Inhibition of cyanide overproduction for the therapy of down syndrome and nonketotic hyperglycinemia

Abstract

The present invention relates to a method of treating a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH) with drugs and pharmaceutical combinations that counteract cyanide overproduction in their cells and tissues.

Description

[0001] INHIBITION OF CYANIDE OVERPRODUCTION FOR THE THERAPY OF DOWN SYNDROME AND NONKETOTIC HYPERGLYCINEMIA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method of treating a subject afflicted with Down syndrome or Nonketotic hyperglycinemia with drugs and pharmaceutical combinations that counteract cyanide overproduction in their cells and tissues.

[0004] BACKGROUND OF THE INVENTION

[0005] The global prevalence of Down syndrome (DS) is significant. It is estimated to affect approximately 1.5-3 million individuals worldwide, with approximately 10,000 babies bom with the condition annually. This creates a significant socioeconomic burden due to a substantial support required from both families and society throughout their lives, as most individuals with DS cannot live independently. The primary obstacle to independence stems from their typically low intellectual capacity, with an IQ ranging between 40-50. While DS presents various comorbidities, some more responsive to therapy than others, the paramount unmet need remains a medication capable of addressing the cognitive impairments associated with the condition.

[0006] The pathophysiological processes in DS are complex, because the extra Chromosome 21 induces complex changes in gene expression, and dysregulation of multiple biochemical pathways. Nevertheless, one hallmark of DS is metabolic dysfunction, which, in essence, is characterized by inhibition of mitochondrial function and suppression of normal physiological generation of the universal metabolic currency adenosine triphosphate (ATP) in DS cells. Bioenergetic dysfunction is considered a fundamental root cause of many manifestations of the condition, including deficiency in neuronal development, neuronal function and diminished exercise capacity. It is logical to assume that therapies that improve the metabolic deficiency of DS cell would be therapeutically useful. Currently, however, therapeutic options for this condition are very limited.

[0007] On the other hand, nonketotic hyperglycinemia (NKH) is a rare genetic disease, an inborn error of glycine metabolism, which leads to the accumulation of glycine in the cells and body fluids. It is caused by defects in the genes GLDC and AMT which encode the protein components of the glycine cleavage enzyme system. Its estimated incidence of 1 :60,000 makes NKH the second most common disorder of amino acid metabolism after phenylketonuria. However, the global prevalence of NKH is estimated only around 500-1,000 people due to the very poor prognosis, with most NKH patients dying within the first year of their life. For example, the United Kingdom currently has 15 documented cases of the disease. The socioeconomic burden of the condition is, nevertheless, substantial for the affected families, given to the fact that most NKH patients cannot live an independent life and need substantial parental and societal support throughout their lives. The main reasons for lack of independence is diminished brain function and myoclonic seizures.

[0008] The pathophysiological processes underlying NKH are complex and are associated with the toxicity associated with the excess of glycine in NKH cells, tissues, cerebrospinal fluid and blood. The mechanisms by which glycine exerts toxicity are incompletely understood, but NKH cells have severe metabolic deficiency, a low division rate and reduced levels of ATP. It is logical to assume that therapies that improve the metabolic deficiency of NKH cell would be therapeutically useful. However, currently, the therapeutic options for this condition are very limited.

[0009] Thus, there is a significant need for an effective method of treating subjects having DS or NKH in order to rescue them from premature death and improve the overall clinical condition of these subjects.

[0010] SUMMARY OF THE INVENTION

[0011] An aspect of the present invention provides at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), wherein the subject has been determined to have an increased level of endogenous cyanide in the blood in comparison to the normal physiological cyanide level in a blood of a healthy non-smoking subject, and wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, a glycine transporter inhibitor, and combinations thereof, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed or lometrexol, and the glycine transporter inhibitor is iclepertin.

[0012] Another aspect of the present invention provides a pharmaceutical combination comprising at least two of a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, and a glycine transporter inhibitor, together with pharmaceutically acceptable excipients, for simultaneous, separate or sequential administration, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed or lometrexol, and the glycine transporter inhibitor is iclepertin.

[0013] Another aspect of the present invention provides a pharmaceutical combination of the present invention for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH).

[0014] A further aspect of the present invention provides an in vitro method for determining whether a subject afflicted with Down Syndrome (DS) or Nonketotic Hyperglycinemia (NKH) will respond favorably to the treatment according to the present invention, the method comprising the steps of: a. comparing cyanide level in a biological sample obtained from the subject with the normal physiological cyanide level in body fluids of a healthy non-smoking subject; and b. determining if the subject will respond favorably to the treatment according to the present invention, wherein if the level of cyanide in the biological sample from the subject is increased in comparison to the normal physiological level, the subject will respond favorably to the treatment, whereas if the level of cyanide in the biological sample from the subject is approximately at or below of the normal physiological cyanide level then the subject may not respond favorably to the treatment. BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 shows that detectable levels of cyanide are generated by homogenates of mouse liver and spleen under baseline conditions. Higher cyanide levels can be detected after supplementation of the homogenate with 10 mM glycine. *p<0.05 shows the significant enhancement of cyanide generation in the presence of glycine.

[0016] Figure 2 shows that detectable levels of cyanide are generated in various cell lines under baseline conditions. Higher cyanide levels can be detected after supplementation of the cell culture medium with 10 mM glycine and lower cyanide levels can be detected when cells are maintained in cell culture medium that does not contain glycine, and its precursor serine. *p<0.05 shows the significant enhancement of cyanide generation in the presence of glycine and #p<0.05 or ##p<0.01 shows the significant suppression of cyanide generation in the absence of glycine and serine.

[0017] Figure 3 shows (A) detection of cyanide generation by cultured HepG2 cells (a human hepatocyte cell line). Black bars show cyanide generation under baseline conditions (standard culture medium contains 0.4 mM glycine and 0.4 mM serine). Lower cyanide levels can be detected when cells are maintained in cell culture medium that does not contain glycine, and its precursor serine (white bars, "-Ser / Gly"). Re-introduction of glycine to the serine / glycine- free medium (0.4 mM) and further elevation of glycine in the serine / glycine-free medium (1 mM, 5 mM, lOmM) induce a gradual increase in cyanide generation to regain basal cyanide generation and to increase it to above baseline levels (gray / white checkered bars). Inhibition of serine hydroxymethyltransferase (SHMT), the enzyme that converts serine to glycine, with a specific inhibitor (iSHMT, SML2699, 100 pM) decreases basal cyanide generation (black / white checkered bars). **p<0.01 shows significant inhibition of cyanide generation after omission of glycine and serine from the medium. *p<0.05 shows significant elevation of cyanide generation over baseline levels when the serine / glycine free tissue culture medium is supplemented with 10 mM glycine. ##p<0.01 shows the significant suppression of cyanide generation when the SHMT inhibitor is added to the cells grown in standard culture medium. (B) Detection of cyanide generation by cultured HepG2 cells (a human hepatocyte cell line) and testing the effect of two different cyanide scavengers, trihistidyl-cobinamide (THC, 10 pM) or dicobalt-edetate (CoE, 10 pM). The left 3 bars show cyanide generation under baseline conditions (standard culture medium contains 0.4 mM glycine and 0.4 mM serine), the right 3 bars in the presence of 10 mM glycine. **p<0.01 shows significant elevation of cyanide generation over baseline levels when 10 mM glycine is added to the medium. #p<0.05, #p<0.01 show the significantly lower cyanide levels in cells treated with the cyanide scavengers.

[0018] Figure 4 shows detection of cyanide generation by cultured HepG2 cells (a human hepatocyte cell line) under baseline conditions (standard culture medium contains 0.4 mM glycine and 0.4 mM serine) and after elevation of the glycine concentration to 10 mM. The two bars on the left show the responses in normal wild-type cells, the middle bars show the responses in cells where TST levels were increased by forced overexpression of this enzyme, and the two bars on the right show the responses in cells where TST levels were decreased by TST gene silencing.

[0019] Figure 5 shows (A) confocal microscopy imaging experiment. Left panel: Detection of cyanide generation by cultured HepG2 cells (a human hepatocyte cell line) under baseline conditions using a fluorescent cyanide probe. Middle panel: localization of the lysosomes in the cells using a fluorescent lysosomal probe. Right panel: Merging of the two images on the left and middle revealing the co-localization of the cyanide and the lysosomal signals; selected examples of the co-localization are shown by the arrows. (B) Cyanide generation by isolated lysosomes and low levels of cyanide detected in isolated cytosolic fraction of mouse liver tissue or HepG2 human hepatocyte cell lines. Note that addition of glycine (Gly, 10 mM) to the lysosomes increases cyanide generation, but addition of glycine does not increase cyanide generation in the isolated cytosol. *p<0.05 shows significant stimulation of cyanide generation after addition glycine and #p<0.05 and ##p<0.01 show that the cytosolic levels of cyanide are lower than the levels produced by the lysosome. (C) Comparison of cyanide generation by intact lysosomes vs. by lysosomal fractions where the integrity of the lysosomes was disrupted by mechanical processes. ##p<0.01 shows significantly lower cyanide generation by disrupted lysosomes than in intact ones. (D) Electron microscopy confirming the disruption of the lysosomes.

[0020] Figure 6 shows modulation of cyanide generation in lysosomes isolated from mouse liver. Baseline levels are shown in the left bar (control, C). Treatments of the lysosomes with the lysosomal proton pump inhibitor bafilomycin (Baf, 1 pM), with the lysosomal pH disruptor hydroxychloroquine (HCQ, 100 pM) or with the lysosomal glycine transport inhibitor Gly-Gly (GlyGly, 150 mM) inhibit cyanide generation. #p<0.05 and ##p<0.01 show significant inhibition of cyanide generation by the various pharmacological modulators. Figure 7 shows that cyanide generation in mammalian cells can be suppressed by treatment of the cells with Iclepertin (10 pM) or Lometrexol (10 pM), both basally, and when cells are exposed to additional extracellular glycine (10 mM). HepG2 cells were grown in culture and exposed to the various pharmacological agents for 6 hours, after which cyanide levels were measured in the supernatant.

[0021] Figure 8 shows that the lysosomal pH disruptor hydroxychloroquine) and cyanide scavengers (trihistidyl-cobinamide or hydroxycobalamin / Vitamin B12a) exert additive effects in the suppression of cyanide generation in mammalian cells. HepG2 cells were grown in culture and exposed to the various pharmacological agents (vehicle or 3 pM hydroxychloroquine; vehicle or 1 or 3 pM trihistidyl-cobinamide; vehicle or 100 or 300 pM hydroxycobalamin / Vitamin B12a) for 6 hours, after which cyanide levels were measured in the supernatant.

[0022] Figure 9 shows increased cyanide generation in DS fibroblasts. Control fibroblasts and fibroblasts obtained from DS individual were grown in culture. (A) Confocal imaging of these cells using a fluorescent cyanide probe. Note the higher white-colored signal in the DS cells indicating more cyanide content. (B) Measurement of cyanide generation by control and DS cells and in DS cells treated with the cyanide scavenger trihistidyl-cobinamide (THC, 10 pM) or the lysosomal pH disruptor hydroxychloroquine (HCQ, 10 pM). *p<0.05 shows significantly higher cyanide generation in DS cells than in control cells; #p<0.05 shows significantly lower cyanide levels in the presence of the various pharmacological modulators in DS cells.

[0023] Figure 10 shows increased cyanide generation in NKH fibroblasts. Control fibroblasts and fibroblasts obtained from NKH patient were grown in culture. (A) Confocal imaging of these cells using a fluorescent cyanide probe. Note the markedly higher white-colored signal in the NKH cells indicating more cyanide content. (B) Measurement of cyanide generation by control and NKH cells and in NKH cells treated with the cyanide scavenger trihistidyl-cobinamide (THC, 10 pM) or the lysosomal pH disruptor hydroxychloroquine (HCQ, 10 pM). *p<0.05 shows significantly higher cyanide generation in NKH cells than in control cells; #p<0.05 shows significantly lower cyanide levels in the presence of the various pharmacological modulators in NKH cells. Figure 11 shows effect of treatment with the cyanide scavenger hydroxycobinamide (1 pM) on (A) the suppressed oxygen consumption rate (OCR), measured by Extracellular Flux Analysis and (B) the suppressed proliferation rate of DS cells, measured by the BrdU incorporation method. *p<0.05 shows significantly lower oxygen consumption rate or proliferation rate in DS cells than in control cells; #p<0.05 shows significant improvement in these parameters in DS cells in response to hydroxycobinamide treatment.

[0024] Figure 12 shows effect of treatment with the lysosomal pH disruptor hydroxychloroquine (HCQ, 10 pM) on (A) cell viability, assessed by the ability of the cells to convert the mitochondrial substrate MTT and on (B) the suppressed proliferation rate of NKH cells, measured by the BrdU incorporation method. *p<0.05 shows significantly lower viability or proliferation rate in NKH cells than in control cells; #p<0.05 shows significant improvement in these parameters in NKH cells in response to hydroxychloroquine treatment.

[0025] Figure 13 shows that DS rats exhibit higher circulating cyanide levels and higher rates of cyanide generation than normal control rats. (A) Measurement of blood cyanide levels in wildtype control rats (CTR) and in DS rats (DS). Cyanide levels were quantified in anticoagulated blood samples using a derivatization / mass spectrometry method based on the reaction of cyanide with naphthalene dialdehyde and taurine. (B) Measurement of cyanide generation in brain homogenates from wild-type control rats (CTR) and in DS rats (DS) in the presence of vehicle ("Vehicle" containing 0.4 mM glycine) or with supplementation with 10 mM glycine ("Glycine") for 75 minutes. 75 pl of the supernatant were then taken and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide levels were measured using a cyanide selective electrode sensor and normalized for mg of protein. *p<0.05 show significantly higher cyanide blood levels in the DS group than in the wild-type control group in (A) and *p<0.05 and *p<0.01 show significant differences between the indicated groups in

[0026] (B)

[0027] Figure 14 shows that two structurally different SHMT inhibitors: lometrexol (1 pM) or 4,4'- [2,4-pyrimidinediylbis(4,l-phenyleneimino)]bis[4-oxo-butanoic acid (iSHMT, 30 pM) reduce cyanide generation and improve the proliferation rate of human DS fibroblasts. Human DS fibroblasts were seeded in 24-well plate at a cell density of 20,000 cells / well in a final volume of 500 pl and incubated overnight in a humidified incubator at 37 °C and 5% CO2 atmosphere. The morning after cells were treated with various test compounds for 72h. (A) For the measurement of cyanide levels, 75 pl of the supernatant were taken and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide levels were measured using a cyanide selective electrode sensor and normalized for mg of protein. (B) For the measurement of cell proliferation, cells were incubated with 10 pM BrdU labelling solution for 3 h at 37 °C and 5% CO2. After removing the culture medium, DNA denaturation-fixation was performed, followed by incubation with the anti-BrdU antibody. Subsequently, the colorimetric substrate reaction was measured. Absorbance was measured at 450 nm with 690 nm as the reference wavelength, using a plate reader. In panel (A) **p<0.01 shows significantly lower cyanide levels in DS cells in the presence of the SHMT inhibitors used than in DS cells treated with vehicle control. In panel (B), *p<0.05 shows significantly higher proliferation rate in DS cells in the presence of the SHMT inhibitors used than in DS cells treated with vehicle control.

[0028] Figure 15 shows that the combination of various cyanide down-regulator pharmacological agents is more effective in reducing cyanide levels in human DS fibroblasts than treatment with each individual modulator. Human DS fibroblasts were seeded in 24-well plate at a cell density of 20,000 cells / well in a final volume of 500 pl and incubated overnight in a humidified incubator at 37 °C and 5% CO2 atmosphere. The morning after cells were treated with various test compounds for 72h. Subsequently, 75 pl of the supernatant were taken and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide levels were measured using a cyanide selective electrode sensor and normalized for mg of protein. (A) shows the effect of the lysosomal deacidification agent hydroxychloroquine (Hcq, 10 pM), the cyanide scavenger Vitamin B12 (VitB12, 1 pM) and the glycine transport inhibitor iclepertin (ICE, 10 pM) as well as the effect of the combination of all 3 agents, which is more effective than either agent applied individually. (B) shows the results of a similar experiment when lower concentrations of each of the tested pharmacological agents were tested: (Hcq, 2 pM), Vitamin B12 (VitB12, 0.2 pM) and iclepertin (ICE, 3 pM). At these lower concentrations, only the combination of all three compounds produced a statistically significant inhibition of cyanide generation, while the individual use of each compound was less effective. *p<0.05 or **p<0.01 show significantly lower cyanide levels in DS cells than in control cells in the presence of the various pharmacological modulators.

[0029] Figure 16 shows that the combination of the cyanide down-regulator pharmacological agents hydroxychloroquine and hydroxycobalamin (Vitamin B12) is more effective than either agent alone in improving the viability of human DS cells. Human DS fibroblasts were seeded in flatbottom 96-well plates and incubated in a humidified incubator at 37 °C and 5% CO2 atmosphere. The morning after cells were treated daily with vehicle, 10 pM of the lysosomal deacidification agent hydroxychloroquine, 1 pM of the cyanide scavenger Vitamin B12a or their combination for 72h. For the detection of cell viability cells were further incubated for 2 h with 0.5 mg / ml MTT; the converted formazan dye was dissolved in DMSO and absorbance was measured at 570 nm and 690 nm (reference wavelength) using a plate reader. Only the combination of both agents produced a statistically significant (*p<0.05) improvement in cell viability.

[0030] Figure 17 shows the lack of beneficial effect of sodium thiosulfate (Na2S20s) on cyanide levels or various functional parameters in human DS fibroblasts. (A) Cellular TST activity was assessed by the degradation of exogenously added cyanide in the lysates of healthy human normal control fibroblasts (CTR) or human DS fibroblasts. In this assay, 500 pg cell lysate was incubated with KCN 100 pM or KCN 100 pM+Na2S20s 1 mM for 60 minutes at 37°C. At the specified time points, 75 pl assay medium was collected and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide was measured using a cyanide selective electrode sensor. Cyanide degradation was accelerated by Na2S20s in CTR cells, but not in DS cells. (B) For the quantification of endogenously generated cyanide levels, healthy human normal control fibroblasts (CTR) or human DS fibroblasts were seeded in 24-well plate at a cell density of 20,000 cells / well in a final volume of 500 pl and incubated overnight in a humidified incubator at 37 °C and 5% CO2 atmosphere. The morning after, cells were treated daily with 100 pM Na2S20s or its vehicle for 72h. Subsequently, 75 pl of the supernatant were taken and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide levels were measured using a cyanide selective electrode sensor and normalized for mg of protein. Treatment with Na2S20s tended to decrease cyanide levels in control cells but tended to increase cyanide levels in DS cells. (C) Comparison of TST expression levels between healthy control fibroblasts and DS fibroblasts. TST expression was measured by Western blotting, and subsequently quantified via densitometry, with beta-actin expression used as the reference control. TST expression levels in DS cells were significantly lower compared to the expression detected in healthy control cells. (D) Cellular bioenergetics was evaluated in healthy normal control fibroblasts (CTR) or DS fibroblasts with extracellular flux analyzer (Seahorse), which enables to measure the oxygen consumption rate (OCR) real time. 5,000 cells / well were seeded and treated every 24h, either with the lysosomal deacidification agent hydroxychloroquine (10 pM Hcq, used as a positive control) or 100 pM Na2S20s for 72h. The mitochondrial oxidative phosphorylation uncoupler FCCP (2 pM) was used to estimate the maximal mitochondrial respiratory capacity. Subsequently, 0.5 pM rotenone and antimycin A were injected to inhibit electron flux through complex I and III, respectively, aiming to detect the extra-mitochondrial OCR. The maximal mitochondrial respiratory capacity was obtained by subtracting OCR values after FCCP with OCR values after rotenone / antimycin A. Data were normalized to total protein content. The data show that DS fibroblasts exhibited a markedly suppressed metabolic activity compared to healthy normal control cells. While hydroxychloroquine improved cell metabolism in DS cells, Na2S20s did not improve cell metabolism, but, instead, it further reduced cellular respiration of the DS cells. (E-F) shows the lack of effect of Na2S20s on DS cell viability (E) or proliferation (F). For these experiments, DS cells were seeded in flatbottom 96-well plates and incubated in a humidified incubator at 37 °C and 5% CO2 atmosphere. The morning after, cells were treated daily withlOO pM Na2S2C>3 or vehicle for 72h. For the detection of cell viability (E) cells were further incubated for 2 h with 0.5 mg / ml MTT; the converted formazan dye was dissolved in DMSO and absorbance was measured at 570 nm and 690 nm (reference wavelength) using a plate reader. For the detection of cell viability (F), cells were incubated with 10 pM BrdU labelling solution (F) for 3 h at 37 °C and 5% CO2. After removing the culture medium, DNA denaturationfixation was performed, followed by incubation with the anti-BrdU antibody. Subsequently, the colorimetric substrate reaction was measured. Absorbance was measured at 450 nm with 690 nm as the reference wavelength, using a plate reader. *p<0.05 show significantly lower TST expression in DS cells than in control cells in (C) and significantly higher cellular respiration in hydroxychloroquine-treated DS cells than in vehicle-treated DS cells in (D). #p<0.05 show significantly lower cellular respiration rate in Na2S2O3-treated DS cells than in control cells in (D).

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] All documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0033] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also, as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of' and / or "consisting essentially of’.

[0034] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0035] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B".

[0036] As used herein, a "subject" or "patient" to be treated by the method and the pharmaceutical combinations of the present invention include a human subject that is suspected to be afflicted with Down Syndrome (DS) or Nonketotic Hyperglycinemia (NKH) or a human subject that is afflicted with Down Syndrome (DS) or Nonketotic Hyperglycinemia (NKH).

[0037] As used herein, "cyanide" in the context of living biological system, such as human body, refers to a mixture of HCN (gas) and CN- (the ionized form). The balance of the two species is dependent on the pH of the tissue; at physiological pH of 7.4, over 95% is in the HCN form. Together, both of these species are called "cyanide".

[0038] Cyanide is produced in bacteria and plants and serves various regulatory functions. It is well known that cyanide is present at low concentrations in human and mammalian blood and several investigators observed or proposed that cyanide can be produced by mammalian cells and tissues. However, the function of cyanide in mammalian cells is not completely clarified. The levels of cyanide in mammalian cells are regulated by enzyme systems that decompose this species. The main enzyme responsible for this process is an enzyme called rhodanese (or thiosulfate sulfurtransferase, TST). This enzyme is physiologically activated by thiosulfate, and decomposes cyanide.

[0039] The inventors found that various mouse tissues produce detectable amounts of cyanide (Fig. 1) and so do various human cells (Fig. 2). The inventors demonstrated that the amino acid glycine stimulates the production of cyanide in these tissues and cells, while omission of glycine and serine from the culture medium suppresses cyanide generation (Figs 1, 2).

[0040] The inventors demonstrated that cyanide levels in mammalian cells can be lowered by various cyanide scavengers, as well as by omission of glycine and serine from the culture medium, as well as by inhibition of serine hydroxymethyltransferase (SHMT), the enzyme that mammalian cells use to produce glycine from serine (Fig. 3). The inventors have confirmed that the endogenous generation of cyanide in mammalian cells is under the control of the cyanide detoxification system of the enzyme TST, because overexpression of TST decreases while silencing of TST increases basal endogenous cyanide generation (Fig. 4).

[0041] The inventors also localized the production of glycine to one specific cellular organelle, the lysosome (Fig. 5). The inventors concluded that the cyanide generating reaction requires glycine and depends on the acidic pH of the lysosome, because pharmacological inhibition of the lysosomal proton pump, or inhibition of the uptake of glycine into the lysosome, or various approaches that decrease the acidity of the lysosomes (including the drug hydroxychloroquine) reduces cyanide production (Fig. 6). Reduction of cyanide can also be achieved by repurposing the clinically approved drugs Iclepertin or Lometrexol (Fig. 7).

[0042] The inventors have also demonstrated that the lysosomal pH disruptor hydroxychloroquine exerts additive or synergistic effects with cyanide scavengers in their ability to suppress cyanide generation in mammalian cells (Fig. 8).

[0043] The inventors have also discovered that cells from DS individual, and - to an even larger extent - cells from NKH individuals produce markedly elevated levels of cyanide. Similar to other mammalian cells, cyanide levels in these cells can be suppressed by hydroxychloroquine, as well as by cyanide scavenging (Fig. 9, 10). The levels of cyanide in cells, tissues or body fluids (such as blood) of DS and NKH subjects are so high that it may impair the function and metabolism of these cells. Indeed, both DS cells and NKH cells have a markedly suppressed metabolic rate - consistently with the known ability of cyanide to inhibit mitochondrial Complex IV and suppress aerobic cell respiration and mitochondrial ATP generation. Cyanide scavenging or lysosomal deacidification approaches improve the function of these cells. The inventors have demonstrated the ability of the cyanide scavenger hydroxy cobalamin (Vitamin B12) to improve the mitochondrial respiration rate and proliferation of DS cells (Fig. 11) and the ability of the lysosomal modulator hydroxychloroquine to improve the viability and proliferation of NKH cells (Fig. 12).

[0044] Further to test if DS is associated with elevated cyanide levels, inventors employed a rat model of DS, which is associated with neurodevel opmental and cognitive defects (as characterized in Redox Biology, 51 : 102233, 2022). Blood levels of cyanide in DS rats were significantly higher than cyanide blood levels in healthy control wild-type rats (Fig. 13A). In addition, brain homogenates from DS rats produced higher levels of cyanide than wild-type control rats (Fig. 13B), and glycine, which is the precursor of cyanide (as shown in Fig. 1 and Fig. 2) induced a significant stimulation of cyanide generation in the brains of DS rats (Fig. 13B).

[0045] Fig. 14A shows that - similar to how they reduce cyanide generation in healthy control cells (Fig- 3) - also in DS cells, two different SHMT inhibitors inhibit cyanide generation. The inventors attribute these effects to the inhibition of the conversion of serine to glycine and subsequent reduction in cyanide biogenesis. Consistent with the well-documented inhibitory effect of cyanide on cell proliferation, the suppressed cell proliferation of DS cells is improved (i.e. enhanced) by treatment of the DS cells with the SHMT inhibitors (Fig. 14B).

[0046] The inventors have also demonstrated that the various agents that can reduce cyanide generation in DS cells work better in combination than alone. For example, Fig. 15A demonstrates that while the lysosomal deacidification agent hydroxy choroquine (Hcq, 10 pM), the cyanide scavenger hydroxy cobalamin (Vitamin Bl 2) (VitB12, 1 pM) and the glycine transport inhibitor iclepertin (ICE, 10 pM) all reduce cyanide generation by DS fibroblasts, the combination of all 3 agents is more effective. When the concentrations of each of these agents is reduced 5 fold (Hcq, 2 pM), Vitamin B 12 (VitB12, 0.2 pM) and iclepertin (ICE, 3 pM), the effect of each agent is reduced, but the combination of all three compounds still produces a marked inhibition of cyanide generation (Fig. 15B). These data indicate that by simultaneously administering various drugs that target different checkpoints in the process of cyanide generation, better efficacy can be achieved. Combination approaches can also produce better functional effects. For instance, the inventors have demonstrated (Fig. 16) that hydroxychloroquine (10 pM) exerts a partial restorative effect on the viability of DS cells, as assessed by the MTT assay, whereas the low concentration of hydroxycobalamin (Vitamin B12) (1 pM) does not have significant effect; however, the combination of the two agents is more effective in improving cell viability than either of these agents on their own. Taken together, the data presented in Fig. 15 and Fig. 16 support the concept that combination of various cyanide neutralizing approaches can be more beneficial than single agent therapy. Combination of various agents can also permit dose reductions, which is expected to produce better side effect profiles and can allow safer long-term therapy.

[0047] Because the inventors have demonstrated that TST (i.e. rhodanese) is important in regulating cellular cyanide levels in mammalian cells (Fig. 4), they also tested if sodium thiosulfate, which is a well-known stimulator of TST activity, is able to enhance cyanide degradation to reduce cellular cyanide levels. When normal control cell homogenates were exposed to exogenously administered cyanide, the addition of sodium thiosulfate increased the degradation of cyanide, consistent with the activation of TST (Fig. 17A). Sodium thiosulfate also reduced levels of cyanide in normal control cells, where cyanide levels were basally low, but within the detection limit of the assay (Fig. 17B). In contrast, in DS cell homogenates, sodium thiosulfate did not accelerate the degradation of exogenously added cyanide (Fig. 17A) and did not decrease cyanide levels in DS cells; in fact, in DS cells, sodium thiosulfate tended to increase cyanide levels (Fig. 17B). These data indicate that - while sodium thiosulfate may be a useful approach to reduce cyanide levels in normal control cells - it does not exert a similar lowering effect in DS cells. The inventors then compared the expression of TST enzyme in normal vs. DS cells and discovered that TST is markedly downregulated in DS cells (Fig. 17C). The inventors conclude that this downregulation is the likely reason why in DS cells sodium thiosulfate is ineffective in reducing cyanide levels. Consistent with its lack of effect on cyanide levels, sodium thiosulfate did not improve cellular metabolism (Fig. 17D) viability (Fig. 17E) or proliferation rate (Fig. 17F) in DS cells. In fact, sodium thiosulfate even had an adverse effect on the metabolic activity of DS cells (Fig. 17D). Thus, unexpectedly, in DS cells, treatment with sodium thiosulfate (or, likely, any other approach based on the pharmacological activation of TST) is unlikely to be efficacious against cyanide toxicity, due to the marked downregulation of TST in this condition. Thus, correction of the cyanide and / or glycine metabolism may be a useful therapeutic approach for DS and NKH. Principally, this can be achieved at several levels. At the level of modulation of cyanide concentrations, approaches include (A) direct neutralization (scavenging) of cyanide and / or (B) enhancement of the degradation of cyanide via activation of endogenous detoxification pathways. At the level of modulation of lysosomal function, approaches include (A) inhibition of glycine uptake into the lysosome and / or (B) inhibition of the lysosomal proton pump to deacidify the lysosome and / or (C) use drugs or pharmacological agents that produce lysosomal deacidification (lysosomal pH modulator). At the level of modulation of glycine availability, approaches include (A) inhibition of glycine transport (uptake) into the cells and / or (B) inhibition of SHMT to suppress the endogenous generation of glycine, and / or (C) use of agents that directly bind to glycine (glycine scavengers). Combination of several of the above approaches will be more effective than individual approaches.

[0048] Some of the above-mentioned therapeutic approaches can be achieved by repurposing clinically used drugs. For lysosomal deacidification (lysosomal pH modulator), hydroxychloroquine or chloroquine can be used. For directly neutralizing of cyanide, various clinically used drugs can be used, including dicobalt edetate or hydroxycobalamin.

[0049] Thus an aspect of the present invention provides a method of treating a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the method comprising administering to the subject a therapeutically effective amount of at least one compound that reduces the level of endogenous cyanide in cells, tissues or body fluids of the subject, wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a compound that activates cyanide decomposition enzymes, a lysosomal pH modulator, and combinations thereof.

[0050] In an embodiment, the invention provides at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a compound that activates cyanide decomposition enzymes, a lysosomal pH modulator, and combinations thereof. According to another embodiment, the method of treating of the present invention further comprises administering to the subject a therapeutically effective amount of at least one compound that reduces the level of glycine in DS cells or NKH cells, wherein the at least one compound that reduces the level of glycine is selected from a group comprising a serine hydroxymethyltransferase inhibitor (SHMT), a glycine transporter inhibitor, a glycine scavenger, and combinations thereof.

[0051] According to a further embodiment of the method of treating of the present invention, the subject has been determined to have an increased level of endogenous cyanide in the blood in comparison to the normal physiological (circulating) cyanide level in a blood of a healthy nonsmoking subject. The normal physiological (circulating) cyanide level in the blood or plasma of the healthy non-smoking subject (preferably adult subject) is approximately 0.1-0.6 pM [1,2], Alternatively, measurement of cyanide levels may also be used for diagnostic purposes in other body fluids (e.g. cerebrospinal fluid or saliva).

[0052] Cyanide in the blood is primarily concentrated in the red blood cells. One reason is that methemoglobin (MetHb, the ferric form of hemoglobin, normally ~1% of total Hb) avidly binds cyanide, forming cyanmethemoglobin. There are additional 'pools' of cyanide bound to blood proteins (in the red blood cells as well as in plasma), and a small amount of cyanide may be also present in the blood in free form. Cyanide can also bind to other proteins (including heme proteins) to form various protein adducts. Cyanide can also react with sulfur-containing molecules to form thiocyanate or protein adducts. Cyanide can also be converted into extremely stable chemical forms: for instance, cyanide can form an irreversible adduct with cysteine residues (as a thiocyanate adduct) or be incorporated into hydroxocobalamin (vitamin B12A as cyanocobalamin). Thiocyanate is considered a principal metabolite of cyanide. Forensic scientists developed many methods over the last three decades to detect cyanide levels in the blood. These methods were aimed at the detection of relatively high levels of cyanide (as it occurs during cyanide poisoning, or after smoking, which also elevates blood cyanide levels). Because the topic of endogenous cyanide production and function is a recent advance, these prior methods have focused primarily on the detection of toxicologically relevant levels. Due to various factors discussed below, each method developed has its own particular characteristic, including differences in the particular "pool" of cyanide it detects, as well as its sensitivity and selectivity. The implication of the above issues is that cyanide blood values generated by different detection methods cannot be directly compared. However, each of the developed and validated cyanide detection methods used remains clinically applicable, as long as it is validated in a clinical lab, and as long as the various samples (e.g. control samples and samples from patients) are analyzed with the exact same method.

[0053] The classical approach for cyanide analysis in blood is the Kbnig reaction, a colorimetric assay. In this method, cyanide (CN") is liberated from the blood by acidification and converted to hydrogen cyanide (HCN), which is then trapped in an alkaline solution and reacted to form a colored complex (often using chlorinating agents like sodium hypochlorite and reagents such as pyridine-barbituric acid). The intensity of the resulting color is measured. Typical detection limits were on the order of a few micromolar, meaning that low endogenous cyanide levels (especially in nonsmokers) often fall below the true detection limit. As a result, early studies sometimes reported “zero” cyanide in nonsmokers simply because the assay couldn’t detect such low concentrations. Nonetheless, the Kbnig method measures essentially the total cyanide in the sample (all free or liberated CN") and is useful for the detection of high blood cyanide levels (e.g. poisoning cases).

[0054] To overcome the limited sensitivity of the colorimetric Kbnig method, researchers have developed fluorimetric detection of the same reaction product. By substituting a fluorometric step for the visible absorbance measurement of the Kbnig dye, Lundquist's group achieved much lower detection limits. This fluorescence-based method still involves liberating cyanide from blood (often by acid treatment similar to the colorimetric method) and forming the colored complex, but measures its fluorescence instead of absorbance. The result is an assay sensitive enough to detect sub-micromolar cyanide - with ~0.1 pM in nonsmokers’ blood using this method. The fluorimetric approach thus enabled measurement of baseline cyanide in blood and showed that even nonsmokers have measurable (although very low) cyanide.

[0055] The need for faster and more sensitive cyanide analysis led to the adoption of gas chromatographic (GC) methods. In these methods, acid is added to the blood sample in a sealed vial, converting all CN" to HCN gas, and an aliquot of the headspace gas is injected into a GC. Traditional GC methods used detectors like the nitrogen-phosphorus detector or electroncapture detector to quantify HCN. Headspace GC with NPD became popular because it is much more sensitive and rapid than colorimetric assays. Typical protocols involve adding a strong acid (e.g. phosphoric or sulfuric acid) to liberate cyanide, sometimes with salt or heat to facilitate HCN volatilization, and then analyzing the headspace. Headspace GC methods could achieve detection limits in the sub -micromol ar range, significantly better than classical spectrophotometry. The GC method also measures “total releasable” cyanide.

[0056] There are also various mass spectrometry methods for cyanide analysis. One approach uses a derivatization method where cyanide is reacted with naphthalene-2,3-dicarboxaldehyde and taurine to form N-substituted l-cyano[f]benzoisoindol, which is then detected by mass spectrometry. Another notable approach is electrospray ionization tandem MS (ESI-MS-MS) after chemical derivatization of cyanide. Minakata developed a method where cyanide in a biological sample is reacted with NaAuCU to form the dicyanogold complex, which is then extracted into an organic solvent and injected directly into an MS / MS instrument. This method has one of the lowest detection limits (-0.04 pM) reported for cyanide in biofluids.

[0057] The term "free cyanide" refers to unbound CN7HCN in the blood. In a living sample, free cyanide is rapidly cleared or bound; at equilibrium, the concentration of truly free cyanide in blood is very low (estimated to be in the nanomolar range in unexposed individuals). Methods that measure free cyanide would need to analyze blood without liberating cyanide from its binding sites. An example for such a method might be an ion-selective electrode directly in plasma, if the method has sufficient sensitivity or a SIFT-MS sampling of blood headspace at physiological pH. These approaches would mostly detect free HCN. But pure “free cyanide” measurement is uncommon because of the extremely low levels and the instability of free cyanide during storage). One scenario where free cyanide might be assessed is immediate analysis of plasma: some investigators advocated measuring cyanide in plasma to avoid cyanide release from red blood cells. Measurement of cyanide in the plasma (if the sample is handled quickly with minimal disturbance) would represent mostly free (and very loosely bound) cyanide. Indeed, plasma cyanide values tend to be much lower than whole blood values when the comparison is conducted in the same blood sample using the same detection method. In sum, measuring free cyanide is challenging and not commonly done in routine practice.

[0058] In practice, however, most analytical methods deliberately convert bound cyanide to free HCN (by acid or chemical reagents) in order to measure the total cyanide. As noted in the prior section, these methods aim to recover cyanide from all pools: free CN", weakly bound CN (e.g. to hemoglobin or albumin), and sometimes even strongly bound CN in certain complexes. The usual procedure is to add an acid (liberating HCN gas) or a strong ligand that out-competes the native binding (e.g. Ag+or Au3+) to release cyanide from its binding sites. Such methods thus aim to measure the sum of free and bound cyanide - effectively the total cyanide content in the blood sample. However, depending on the method used and the particular conditions applied, the cyanide release from bound pools may be incomplete or complete, which may produce different absolute values between different methods. Some harsh release conditions may even liberate cyanide from stable forms (such as thiocyanate adducts or from Vitamin B12a as cyanocobalamin).

[0059] Even within the same general technique, small methodological differences can greatly affect cyanide measurements. Cyanide is a volatile and reactive analyte, and its measurement is prone to loss or artifact if conditions are not carefully controlled. The way the blood sample is collected, transported and stored may also have an effect on the cyanide value that will be ultimately detected and reported. Differences in sampling vials (open tube vs. sealed vacutainer) and delays before analysis can drastically change results. Moreover, small differences in pH adjustment or reagent additives can produce results that may either be falsely low (e.g. due to lost HCN) to falsely high (e.g. due to artifactual CN released from stable pools such as SCN). Optimizing these conditions is why protocols differ: e.g. some use phosphoric acid (a moderate acid) instead of a strong oxidizing acid, to gently release CN without excessive side reactions.

[0060] When liberating cyanide from blood, how long and at what temperature the sample is incubated can affect yield. A method might incubate at room temperature for 5 minutes versus at 60 °C for 15 minutes (as in a forensic HS-GC-MS method). Longer or hotter incubation generally ensures more complete release of cyanide from binding sites and better equilibration of HCN in the headspace. Thus, timing differences can cause variability. The choice of the detection mode matters as well: GC nitro-phosphorus (NPD) detectors might respond non-linearly at low levels or be subject to quenching by co-eluting substances, whereas MS detection tends to be more linear. Minor differences in the calibration (standard curve preparation) can also contribute to differences in the reported values. Methods that do not use an internal standard must rely on external calibration curves, which can introduce error if matrix effects occur. Using an isotopic internal standard (like13C15N~) helps standardize recovery between samples. Methods that did not use such techniques might have under- or over-estimated cyanide due to losses in sample preparation that were unaccounted for. Thus, two methods might both liberate all cyanide, but if one loses 10% to evaporation in transfer and doesn’t correct for it, it will report a lower value. The one using internal standard (or a complete recovery study) will correct that loss. This is a subtle methodological point that nonetheless leads to systematic differences in reported concentrations between methods.

[0061] In conclusion, the variability in blood cyanide data across different studies often boils down to various methodological issues. A small change in procedure - collection, storage, transport and the various aspects of the analysis including acid strength, presence of a reductant, incubation time, calibration approach and detection instrument - may have an effect on the reported cyanide level. That is why older literature shows a wide range of “normal” (baseline) cyanide values and why inter-laboratory and inter-assay comparisons are difficult.

[0062] Thus, cyanide can be measured in blood by a variety of analytical techniques, and each technique interacts differently with cyanide’s various forms in the blood. A critical insight is that most cyanide in blood is not “free” - it is temporarily bound to various blood components. Methods that aggressively liberate all bound cyanide (for example, by strong acid or chemical reagents) will therefore report a higher, more complete cyanide level than gentler methods that might capture only free cyanide. This explains why the same blood sample can yield different numeric results: a method measuring only plasma (free cyanide) might find a low value, whereas a whole-blood acid treatment may produce a much higher value due to releasing cyanide from bound pools. Even within the same technique, small procedural tweaks can greatly influence the outcome.

[0063] Nevertheless, while the above particulars make it difficult if not impossible to directly compare cyanide blood values reported using different methods and different analytical sites, it remains entirely possible and feasible to detect "high cyanide producer" individuals, when the blood of the control (healthy subjects) and the patient samples (potential high cyanide producers) are analyzed using the exact same method. During such analysis, attention must be also paid to determine if the patient sample comes from a smoker or a non-smoker, because smokers have higher cyanide levels (in clinical trials, in order to reduce confounding variables, smokers may be excluded). Ultimately, the important point is not the absolute numeric cyanide value reported, but to determine whether a given patient's circulating cyanide level is higher than the healthy control baseline (and, subsequently, whether this elevated level can be normalized, suppressed, via the cyanide mitigating approaches outlined in the patent application). In this respect, the method used in the present disclosure is a cyanide release / headspace derivatization / mass spectrometry method [3, 4], which is able to detect baseline cyanide levels in healthy humans and animals [5] and is able to consistently show increased blood cyanide levels, for example, in Down syndrome rats, compared to healthy control rats (see the below section Examples). This is a clinically useful and validated method and could serve as a prime candidate as a central method for future clinical trials in the context of the current patent application and its clinical translation.

[0064] Validated blood cyanides analysis, for example, can be performed in a dedicated central laboratory, where all samples are collected, stored and transferred in the same way, and the processing and analysis is conducted in a highly standardized and validated manner. This approach, in fact, is often used when rare metabolites and analytes are measured with specialized methods that are not available as part of the routine analysis of most clinics and hospitals.

[0065] In some embodiments of the present invention, the body fluids are selected from the group comprising blood, saliva and cerebrospinal fluid; preferably selected from blood and cerebrospinal fluid. In a preferred embodiment, the body fluid is blood. In other preferred embodiment, the body fluid is cerebrospinal fluid.

[0066] The terms "treat", “treating”, “treated” or "treatment", as used in the context of the present invention, refer to therapeutic intervention wherein the aim is to eliminate or at least to lessen symptoms. Beneficial or desired clinical results include, but are not limited to, elimination of symptoms, alleviation of symptoms, dimini shm ent of extent of DS orNKH, stabilized (i.e., not worsening) state of DS or NKH, delay or slowing of progression of DS or NKH.

[0067] A therapeutically effective amount can be readily determined by the attending healthcare professional, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: subject size, age, and general health; the degree of involvement or the severity of DS or NKH; the response of the subject to the particular compound administered; the mode of administration; the bioavailability of the preparation administered; the dose regimen selected; the use of concomitant medication(s); and other relevant circumstances. As used herein, a "therapeutically effective amount" refers to an amount which is effective in reducing, eliminating, treating, or managing the symptoms of DS or NKH. The term "managing" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of DS or NKH, but does not necessarily indicate a total elimination of all symptoms, and is intended to include chronic use as well.

[0068] Another aspect of the present invention provides a pharmaceutical combination comprising at least two of a cyanide scavenger, a compound that activates cyanide decomposition enzymes, and a lysosomal pH modulator, together with pharmaceutically acceptable excipients, for simultaneous, separate or sequential administration, wherein the cyanide scavenger, the compound that activates cyanide decomposition enzymes, and the lysosomal pH modulator reduce a level of endogenous cyanide in cells, tissues or body fluids of a subject afflicted with DS or NKH

[0069] According to an embodiment, the pharmaceutical combination of the invention further comprises at least one compound that reduces a glycine level in DS cells or NKH cells, wherein the at least one compound that reduces the glycine level is selected from a group comprising a SHMT inhibitor, a glycine transporter inhibitor, a glycine scavenger, and combinations thereof.

[0070] The compounds of the pharmaceutical combinations of the present invention may be administered simultaneously, separately or sequentially. Preferably, the compounds that are independently selected from at least two of a cyanide scavenger, a lysosomal pH modulator, a SHMT inhibitor and a glycine transporter inhibitor are administered within 6 hours, more preferably 4 hours, more preferably 2 hours, 1 hour or 30 minutes of one another. In one embodiment the compounds are administered simultaneously. For example, the two or three compounds may be administered simultaneously by oral administration.

[0071] For purposes of the present invention, when administered simultaneously, such administration may also be termed "co-administration of' and "co-administering" the compounds of the pharmaceutical combination, and may refer to any administration of the compounds, either separately or together, where the compounds are administered as part of an appropriate dose regimen designed to obtain the benefit of the combination therapy. Thus, the compounds can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. Each compound can be administered prior to, at the same time as, or subsequent to administration of another compound in the combination, or in some combination thereof. Where one compound is administered to the subject at repeated intervals, the other compound(s) can be administered prior to, at the same time as, or subsequent to, each administration of the first compound, or some combination thereof, or at different intervals in relation to the first compound, or in a single dose prior to, at any time during, or subsequent to the course of treatment with the first compound.

[0072] The pharmaceutical combinations and compounds of the present invention will typically be administered to the subject in a dose regimen that provides for the most effective treatment of Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), (from both efficacy and safety perspectives), as known in the art, and as disclosed herein. In conducting the treatment method of the present invention, the compounds in the pharmaceutical combination can be administered in any effective manner known in the art, such as by oral, topical, intravenous, intra-peritoneal, intramuscular, intra-articular, subcutaneous, intranasal, intra-ocular, vaginal, rectal, or intradermal routes, depending upon the type of condition being treated, the type of compound being used, and the medical judgement of the prescribing physician as based, e.g., on the results of published clinical studies.

[0073] Compounds provided herein can be formulated into pharmaceutical compositions or pharmaceutical combinations of the present invention, optionally by admixture with one or more pharmaceutically acceptable excipients.

[0074] Such compositions and combinations may be prepared for use in oral administration, particularly in the form of tablets or capsules, in particular orodispersible (lyoc) tablets, or parenteral administration, particularly in the form of liquid solutions, suspensions or emulsions.

[0075] It may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington: The Science and Practice of Pharmacy, 20th ed.; Gennaro, A. R., Ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2000. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Oral compositions will generally include an inert diluent carrier or an edible carrier. They can be administered in unit dose forms, wherein the term “unit dose” means a single dose which is capable of being administered to a subject, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active compound itself, or as a pharmaceutically acceptable composition.

[0076] The tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal.

[0077] Liquid preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Nonaqueous solvents include alcohols, propylene glycol, polyethylene glycol, acrylate copolymers. Aqueous carriers include mixtures of alcohols and water, hydrogels, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxy ethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.

[0078] According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the cyanide scavenger is selected from the group comprising hydroxycobalamin (Vitamin B12), cobalamin, cobinamide, nitrocobinamide, aquohydroxocobinamide, trihistidylcobinamide, dicobalt edetate, and dimethyl trisulfide. Preferably, the cyanide scavenger is hydroxycobalamin (Vitamin B 12). According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the compound that activates endogenous cyanide decomposition enzymes is 3 -mercaptopyruvate, and wherein the enzymes that decompose cyanide are selected from the group comprising thiosulfate sulfurtransferase (TST) and 3 -mercaptopyruvate sulfurtransferase (3-MST).

[0079] According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the lysosomal pH modulator is selected from the group comprising hydroxychloroquine and chloroquine. Preferably, the lysosomal pH modulator is hydroxychloroquine.

[0080] According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the SHMT inhibitor is selected from the group comprising lometrexol ([(2S)-2-[[4-[2-[(6R)-2-amino-4-oxo-5,6,7,8-tetrahydro-lH- pyrido[2,3-d]pyrimidin-6-yl]ethyl]benzoyl]amino]pentanedioic acid)]), pemetrexed ([(2S)-2- {[4-[2-(2-amino-4-oxo-l,7-dihydropyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]amino}pentanedioic acid]), and combination thereof. Preferably, the SHMT inhibitor is lometrexol or pemetrexed.

[0081] According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the glycine transporter inhibitor is selected from the group comprising iclepertin ([[5-(Methylsulfonyl)-2-[(lR)-2,2,2-trifluoro-l- methylethoxy]phenyl][(lR,5R)-l-[5-(trifluoromethyl)-3-isoxazolyl]-3-azabicyclo[3.1.0]hex- 3 -yl] methanone]) and PF-03463275 ([N-[(3-chloro-4-fluorophenyl)methyl]-l-methyl-N- [[(lS,5R)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl]methyl]imidazole-4-carboxamide]).

[0082] Preferably, the glycine transporter inhibitor is iclepertin ([[5-(Methylsulfonyl)-2-[(lR)-2,2,2- trifluoro- 1 -methylethoxy]phenyl] [(1R,5R)- 1 -[5-(trifluoromethyl)-3-isoxazolyl]-3- azabicyclo[3.1 ,0]hex-3-yl]methanone]).

[0083] According to an embodiment of the method of treating of the present invention and of the pharmaceutical combination of the present invention, the glycine scavenger is sodium benzoate. An aspect of the present invention provides a method of treating a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the method comprising administering to the subject a therapeutically effective amount of at least one compound that reduces the level of endogenous cyanide in cells, tissues or body fluids of the subject, wherein the subject has been determined to have an increased level of endogenous cyanide in the blood in comparison to the normal physiological cyanide level in a blood of a healthy non-smoking subject, and wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, a glycine transporter inhibitor and combinations thereof, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed, or lometrexol, and the glycine transporter inhibitor is iclepertin.

[0084] In an embodiment, the present invention provides at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), wherein the subject has been determined to have an increased level of endogenous cyanide in the blood in comparison to the normal physiological cyanide level in a blood of a healthy non-smoking subject, and wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, a glycine transporter inhibitor, and combinations thereof, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed, or lometrexol, and the glycine transporter inhibitor is iclepertin. l ' l

[0085] In a preferred embodiment of the treatment of the subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the combination of hydroxycobalamin (Vitamin B 12) and hydroxychloroquine is administered to the subject.

[0086] In another preferred embodiment of the treatment of the subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the combination of hydroxycobalamin (Vitamin B12), hydroxychloroquine and iclepertin is administered to the subject.

[0087] In another preferred embodiment of the treatment of the subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the combination of hydroxycobalamin (Vitamin B12), hydroxychloroquine, iclepertin and pemetrexed is administered to the subject.

[0088] In another preferred embodiment of the treatment of the subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the combination of hydroxycobalamin (Vitamin B12), hydroxychloroquine, iclepertin and lometrexol is administered to the subject.

[0089] When the combinations of two, three or four above-mentioned compounds are used in the treatment of the subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH) of the present invention, the above-mentioned compounds may be administered simultaneously, separately or sequentially. In a preferred embodiment, the compounds are administered separately within 6 hours, more preferably 4 hours, more preferably 2 hours, 1 hour or 30 minutes of one another. In another preferred embodiment the compounds are administered simultaneously. For example, the two compounds, or the three compounds, or the four compounds are administered simultaneously and / or separately. The preferred administration is oral administration.

[0090] Another aspect of the present invention provides a pharmaceutical combination comprising at least two of a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, and a glycine transporter inhibitor, together with pharmaceutically acceptable excipients, for simultaneous, separate or sequential administration, preferably simultaneous and / or separate administration, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed, or lometrexol, and the glycine transporter inhibitor is iclepertin.

[0091] In a preferred embodiment, the pharmaceutical combination of the invention comprises hydroxy cobalamin (Vitamin Bl 2) and hydroxychloroquine, together with pharmaceutically acceptable excipients.

[0092] In another preferred embodiment, the pharmaceutical combination of the invention comprises hydroxycobalamin (Vitamin B12), hydroxychloroquine and iclepertin, together with pharmaceutically acceptable excipients.

[0093] In another embodiment, the pharmaceutical combination of the invention comprises hydroxycobalamin (Vitamin B12), hydroxychloroquine, iclepertin and pemetrexed, together with pharmaceutically acceptable excipients.

[0094] In another embodiment, the pharmaceutical combination of the invention comprises hydroxycobalamin (Vitamin B12), hydroxychloroquine, iclepertin and lometrexol, together with pharmaceutically acceptable excipients.

[0095] Another aspect of the present invention provides a method of treating a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical combination of the present invention.

[0096] In an embodiment, the present invention provides a pharmaceutical combination of the invention for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH).

[0097] Another aspect of the present invention provides a method for determining whether a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH) will respond favorably to the method of treatment according to the present invention, the method comprising the steps of: a. comparing cyanide level in a biological sample obtained from the subject with the normal physiological cyanide level in body fluids (preferably blood) of a healthy non-smoking subject; and b. determining if the subject will respond favorably to the method of treatment according to the present invention, wherein if the level of cyanide in the biological sample from the subject is increased in comparison to the normal physiological level, the subject will respond favorably to the method of treatment, whereas if the level of cyanide in the biological sample from the subject is approximately at or below to the normal physiological cyanide level then the subject may not respond favorably to the method of treatment.

[0098] In a preferred embodiment of the method for determining is an in vitro method.

[0099] In another embodiment, the biological sample is a cell sample, a tissue sample, a cerebrospinal fluid sample or a blood sample.

[0100] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0101] The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the application and the scope of the invention.

[0102] EXAMPLES

[0103] Measurement of cyanide levels in tissue homogenates and cell culture supernatants. C57BL / 6J mice were sacrificed and various organs were obtained and homogenized on ice. HepG2 cells, human fibroblast cell lines or other cell lines were grown in DMEM culture medium in the presence of 10% heat-inactivated fetal bovine serum (FBS), 100 units / ml of penicillin and 100 pg / ml of streptomycin. HCN levels in the supernatants were measured using a CN' selective electrode (Lazar Research Labs, Inc., LIS-146CNCM-XS micro ion). Prior to measuring, all samples were prepared by diluting 1 : 1 (v / v) in 1 M NaOH (0.5 M, final concentration) and incubated at room temperature for at least 30 min, thus favoring the partition of HCN to CN'. The CN' electrode was fully soaked within the sample and voltage (mV) was acquired until the signal was stabilized. The value of the blank sample was subtracted from all measurements. CN' concentration was calculated against a standard curve; absolute concentrations were typically in the range of 1-20 pM. Data were normalized to total mg of protein obtained for cells and lysosomes, or total wet weight for mouse tissues and expressed as cyanide generation rate (nmoles cyanide / mg protein h for cells and lysosomes or nmoles cyanide / mg tissue h for wet tissues).

[0104] Measurement of cellular cyanide content using a fluorescent probe; co-localization of lysosomes and cyanide in living cells.

[0105] Cells were seeded on poly-L-ornithine-coated glass-bottom microscopy chamber at a density of 300,000 cells / well and incubated overnight. Before the experiment, cells were incubated for 1 hour with 10 pM the fluorescent HCN probe CSP, a spiropyrane derivative of cyanobiphenyl which undergoes a turn-on fluorescence in the presence of HCN with the following excitation and emission spectra: Ex 405 nm / Em 495 nm. For co-localization experiments cells were also incubated together with cell-permeant dye LysoTrackerGreen (50 nM, which detects lysosomes with the following excitation and emission spectra: Ex 488 nm / Em 517 nm) at 37°C and 5% CO2. At the end of the incubation, cells were washed 3-times and visualized using confocal microscope Leica SP5 using a 40x oil-immersion APO Plan objective. The parameters of acquisition were as follows: image format of 1,024 x 1,024 pixels, 200 Hz scan speed. Cell fluorescence was measured using ImageJ using the CTCF formula where CTCF is defined as Integrated density-(area of selected cell x mean fluorescence of background reading) in order to calculate the corrected total cell fluorescence.

[0106] Isolation and disruption of lysosomes and measurement of cyanide generation.

[0107] Lysosomes from mouse livers or from HepG2 cells were isolated using the lysosome isolation kit (Sigma-Aldrich, MAK405). After homogenization and centrifugations, the layer corresponding to the lysosomal fraction was marked as Lyso, while the other fractions were pooled together and marked as extra-lysosomal fraction (Extra-Lyso). Both Lyso and Extra- Lyso fractions were resuspended in 200 pl of Suspension Buffer (10 mM HEPES, pH 7.4, 150 mM NaCl supplemented with phosphatase / protease inhibitor cocktail Halt, 1861281). Lysosome disruption was achieved by exposing the Lyso fraction to 5 cycles of 30 s pulse followed by 30 s pause on ice (using an ultrasonic bath sonicator), followed by 3 cycles of freeze (10 min at -20°C) and thaw (3 min at 37°C). Lysosomal disruption was confirmed by electron microscopy. The various fractions were then incubated with glycine or vehicle and various pharmacological agents, and cyanide generation was measured using a CN’ selective electrode (Lazar Research Labs, Inc., LIS-146CNCM-XS micro ion).

[0108] Measurement of cell viability.

[0109] Cell viability was measured by quantifying the mitochondrial-dependent ability of the cells to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Cells were incubated 3h at 37°C and 5% CO2 with 50 pl / well of MTT reagent. Formazan produced by cells with active metabolism was solubilized in 150 pl / well of MTT solvent by mixing well and shaking in an orbital way for 15 minutes at room temperature protected from light. The absorbance was measured at 590 nm using a microplate reader.

[0110] Measurement of cell proliferation.

[0111] Cell proliferation was analyzed using the BrdU method by a commercially available kit (Merck, 11647229001). Cells were incubated with 10 pM BrdU labelling solution for Ih at 37°C and 5% CO2. Subsequently cells were fixed for 25 min using the fixation buffer provided with kit. Fixed cells were incubated for 1 h with an anti-BrdU antibody, washed 3 times in PBS and incubated for 5 min with a colorimetric substrate. Absorbance was measured at 450 nm with 690 nm as the reference wavelength using a microplate reader.

[0112] Measurement of cellular oxygen consumption rate.

[0113] The Seahorse XFe24 flux analyzer (Agilent) was used to estimate cellular bioenergetics of fibroblasts. Cells were seeded at a density of 20,000 cells / well on Agilent Seahorse XF24 cell culture microplates in the absence or presence of various pharmacological agents, and oxygen consumption of the cells was measured by the analyzer.

[0114] Measurement of whole blood cyanide levels Quantification of cyanide proceeded via active microdiffusion, chemical modification of HCN using NDA and taurine, and LC-MS / MS analysis of the CN-NDA-taurine compound. Briefly, ND A (2 mM), taurine (100 mM), and NaOH (10 mM), 200 pl each, were added to the reagent chamber of a two-chamber sample preparation cartridge which allows active airflow from the sample chamber to the reagent chamber. Rat blood (25 pl) was placed in the sample chamber and diluted with 80 pl of deionized water. H2SO4 was added to the sample chamber to ensure that cyanide was in the gaseous (HCN) form. The sample and reagent chambers were immediately capped. Carrier gas (i.e., room air at 200 ml / min) was pumped through the sample chamber into the capture chamber to transfer the HCN gas to the capture solution. In the capture chamber, the NDA and taurine reacted with HCN to form a CN-NDA-taurine complex. An aliquot (250 pl) of the cyanide capture chamber solution was filtered with a 0.22-pm polytetrafluoroethylene into a 300-pl glass insert placed in a 2 ml HPLC vial for subsequent HPLC-MS / MS analysis. Prepared samples were analyzed using HPLC. Separation was achieved by reversed-phase chromatography. MS / MS with multiple reaction monitoring was used to detect the CN-NDA-taurine complex using electrospray ionization operated in negative ion mode.

[0115] Measurement of TST activity in cell homogenates

[0116] TST activity was assessed by the degradation of exogenously added cyanide in cell lysates. 500 pg cell lysate was incubated with KCN 100 pM or KCN 100 pM+Na2S2O3 1 mM for 60 minutes at 37°C. At various time points, 75 pl assay medium was collected and incubated with 75 pl of NaOH IM at room temperature for 30 minutes. Cyanide was measured using a cyanide selective electrode sensor as described above.

[0117] Measurement of TST expression in cell homogenates

[0118] Twenty pg of total protein from whole cell extracts was separated on a 4-12% Bis-Tris Plus Gels and then transferred onto a polyvinylidene difluoride membrane by a dry transfer using the iBlot 2 system (Invitrogen). After blocking with 5% non-fat milk in TBS-Tween20 for 1 h, membranes were incubated with specific antibodies overnight at 4°C. The following primary antibodies and dilutions were used: anti-TST rabbit polyclonal antibody (Abeam, #231248, 1 : 1,000) and anti-P-actin mouse monoclonal antibody (#AC-15) (Sigma-Aldrich, 1 : 1,000). After incubation, the membranes were briefly washed 3 times with TBS and incubated for 1 h in room temperature with secondary antibodies either anti-rabbit IgG or anti-mouse IgG, HRP- linked antibody diluted at 1 :3,000 in TBS-T / 5% milk. Membranes were washed twice with TBS-T and once with TBS and the detection was performed using Radiance plus femtogram HRP substrate (Azure Biosystems, AC2103). Chemiluminescence was captured using Azure Imaging System 300 (Azure Biosystems).

[0119] References

[0120] 1. Tsuge K, Kataoka M, Seto, Y. Cyanide and thiocyanate levels in blood and saliva of healthy adult volunteers. J Health Sci, 46, 343-50, 2000.

[0121] 2. Vinnakota CV, Peetha NS, Perrizo MG, Ferris DG, Oda RP, Rockwood GA, Logue BA. Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers. Biomarkers. 17:625-33, 2012.

[0122] 3. Vinnakota CV, Peetha NS, Perrizo MG, Ferris DG, Oda RP, Rockwood GA, Logue BA. Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers. Biomarkers. 17: 625-33, 2012.

[0123] 4. Bhandari RK, Manandhar E, Oda RP, Rockwood GA, Logue BA. Simultaneous high- performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analysis of cyanide and thiocyanate from swine plasma. Anal Bioanal Chem. 406: 727-34, 2014.

[0124] 5. Zuhra K, Petrosino M, Janickova L, Petrie J, Ascengao K, Vignane T, Khalaf M, Philipp TM, Ravani S, Anand A, Martins V, Santos S, Erdemir S, Malkondu S, Sitek B, Kelestemur T, Kieronska-Rudek A, Majtan T, Filgueira L, Marie D, Chlopicki S, Hoogewijs D, Hasko G, Papapetropoulos A, Logue BA, Boss GR, Filipovic MR, Szabo C. Regulation of mammalian cellular metabolism by endogenous cyanide production. Nat Metab. 7: 531-555, 2025.

Claims

CLAIMS1. At least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH), wherein the subject has been determined to have an increased level of endogenous cyanide in the blood in comparison to the normal physiological cyanide level in a blood of a healthy non-smoking subject, and wherein the at least one compound that reduces the level of endogenous cyanide is selected from a group comprising a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, a glycine transporter inhibitor, and combinations thereof, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed or lometrexol, and the glycine transporter inhibitor is iclepertin.

2. The at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use according to claim 1, wherein the at least one compound that reduces the level of endogenous cyanide is a combination of the cyanide scavenger and the lysosomal pH modulator, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), and the lysosomal pH modulator is hydroxychloroquine.

3. The at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use according to claim 1, wherein the at least one compound that reduces the level of endogenous cyanide is a combination of the cyanide scavenger, the lysosomal pH modulator, and the glycine transporter inhibitor, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, and the glycine transporter inhibitor is iclepertin.

4. The at least one compound that reduces a level of endogenous cyanide in cells, tissues or body fluids of a subject for use according to claim 1, wherein the at least one compound that reduces the level of endogenous cyanide is a combination of the cyanide scavenger, the lysosomal pH modulator, the glycine transporter inhibitor, and the serine hydroxymethyltransferase (SHMT) inhibitor, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the glycine transporter inhibitor is iclepertin, and the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed or lometrexol.

5. A pharmaceutical combination comprising at least two of a cyanide scavenger, a lysosomal pH modulator, a serine hydroxymethyltransferase (SHMT) inhibitor, and a glycine transporter inhibitor, together with pharmaceutically acceptable excipients, for simultaneous, separate or sequential administration, wherein the cyanide scavenger is hydroxy cobalamin (Vitamin B 12), the lysosomal pH modulator is hydroxychloroquine, the serine hydroxymethyltransferase (SHMT) inhibitor is pemetrexed or lometrexol, and the glycine transporter inhibitor is iclepertin.

6. The pharmaceutical combination of claim 5, comprising hydroxycobalamin (Vitamin B12) and hydroxychloroquine, together with pharmaceutically acceptable excipients.

7. The pharmaceutical combination of claim 5, comprising hydroxycobalamin (Vitamin B12), hydroxychloroquine and iclepertin, together with pharmaceutically acceptable excipients.

8. A pharmaceutical combination of any one of claims 5-7 for use in the treatment of a subject afflicted with Down syndrome (DS) or Nonketotic hyperglycinemia (NKH).

9. An in vitro method for determining whether a subject afflicted with Down Syndrome (DS) or Nonketotic Hyperglycinemia (NKH) will respond favorably to the treatment according to claim 1 or claim 8, the method comprising the steps of:a. comparing cyanide level in a biological sample obtained from the subject with the normal physiological cyanide level in body fluids of a healthy non-smoking subject; and b. determining if the subject will respond favorably to the treatment according to claim 1 or claim 8, wherein if the level of cyanide in the biological sample from the subject is increased in comparison to the normal physiological level, the subject will respond favorably to the treatment, whereas if the level of cyanide in the biological sample from the subject is approximately at or below of the normal physiological cyanide level then the subject may not respond favorably to the treatment.

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