydrogen sulfide with excess MBB (monobromobimane) in pH 9.5 50 mM Tris-HCl buffer containing 0.1 mM DTPA for 30 min in 1% 02 at room temperature. The reaction was stopped by adding 200 mM sulfosalicylic acid and placing the sample on ice for 10 min. For the second measurement, a combination of the free and acid labile pools, the sample was incubated with 100 mM phosphate buffer (pH 2.6) with 0.1 mM DTPA, to release the acid labile sulfur in an enclosed system to contain volatilized H2S. All reactions were performed in BD vacutainer tubes with rocking to facilitate H2S gas release. After removal of the liquid solution with a syringe equipped with a long needle, trapping buffer (100 mM Tris- HCl, pH 9.5, 0.1 mM DTPA) was added into the vacutainer tube and rocked for 30 min. Sulfide levels of these solutions were measured by derivatization of hydrogen sulfide as above for the free sulfide alone. Finally, the total sulfide measurement, which includes the free, acid labile and bound sulfide components, was processed as above, with the exception that 1 mM tns(2- carboxyethyl)phosphine hydrochloride (TCEP), which releases the bound sulfide, was added to the 100 mM phosphate buffer (pH 2.6) with 0.1 mM DTPA. For all measurements, the fluorescent product sulfide-dibimane was then measured by RP-HPLC with fluorescence detector using an Eclipse XDB-C18 (4.6 x 250 mm) column and a gradient elution by 0.1% (v / v) trifluoroacetic acid (TFA) in acetonitrile and water at a flow of 0.6 ml / min.

[0158] The total sulfide and individual pools were calculated as follows. The total sulfide was obtained directly from the total sulfide measurement. The free sulfide was obtained directly from the free sulfide measurement. The bound sulfide was calculated by subtracting the combined free sulfide and acid labile sulfide measurement from the total sulfide measurement. Finally, the acid labile sulfide level w as calculated by subtracting the free sulfide measurement from the combined free sulfide and acid labile sulfide measurement.

[0159] 2,4, Quantification and statistical analysis

[0160] Statistical analysis was performed in SPSS version 28. Multivariate analysis of covariance with age and education as covariates, was used to evaluate differences between groups. Pearson correlation was used to analyze the relationships between plasma enzyme levels, and cognitive outcome measures. An alpha level of p<0.05 was used to determine significance.

[0161] Results

[0162] All participants were evaluated for cognitive function and assigned to the ADRD (ADAS Cog >17; N = 35) or control (N = 29) groups. Multivariate ANCOVA revealed that there were significant differences by group for age (F(3, 61) = 4.7, p=0.034; eta squared = 0.070 and education (F(3, 61) = 17.108, p < 0.001; eta squared = 0.20; Table 1). Subjects in the ADRD group were older and had, on average, significantly fewer years of education. ADRD subjects also had significantly worse cognitive performance on the ADAS-cog compared to controls (F(3. 61) = 99.83, p < 0.001; eta squared 0.63).

[0163] Table 1. Participant Demographic Variables by Group

[0164] We observed elevated plasma CBS in the ADRD group compared to controls (F(3. 60) = 24.7, p = < 0.001; Eta Squared = .29). We saw no significant difference in plasma CSE (F(3, 60) = 0.732, p = 0.396) or MST (F(3, 60) = 0.329, p = 0.569) between groups (FIG. 2). We also found an increase in plasma BA in ADRD participants compared to controls (F(3.60) = 7.335, p = 0.009). For all subjects, poorer performance on the ADAS-Cog was associated with elevated plasma CBS (r(l,63) = .534, p < 0.000). Cognitive performance was not associated with plasma CSE (r(l .63) = -.174, p = 0.175), plasma MST (r(l ,63) = -0.034, p = 0.695), or plasma BA (r(l,63) = 0.171, p = 0.183; FIG. 3). Plasma CSE was significantly and negatively associated with age (r(63) = -.310, p = .013). No significant association with age was observed for plasma CBS, (r(63) = 0.228, p = 0.070), plasma MST (r(63) = -0.118, p = 0.355), or plasma BA (R = -0.025, p = 0.843). (FIG. 4) Both plasma CBS (r(l,63) = .254, p =.043) and plasma MST (r(l,63) = 0.619, p < 0.000; FIG. 5) were significantly related with elevated plasma BA. Plasma CSE was not associated with plasma BA levels. (r( 1 ,63) = .06, p = .639).

[0165] Acid labile plasma sulfide was elevated in the ADRD group compared to controls (F(3,57) = 7.72, p = 0.007; Table 2). There were no differences across groups for free (F(3 ,57) = 0.55, p = 0.456), bound (F(3,57) = 0.28, p = 0.598) or total sulfides (F(3,57) = 1.53, p = 0.221). There were no significant correlations among plasma sulfide levels and plasma enzyme levels.

[0166] Table 2. Plasma sulfide levels (pm) across disease groups. Count or mean (SD) *p < 0.01. Acid labile sulfide was elevated in the ADRD group.

[0167] Plasma MST (r(l,63) = .619, p < 0.0001; FIG. 5) was significantly related with elevated plasma BA. Both plasma CBS (r(l,63) = .241, p =.055) and Plasma CSE (r(l,63) = .001. p = .993) were not associated with plasma BA levels. Beta-amyloid was also correlated with acid labile sulfide (r(l ,63) = .282, p=0.028; FIG. 6), but not free, (r(l ,63) = .282, p=0.028), bound (r(l,63) = . 282, p=0.028) or total sulfides (r(l,63) = .282, p=0.028).

[0168] Discussion

[0169] The role of sulfides in regulating cerebrovascular and neuronal homeostasis in health and ADRD is complex. There are several relevant sources of sulfides in the brain. CBS has been suggested to be exclusively localized to neurons, but also endothelial cells [Yuan et al., 2016], CBS is expressed with highest levels in Purkinje cells and in the hippocampus (Robert et al.. 2003), Bergmann glia and astrocytes [Morikawa et al., 2012. Enoikido et al., 2005] and brain endothelial cells [Veerareddy et al., 2021], 3-MST has also been localized to neurons [Shibuya et al., 2009] and brain endothelial cells [Veerareddy et al., 2021] in both mitochondria and in the cytoplasm. EES, or free hydrogen sulfide, has been shown to be neuroprotective at normal levels in models of AD (Giovinazzo et al., 2021, Vandini et al., 2019) However we have shown that in human ADRD. H2S levels were similar across disease groups, while sulfide metabolites were elevated in AD (Disbrow et al., Reekes et al). Here we found that acid labile sulfides were elevated in the ADRD compared to control groups, and acid labile sulfides were significantly positively correlated with beta-amyloid levels. While plasma sulfide levels do not necessarily directly parallel levels of sulfide generating enzyme levels, our finding that plasma CBS was significantly increased in ADRD, even after correction for age, is consistent with elevated plasma CBS in ADRD, and elevated plasma CBS was associated with poorer cognitive performance. This sulfide increase may be indirectly, at least in part, a CBS mediated increase in free plasma H2S. Plasma beta-amyloid was significantly increased in ADRD, but more modestly increased than CBS. by 19%. and was positively correlated with plasma acid labile sulfide and plasma MST levels. Our findings indicate that plasma is an accessible and apparently sensitive depot for the sulfide generating enzymes CSE, 3-MST and CBS.

[0170] CBS Dysregulation

[0171] Elevated circulating levels of CBS may reflect increased rates of protein synthesis and shedding, and it is not clear whether such elevation is adaptive or harmful. For example,lower levels of CBS, as well as inhibition of CBS can lead to accumulation of homocysteine (Hey) which can drive accumulation of S-adenosylmethionine. a powerful inhibitor of methylation reactions (Hoffman et al., 1979). This disturbance can affect DNA and histone methylation to modulate epigenetic patterns, signal transduction and activation of signaling modules like kinases and phosphatases, which are sensitive to methylation (Gao et al., 2018). It is also worth noting that genetic deletion of CBS in mice increases coagulation (Maclean et al.. 2010) which could predispose individuals with low vascular CBS to developing thrombi. (Kalariaet al., 2021) a phenomenon which contributes to small vessel disease in AD. In familial homocysteinuria (hHcyuria) a disease of defective CBS activity7, elevated levels of plasma and urine Hey due to reduced recycling of sulfides has negative effects on cognition (Suri et al., 2014). Interestingly, CBS deficiency also increases insulin insensitivity, another risk factor for AD progression (Cruciani-Guglielmacci et al., 2022).

[0172] CBS is increased in ADRD and decreased in MS. In 2021, we previously reported that in multiple sclerosis (MS), there was a significant decrease in circulating levels of plasma CBS. MS is a disease of the central nervous system (CNS) that affects the brain, spinal cord, and optic nerves which is primarily characterized by damage to myelin sheaths, the protective covering surrounding myelinated nen e fibers. The pathophysiology7of MS differs from that of AD in that MS is largely considered to be an inflammatory7demyelinating disease (Zhao and Jacob, 2023), By comparison, AD is largely considered to be a gray matter disease (Swinford et al., 2023); consequently the mechanisms underlying these conditions may be different. While damage to myelinated structures can occur in both gray and white matter, it has been traditionally thought that MS is a disease primarily affecting white matter, which transmits signals within the brain and to via myelinated nerves to he periphery7. Recent studies have shown that gray matter damage can occur in MS, particularly in the cerebral cortex, which can influence memory, attention, and language. Therefore, while MS is often described as a white matter disease, this monolithic model may' inappropriately7marginalize important contributions from gray matter injury7.

[0173] We have previously reported that human brain endothelial cells which are exposed to inflammatory stimuli increase the shedding of CBS within apical extracellular vesicles, which matches the appearance of plasma CBS in the vascular compartment in ADRD (Veerareddy et al., 2021). While the exact likelihood of CBS shedding by endothelial cells is unclear, it is possible that removal of CBS from the endothelial cells by the process of extracellular vesicle shedding could have negative vascular consequences. For example, decreased vascular CBS can predispose towards endothelial senscence via mitochondrial stressand increased sensitivity to homocysteine (Albertini et al., 2012). These effects of CBS depression have been shown to be mediated by elevated ROS production, to reduced mitofusin 2 (MFN2) expression, decoupled endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death (Rao et al., 2020), effects which can be ‘rescued’ by administration of H2S donors. However, vascular responses to H2S are complex, species and anatomy specific reflecting different sources of H2S and its interactions with other signaling modules, especially endothelial nitric oxide synthase (eNOS), nitric oxide (NO), protein kinase G and cGMP, as well as several other novel second messengers.

[0174] The role of CSE in H2S production

[0175] As part of the transsulfuration pathway, CBS is responsible for converting the amino acid homocysteine (with serine) into cystathionine, ultimately leading to the synthesis of cysteine which can yield hydrogen sulfide. Another pathway for H2S formation involves cystathionine, (which is produced by CBS), that can be further metabolized by CSE to produce H2S, a-ketobutyrate. and ammonia. When pyroxidal is limited, homocysteine is converted to cystathionine more abundantly when CSE activity is reduced. Homocysteine elevation, often seen in ADRD, does not typically reflect genetic patterns in CBS expression, but rather low levels of cofactors for CBS and CSE e.g. pyroxidal which are needed for normal CBS and CSE metabolism. Because CSE has a greater requirement for pyroxidal than CBS, pyroxidal limitation has a net greater depressive effect on CSE action. In this scheme, it is interesting that hyperhomocysteinemia / uria is treated with folic acid and cyanocobalamin (Angelini et al., 2021). If elevated sulfides in ADRD can be convincingly demonstrated to be mediated by disturbances in CBS metabolism, it is possible that folic acid and cyanocobalamin supplementation may represent a potential means of reducing sulfides which may contribute to vascular cognitive impairment in this condition.

[0176] 3-MST

[0177] 3-MST is most highly expressed in the kidney, liver, testes, large intestine, and brain and is most abundant within the endocrine system [Nagahara et al., 1998], 3-MST participates in converting 3-mercaptopyruvate (with dihydrolipoic acid and thioredoxin) to H2S. 3-MST may help mediate H2S dependent memory and cognition. For example, in the symptomatic transgenic APP / PS1 mouse model of Alzheimer disease pathology, Rao et al. (2022), found a significant reduction in 3-MST activity, in the cortex and hippocampus of APP / PS1 mice. We found that beta amyloid was correlated with 3-MST levels indicating that 3-MST may play a role in controlling beta-amyloid generation in AD.

[0178] H2S mediated vasoconstriction as a contributor to AD

[0179] H2S effects on vascular tone may be vasodilatory or vasoconstrictive depending on the concentration, species and anatomy under consideration. Further, the relationship among pools of H2S and its metoblites is not well understood, and the vasodilator / constrictor effects of acid labile and bound sulfides are unclear. At higher concentrations, Kubo et al., 2017 suggested that elevated H2S can suppress eNOS leading to vasoconstriction. This effect would be anticipated to reduce blood flow, and could intensify ADRD through cerebrovascular ischemic stress. In the rat, sodium sulfide effects on aortic tone were enhanced and partially blocked by glibenclamide, a KATP channel inhibitor; this was not seen in mouse aortas. In KCl-pre-contracted rat aortas, sodium sulfide provoked a glibenclamide-resistant contraction and relaxation. When endothelial cells were removed, sodium sulfide induced only relaxation suggesting that endothelial-dependent vasodilation is impaired by H2S. Mechanistically, Lim et al., suggested that such sulfide mediated vasoconstriction may reflect H2S mediated suppression of eNOS and reduced nitric oxide which involves cyclic AMP, potentially via increased activation of phosphodiesterases (Lim et al., 2008). Additionally, sodium sulfide pretreatment significantly reduced relaxation induced by acetylcholine, but not by NO donors inhibited conversion of arginine into citrulline by eNOS consistent with depression of eNOS by H2S. Therefore high rates of H2S formation or higher H2S may actually promote vasoconstriction and suggests that the elevated CBS elevations seen in ADRD might produce sufficiently high levels of sulfide to cause this. Additionally, Capmda et al., (2017) showed that in rabbits, sodium sulfide, a relatively weak vasodilator at low concentrations was actually a vasoconstsrictor at higher concentrations. In the aorta, a single exposure to sodium sulfide first dilated, then contracted the aorta. After treating the aorta with acetylcholine to maximally dilate it, sodium sulfide produced a strong constriction. While the sources of sulfides which drive vasoconstriction are still unclear, in addition to CBS, Miditen et al. (2021) suggested that CSE-derived sulfide may promote vasoconstriction via formation of cyclic inosine monophosphate (cIMP), and the contractile effect of H2S reflects a brief drop in cGMP and cAMP mediated by PDE5 and PDE4A activation respectively, which unmasks the proconstrictor effects of cIMP.

[0180] Conclusions

[0181] Based on their presence in the circulation, it is possible that changes in sulfide generating enzymes may impact vascular mechanisms active in AD. We cannot yet exclude contributions of other sources, like neurons, glia and astroctyes whose products may reach the vascular compartment and cause effects on the vasculature. Irrespective of theseconsiderations, measurement of these enzymes appears to represent be a useful approach for diagnosing, staging and gauging therapeutic responses in AD.

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[0213] Increased Plasma Cystathionine Beta-Synthase in Alzheimer’s Disease and Related Dementias (ADRD): Inverse Correlation with Cognitive Performance

[0214] The "vascular hypothesis7’ of Alzheimer’s disease (AD) states that neurodegenerative proteinopathy is preceded by cerebral hypoperfusion and brain microvascular abnormalities. Hydrogen sulfide and its metabolites are important in neuromodulation and vasoregulation, and we have described a link between redox-related disturbances in sulfide metabolism and measures of cerebrovascular disease, brain atrophy and cognitive disturbance in human AD and related dementias (ADRD). However, the tissue andenzymatic sources of these elevated sulfides still remain unclear. Using immunoblotting, we compared levels of 3 enzymatic sources of plasma sulfides, cystathionine beta synthase (CBS), cystathionine gamma-lyase (CSE), 3 -mercaptopyruvate sulfurtransferase (3-MST) as well as plasma beta-amyloid. Neuropsychological test results, demographic data, and whole blood samples were collected from healthy controls (N = 27; mean age 71.3 (SD = 6.3) years) and individuals who met the criteria for ADRD (AD Dementia Assessment Scale-cognitive subscale score >=17; N = 37; mean age 74.3 (SD = 6.80) years). Plasma (0.5ul) CBS, MST. CSE or beta amyloid was immunoblotted and imaged using HRP / ECL reagent on a Chemidoc Image Analyzer. Plasma levels of free, acid-labile, bound, and total hydrogen sulfide were evaluated using the monobromobimane method. Plasma CBS was increased in ADRD (2.43- fold higher, p < 0.001) vs. controls. Plasma beta amyloid was also significantly increased in ADRD (19%, p=0.0121). CBS was positively associated with poorer ADAS-Cog scores (R=0.455, p<0.0001). Plasma beta amyloid was significantly correlated with acid-labile sulfide (R = 0.265, p = 0.041), plasma CBS (R = 0.254, p = 0.043), and plasma MST (R = 0.619, p < 0.001). While cellular sources of these sulfides remains to be determined, the cellular source of CBS may be endothelial cells which he at the interface of the brain and blood and express CBS.

[0215] A better understanding of the underlying vascular pathomechanisms and consequences that confer greater risk due to excess sulfide burden may lead to improved detection and treatment of ADRD.EXAMPLE 4

[0216] Plasma CBS in AD

[0217] CBS was assessed in plasma from subjects with Alzheimer's disease and control subjects without Alzheimer’s disease. Plasma CBS was 502.964 + / - 62.5 ng / ml in AD and 343.083ng / ml + / - 45 in control. Therefore, a significant (p=0.042) difference was observed between AD and Control plasma. There was a 47% increase in AD CBS plasma compared to control (FIG. 18).EQUIVALENTS

[0218] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

What is claimed:

1. A method of identifying and / or treating a subject afflicted with Alzheimer's Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD), the method comprising: determining a level of one or more H2S-generating enzymes in a biological sample obtained from a subject, wherein the level of one or more H2S -generating enzymes is indicative of a subject afflicted with AD / ADRD; and treating the subject afflicted with AD / ADRD by administering an H2S- generating enzyme inhibitor, an H2S -generating enzyme substrate, H2S -generating enzy me genetic augmentation, or a combination thereof.

2. The method of claim 1 , further comprising obtaining the biological sample from the subj ect.

3. The method of claim 1, wherein a level of the one or more H2S -generating enzy mes in the biological sample at least 15% higher or lower than the level of the one or more H2S- generating enzymes in an age-matched control sample is indicative of a subject afflicted with AD / ADRD.

4. The method of claim 1, wherein the one or more H2S-generating enzy mes comprises Cystathionine beta synthase (CBS), Cystathionine gamma-lyase (CSE), or both.

5. The method of claim 4, wherein if the one or more H2S generating enzymes is CBS, a level of about two times higher than the level of CBS in an age-matched control sample is indicative of AD / ADRD.

6. The method of claim 4, wherein if the one or more H2S generating enzymes is CSE, a level of about 20% lower than the level of CSE in an age-matched control sample is indicative of AD / ADRD.

7. The method of claim 1, wherein the EI2S generating inhibitor comprises hydroxylamine (EIA), aminooxy acetic acid (AOAA), or both.

8. A method for screening the presence of an Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) signature, the method comprising:obtaining a biological sample from a subject; detecting the presence of at least one AD / ADRD biomarker in a biological sample from the subject, wherein the level of the AD / ADRD biomarker in the biological sample is higher or lower than the level of AD / ADRD biomarker in an age-matched control sample, wherein the AD / ADRD biomarker is indicative of a subject afflicted with AD / ADRD, wherein the AD / ADRD biomarker comprises one or more H2S -generating enzymes; and treating the subject afflicted with AD / ADRD, wherein treating comprises administering a pharmaceutically effective amount of a therapeutic, wherein the therapeutic comprises a H2S -generating enzyme inhibitor, an additional H2S- generating enzyme substrate, H2S-generating enzyme genetic augmentation, or a combination thereof.

9. The method of claim 8, wherein the one or more H2S-generating enzymes comprise Cystathionine beta synthase (CBS), Cystathionine gamma-lyase (CSE), or both.

10. The method of claim 1 or 8, wherein detecting comprises performing an assay to determine the protein level of one or more H2S -generating enzymes in the biological sample.

11. The method of claim 10, wherein the assay comprises an immunoassay, a colorimetric assay, a fluorometric assay, or a combination thereof.

12. The method of claim 11, wherein the immunoassay comprises a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or a combination thereof.

13. The method of claim 1 or 8. further comprising incubating the biological sample with an agent that binds to the one or more H2S-generating enzymes.

14. The method of claim 13, wherein the agent comprises an antibody.

15. The method of claim 14, wherein the antibody comprises an anti-CBS antibody or an anti- CSE antibody.

16. The method of claim 1 or 8, wherein the biological sample comprises plasma, blood, urine, or saliva.

17. The method of claim 1, further comprising isolating extracellular vesicles and / or microparticles from the biological sample to determine the level of the one or more H2S- generating enzymes.

18. The method of claim 1 or 8, wherein the therapeutic comprises a CBS inhibitor if the one or more H2S-generating enzymes is CBS.

19. The method of claim 1 or 8, wherein the therapeutic comprises a CSE substrate if the one or more H2S -generating enzymes is CSE.

20. A method of treating a subject afflicted with Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) comprising administering an H2S-generating enzyme inhibitor, an H2S-generating enzyme substrate, and H2S-generating enzyme genetic augmentation, or any combination thereof.

21. The method of claim 20, wherein the one or more H2S-generating enzymes includes CSE, and the therapeutic includes a CSE inhibitor.

22. The method of claim 20, wherein the one or more H2S -generating enzymes includes CBS, and the therapeutic includes one of a CBS substrate, a CBS genetic augmentation, or both.

23. The method of claim 20. wherein the one or more H2S -generating enzymes includes CSE and CBS, and the therapeutic includes (i) a CSE inhibitor and (ii) a CBS substrate, a CBS genetic augmentation, or both.

24. The method of claim 20. further comprising administration of a further therapeutic.

25. The method of claim 24, wherein the further therapeutic includes one of a biologic and / or an immunotherapy.

26. A method of identifying a subject responsive to one or more CBS inhibitors, the subject afflicted with Alzheimer's Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD), the method comprising: determining H2S levels in a first biological sample obtained from the subject; administering to the subject a therapeutically effective dose of the one or more CBS inhibitors; and determining H2S levels in a second biological sample obtained from the subject; wherein the subject is responsive to the one or more CBS inhibitors if H2S levels in the second biological sample are lower than the H2S levels in the first biological sample.

27. The method of claim 26, wherein H2S levels in the second biological sample are at least and / or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, 100% lower than the H2S levels in the first biological sample.

28. The method of claim 26, further comprising determining CBS levels in the first biological sample.

29. The method of claim 28, wherein a CBS level in the first biological sample is at least and / or about two times higher than the level of CBS in an age-matched control sample is indicative of AD / ADRD.

30. The method of claim 26. wherein the one or more CBS inhibitors comprise hydroxylamine (HA).

31. The method of claim 26, wherein the the one or more CBS inhibitors comprise aminooxyacetic acid (AOAA).

32. The method of claim 26, wherein the second biological sample is obtained within about 1-60 minutes, 1 to 24 hours, 1 to 30 days after administering to the subject the CBS inhibitor.

33. A method of delaying in a subject the onset of Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD), the method comprising administering to the subject a therapeutically effective dose of one or more CBS inhibitors.

34. The method of claim 33, wherein the one or more CBS inhibitors comprises hydroxylamine (HA).

35. The method of claim 33, wherein the one or more CBS inhibitors comprises aminooxyacetic acid (AOAA).

36. The method of claim 33, wherein the subject has not been diagnosed with Alzheimer’s Disease or Alzheimer’s Disease and Related Dementias (AD / ADRD) before administering the one or more CBS inhibitors.

37. The method of claim 33, wherein CBS levels in a biological sample obtained from the subject before the administering the therapeutic dose of the one or more CBS inhibitor are higher than about 0. 1 , 0.2, 0.3, 0.4, 0.5, one or two times the level of CBS in an age-matched control sample.

38. A method of identifying a subject responsive to one or more CBS inhibitors, the method comprising administering to a subject one or more CBS inhibitors, wherein subject’s H2S plasma levels are determined before and after administering the oner or more CBS inhibitors, and wherein the subject is responsive to the one or more CBS inhibitors if H2S levels after the administering of the one or more CBS inhibitor are lower than H2S levels before the administering of the one or more CBS inhibitor.

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