Preparation of polybasic agents for reducing multi-organ toxicity

Abstract

The present invention relates to compositions and formulations of polybasic drugs to reduce multi-organ toxicity by creating supramolecular cationic complexes without the formation of covalent bonds and without chemical modification of the polymeric entities used for conjugation. The compositions and formulations made therefrom act by multiple mechanisms simultaneously to reduce the toxicity of cationic antibiotic drugs.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to a composition for reducing multi-organ toxicity caused by polybasic antibiotic agents achieved by supramolecular cationic complexes and a preparation made therefrom. Compounds of such compositions and preparations made therefrom act simultaneously by multiple mechanisms to establish homeostasis in multiple organs to prevent toxicity. In particular, the present invention relates to the optimization and delivery methods of formulations of polybasic agents for preventing nephrotoxicity, neurotoxicity, and ototoxicity.

[0002] Aminoglycosides and polymyxins, generally known as polybasic agents, are broad-spectrum antibiotics commonly used in the treatment of severe bacterial infections. If the kidneys cannot excrete aminoglycosides and polymyxins, drug accumulation and / or high blood concentrations in other parts of the nephron, even at therapeutic doses, or high intracellular drug concentrations can occur, causing further damage to the kidneys and vestibule. To avoid problems related to multi-organ toxicity, extensive research has been conducted and is still ongoing to improve the structure of compounds that overcome toxicity.

[0003] In Patent WO2013 / 191550 A1, a novel one-step regioselective chemical diazotization of the C3-amine functional group of the 2-DOS ring is presented for modifying aminoglycosides. In Patent US2013 / 03455411 A1, a novel aminoglycoside albecacin is being developed by a multi-step strategy involving the use of basic carbohydrate building blocks and a glycosylation reaction between the 1'' position of ring III and the O6 of ring II.

[0004] In Patent WO2011 / 143497 A1, 140 monofunctional and bifunctional derivatives of gentamicin were synthesized by modifying its N1 and / or N6' positions.

[0005] To expand the scope of structural modification of parent aminoglycosides, in Patent WO2014 / 013495 A1, without using a vast number of synthetic steps targeting the negatively charged lipopolysaccharides present in the bacterial cell wall through ionic interactions, the work of synthesizing cationic amphiphilic derivatives of various aminoglycoside drugs was carried out.

[0006] In Patent WO2011 / 044501 A2, to accelerate the development process of new antibiotics, a chemical strategy for synthesizing various neomycin analogs was developed.

[0007] Subsequently, derivatives of the novel plazomycin of Patent WO2011 / 044501 A2 were synthesized by Achaogen. Pharmacokinetic evaluation and safety monitoring in healthy subjects receiving plazomycin injection showed no nephrotoxicity and ototoxicity in humans.

[0008] In Patent WO2014 / 1454713 A2, a novel chemical synthesis approach was used to develop sisomicin analogs for alleviating the ototoxicity of aminoglycosides while maintaining antibacterial activity. Recent evidence has suggested that mitochondrial protein synthesis is an important factor in the ototoxicity of aminoglycosides, and defects in mitochondrial function lead to the generation of reactive oxygen species (ROS) that cause ototoxicity.

[0009] In Patent WO2013 / 170985 A1, the activities and ototoxicities of various drugs, particularly apramycin, are disclosed. In Patent WO2011 / 124986 A2, the addition to aminoglycoside-lipid conjugates is presented.

[0010] In Patent WO2012 / 097454 A1, a class of acetyltransferase (AAC) inhibitors composed of pantetheine conjugated to the 6'-amine of aminoglycosides is being studied. In Patent US2014 / 0357590 A1, newer analogs were synthesized by modifying rings I, II, and III of the pseudotrisaccharide.

[0011] Another approach used in Patent US2014 / 0243280 A1 is the use of a new class of supramolecular protecting groups (SPGs) based on host-guest interactions that provide an attractive new perspective for single-step modification based on oligonucleotides.

[0012] Another effort that has been made is the use of amiloride as a substance secreted by the kidneys to prevent toxicity. US5,691,304 enumerates an improved process for preparing a polymyxin B / dextran conjugate in which dextran covalently binds to polymyxin B via an amine bond.

[0013] Aminoglycosides and polymyxins appear to generate free radicals within the inner ear, which then cause permanent damage to sensory cells and neurons, leading to permanent hearing loss. The nephrotoxicity induced by aminoglycosides and polymyxins presents clinically as non-oliguric renal failure, accompanied by a slow rise in serum creatinine and the occurrence of low-osmolar urine volume after several days of treatment. Aminoglycosides exhibit nephrotoxicity because a significant proportion (≈5%) of the dose, although small, is retained by the epithelial cells covering the S1 and S2 segments of the proximal renal tubule after glomerular filtration.

[0014] Currently, two approaches are used. One of the simplest and most fundamental approaches to reducing the nephrotoxicity of polybasic drugs that will lead to success is to reduce or prevent the accumulation of the drug at the organ site. The accumulation of aminoglycoside / polymyxin can be reduced by inhibiting its uptake or by promoting its release. The reduction in uptake has been achieved by two strategies.

[0015] The first aims to complex extracellularly by conjugating the polybasic drug, but conjugation makes the drug heavier, reduces excretion, and also causes toxicity to the kidneys.

[0016] Second, it aims to compete with or reduce the binding of drugs to the brush border membrane, but since the uptake into renal tubular cells saturates, some polybasic drugs can themselves act as competitors, leaving problems unresolved.

[0017] In such cases, drugs passing through the lumen are not reabsorbed if their concentration is too high. Therefore, reducing the dosing frequency of such drugs reduces toxicity. This leads to the conclusion of reducing the dosing cycle of these polybasic drugs from three or two times a day to once a day, which would be the only approach to reducing toxicity.

[0018] The problem of toxicity reduction has remained largely unaddressed. Therefore, this research of the present invention was taken up to address a new approach of supramolecular cationic complex formation without conjugation, without covalent bond formation, and without micelle formation, involving multiple mechanisms.

[0019] (Objectives of the Invention) One of the main objectives of the present invention is to identify compounds that can help reduce multi-organ toxicity and act through multiple mechanisms.

[0020] Another objective is to establish the concentration optimization of such each compound at a predefined ratio to achieve the goal.

[0021] Yet another objective is to optimize the formulation process to establish homeostatic conditions such that toxicity is minimal or not observed at all in vivo.

[0022] Yet another objective is to establish a dosing method for polybasic drugs.

[0023] A further objective of the present invention is to provide a hydrophilic formulation of a polybasic drug for reducing multi-organ toxicity in a mammalian subject that requires no conjugation, no covalent bond formation, and no micelle formation.

Summary of the Invention

[0024] This abstract is provided to introduce a selection of concepts in a simplified form that will be further described in the "Detailed Description" section below. This abstract is not intended to identify the key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0025] One aspect of the present invention is to provide compositions and formulations for reducing multi-organ toxicity associated with polybasic drugs by preparing supramolecular cationic complexes. The compositions and formulations of the present invention reduce ototoxicity, neurotoxicity, and nephrotoxicity associated with polybasic drugs.

[0026] Another aspect of the present invention relates to a process for preparing the supramolecular cationic complex, hereinafter referred to as the SMCC composition and formulation. The SMCC hydrophilic compositions and formulations of the present invention are usefully employed to reduce multi-organ toxicity associated with polybasic / cationic drugs in mammals when administered.

[0027] In another aspect of the present invention, the supramolecular cationic complex comprises a polybasic / cationic drug selected from the group of aminoglycoside or polymyxin antibiotics.

[0028] In another aspect of the present invention, the cationic compound is selected from ethoxylated amines, quaternary ammonium compounds, the amino acids l-arginine, l-lysine, histidine.

[0029] In another aspect of the present invention, natural polysaccharides are used as a scaffold basis for the supramolecular cationic complex.

[0030] In another aspect of the present disclosure, for the preparation of the present invention, a polybasic / cationic drug to cationic compound to natural polysaccharide in a ratio of 1:0.1:0.1 to 1:3:1 is required.

[0031] In another aspect of the present invention, the supramolecular cationic complex of the polybasic agent is formed without any chemical crosslinking via electrostatic interaction due to a specific charge-to-molecular weight relationship.

[0032] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings and figures.

Brief Description of the Drawings

[0033] The embodiments of the present specification will be better understood with respect to their characteristics and effectiveness from the following detailed description with reference to the figures showing various test results.

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] (DETAILED DESCRIPTION OF THE INVENTION) Embodiments of the present specification and their various features and advantageous details are shown in the accompanying drawings and tables and will be more fully described with reference to the non-limiting embodiments detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as not to unnecessarily obscure the embodiments of the present specification. The examples used herein are merely intended to facilitate understanding of how the embodiments of the present specification can be implemented and to enable those skilled in the art to implement the embodiments of the present specification. Therefore, the examples should not be construed as limiting the scope of the embodiments of the present specification.

[0039] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where the definition or use of a term in an incorporated reference conflicts with or contradicts the definition of that term as defined herein, the definition of that term as defined herein shall apply and the definition of that term in that reference shall not apply.

[0040] Various terms used herein are defined below. Unless a term used in a claim is defined below, it shall be given the broadest definition given to that term by a person skilled in the relevant art as reflected in publications printed at the time of filing and issued patents. Where the definition or use of a term in an incorporated reference conflicts with or contradicts the definition of that term as defined herein, the definition of that term as defined herein shall apply and the definition of that term in that reference shall not apply.

[0041] It should also be understood that the present disclosure can be implemented in a number of ways, such as as a composition, formulation, treatment method, or preparation method. In this specification, these embodiments, or other forms that the present invention can take, may be referred to as compositions and formulations. Generally, the composition or formulation can be modified within the scope of the present invention.

[0042] For the purposes of the present invention, the term "multiple organ toxicity" is understood to refer to systemic toxicity related to damage, cell death, and renal failure (nephrotoxicity), neurotoxicity (neuromuscular blockade, peripheral neuropathy, sensory impairment, and encephalopathy induced by polymyxins / aminoglycosides), and auditory impairment (ototoxicity) to one or more organs, such as the renal system leading to acute kidney injury (AKI), due to the toxicity of polybasic agents.

[0043] As used herein, the term "supramolecular cationic complex (SMCC)" refers to the process of preparing a complex in which the association of like charges occurs through electrostatic interactions, whereby cationic compounds / drugs are complexed with like-charged molecules by changing their properties from basic to acidic or by the simultaneous physical capture of complexes formed on a cation-π interaction and a polymeric scaffold.

[0044] Here, it is important to state that SMCC has nothing to do with supramolecular structures, which are large molecules formed by grouping or binding smaller molecules and often enable the development of molecules with desired shapes or functionalities, and thus belong to the field of nanoscience.

[0045] As used herein, the term "polymer" refers to large organic molecules composed of monomer units, such as polysaccharides, lipids, proteins, nucleic acids, etc.

[0046] As used herein, the term "polybasic / cationic" refers to an antibiotic cationic drug compound having two or more atoms of replaceable hydrogen, including but not limited to, gentamicin, tobramycin, amikacin, plazomycin, streptomycin, neomycin, paromomycin, ribostamycin, albekacin, dibekacin, gentamicin, kanamycin A and B, aprotinin, aristolochic acid, tricosanthin, etimicin, netilmicin, sisomicin, and apramycin, and drugs containing as part of the molecule amino-modified glycosides (sugars) called polymyxin b, colistin, and aminoglycosides, including but not limited to polymyxins such as polymyxins A through E, for example, non-ribosomal cyclic lipopeptide antibiotics.

[0047] As used herein, the term "adverse drug reaction (ADR)" refers to any adverse medical event associated with the use of a drug in humans, which can result in disorders, toxicity, tissue or cell damage, and adverse reactions caused by taking the drug. ADRs can occur due to single or long-term administration of a drug, are related to organ-specific toxicity, and can affect multiple organs. Here, ototoxicity refers to toxicity in ear cells and related symptoms such as hearing impairment, neurotoxicity refers to damage to nerve cells and related symptoms, and nephrotoxicity refers to cell damage and kidney system disorders in the kidneys and the kidney system.

[0048] The present invention relates to the systemic delivery of an SMCC formulation of a polybasic agent for reducing multi-organ toxicity in mammals after administration. The systemic use of some polybasic antibiotic agents such as aminoglycosides, polymyxins A to E is associated with the risk of nephrotoxicity, neurotoxicity and ototoxicity. Signs of toxicity appear immediately after high-dose administration or within 24 hours after drug administration, regardless of the dosing schedule. Frequent administration causes more toxicity and more severe ADRs, especially in severe cases such as sepsis or ICU patients. Many studies have been conducted to find ways to reduce toxicity, but with limited success. Currently used polybasic antibiotic products (hereinafter referred to as reference products) are still reported to have signs of toxicity, indicating that previously employed methods were not sufficient or that there were several other issues that prevented them from reaching the market. The main reason for the incomplete success of toxicity reduction solutions is the existence of multiple mechanisms, and it is now established that a single approach cannot address toxicity reduction. Therefore, the present invention is designed to address the orchestration of cellular mechanisms rather than focusing on a single mechanism involved in its etiology.

[0049] The disclosure of the present invention relates to the reduction of multi-organ toxicity, including the reduction of the nephrotoxicity of polybasic antibiotic agents known to cause acute kidney injury (AKI). More specifically, the present invention relates to compositions and formulations made therefrom for the safer management of ototoxicity, neurotoxicity and nephrotoxicity in critically ill patients or mammals.

[0050] According to one embodiment, one of the important issues in multi-organ toxicity reduction is the prevention of drug accumulation within organs, for example, for nephrotoxicity reduction, and the prevention of drug accumulation within kidney cells should be achieved.

[0051] One of the main causes of polymyxin and aminoglycoside nephrotoxicity has been elucidated to their binding to megalin and cubilin receptors present in the proximal renal tubules of the kidney or the apical brush border membrane of the cochlea of the inner ear. As defined above, polybasic drugs have a high binding affinity for megalin, leading to drug accumulation that results in ischemia and cell death. These drugs, which are essentially basic, are known to have an affinity for deposition in kidney / ear / nerve cells. As the drug passes through the kidney during the excretion process, it binds to these receptors, is internalized into epithelial cells, and deposits there.

[0052] Megalin, an endocytosis receptor in the proximal renal tubule, is the main route of aminoglycoside / polymyxin accumulation in the kidney and is involved in the development of nephrotoxic acute kidney injury (AKI) by mediating the tubular uptake of nephrotoxic substances. The accumulation of aminoglycosides and polymyxins in lysosomes and subsequent vesicle rupture are considered the main mechanisms causing nephrotoxicity in animals and humans. Tubular changes are associated with the occurrence of focal necrosis and apoptosis in the tubular epithelium, along with extensive proliferation of tubular and peritubular cells without obvious changes in renal function. These signs of toxicity can be measured by measuring blood biochemical parameters such as BUN and creatinine, biomarkers such as KIM-1 and Custatin-C, and performing histopathology of the relevant body parts. Figure 1 clearly emphasizes a histopathological study showing very little (near-normal) damage to renal tubular cells by the formulation of the present invention.

[0053] Figure 1(A): Shows acute tubular necrosis with patchy or diffuse exposure of renal tubular cells with loss of brush border. Tubular dilation, intratubular cast formation, vacuolization (arrowhead with circle), congestion (arrowhead with line), and flattening of renal tubular cells by eosinophilic cytoplasm (arrowhead with square) are seen. Interstitial mononuclear cell infiltration (IM) and small nuclear condensation nuclei (arrow) in the kidney of a control polymyxin-treated rat indicate significant damage.

[0054] Figure 1(B): Shows the histological images of normal glomeruli and renal tubules. Slight changes in the inner layer of the tubular epithelium (arrowhead with line), small condensed nuclei (arrowhead with circle). The renal tubules show changes of regeneration (arrowhead with square) in the rat kidneys treated with formulation F30, indicating slight toxicity.

[0055] Figure 1(C): Shows the histological image of normal glomeruli. Slight disappearance of renal tubular epithelial cells, slight congestion (arrow with circle). In the kidneys of rats treated with formulation F108, the renal tubules show changes of regeneration (arrow with line), indicating very low toxicity.

[0056] Figure 1(D): Shows the histological image of normal glomeruli. Slight disappearance of renal tubular epithelial cells, irregular lumen dilation (arrowhead with circle). In the kidneys of rats treated with F57, the renal tubules show changes of regeneration (arrowhead with line), indicating very slight toxicity.

[0057] Figure 1(E): Shows the enlargement of glomeruli with loss of brush border and acute renal tubular necrosis. Flattening of renal tubular cells due to renal tubular dilation (arrowhead with circle), cast formation in the renal tubules (arrowhead with square), vacuolization of the renal tubules (arrowhead with V), congestion (arrow with line) in the control amikacin-treated rat kidneys indicate damaged organs. All three drug groups treated with the new formulations of the present invention (B: polymyxin F30, C: colistin F108, and D: amikacin F57) showed minimal toxicity even with TID administration when compared with their respective control (reference) formulations.

[0058] One embodiment of the present invention is to identify a compound that can provide competitive binding and limit the accumulation of polybasic drugs at organ sites.

[0059] Another problem is the management of neurotoxicity that causes abnormal neurobehavioral changes including sensory and motor dysfunction. Neurotoxicity is one of the major adverse drug reactions (ADRs) associated with polymyxin therapy, caused by polymyxin-induced nerve damage and mainly related to oxidative stress and mitochondrial dysfunction. The central nervous system is very sensitive to oxidative damage due to its obligatory high oxygen demand. Mitochondria are essential for maintaining basic cellular functions such as energy metabolism and ATP production. Therefore, the present invention carefully manages the reduction of mitochondrial dysfunction by administration of SMCC in the subject in need.

[0060] Apoptosis plays an important role in maintaining brain homeostasis in response to drug-induced toxicity. Autophagy involves cellular proteins and organelles that are engulfed by autophagosomes, digested by lysosomes, and recycled to maintain cellular homeostasis in the face of various stresses including nutrient deprivation, hypoxia, oxidative stress, and DNA damage. Autophagy is known to be involved in maintaining neuronal homeostasis, particularly in response to drug-induced oxidative stress and mitochondrial dysfunction. Two enzyme families, NOX and NOS, are major sources of reactive oxygen species (ROS) / reactive nitrogen species (RNS) / nitrosative stress due to the overproduction of nitric oxide (·NO), which act together to disrupt homeostasis and damage cells. Therefore, the reduction of oxidative stress is another important factor managed by the present invention by formulating SMCC to maintain homeostasis. The use of appropriate polymers that can regulate NO production was another important feature of the present invention.

[0061] Catalase, superoxide dismutase, and plasma ferric reducing ability are known oxidative stress markers studied in a rat model. A shift to negative values in the control group indicates an increase in oxidative stress. When tested, the compositions of the present invention and the formulations made therefrom showed a significant increase in the antioxidant levels of the three parameters tested, as shown in the examples.

[0062] Yet another problem is the control of relative cellular hypoxia as a major activator of hypoxia-inducible factors, which is detectable in neurotoxic and chronic kidney disease tissues regardless of etiology and results from a combination of structural and functional changes including: decreased peritubular blood flow associated with glomerular injury, capillary rarefaction, vasoconstriction, luminal narrowing of atherosclerotic vessels, increased oxygen demand from filtration and tubular hypertrophy, limitation of oxygen diffusion as a result of extracellular matrix expansion, and renal anemia.

[0063] Hypoxia is associated with a significant increase in blood lactate and severe systemic acidosis as a direct effect of anaerobic metabolism. Thus, effective management of hypoxia by regular oxygen supply is also innovatively managed by the present technique of SMCC formation and administration to the subject in need.

[0064] Another problem of the present invention is to identify the appropriate balance of compounds that can significantly prevent injury or cell damage and maintain homeostasis when formulated with nephrotoxic polybasic / cationic agents.

[0065] According to a preferred embodiment of the present invention, for successful competitive inhibition, the cationic compound is selected to pair with basic agents such as polymyxin and aminoglycoside. Here, the cationic compound is selected from the group including ethoxylated amines, quaternary ammonium compounds, the amino acids l-arginine, l-lysine, and histidine. According to a preferred embodiment of the present invention, the cationic compound is an amino acid.

[0066] Another problem innovatively overcome in the present invention is the complex formation of similarly charged molecules because both the drug compound and the competitive inhibitor compound are essentially cationic. This problem was overcome by experimentally changing the pH of the solution to make the cationic amino acids anionic due to the zwitterionic nature. Amino acids can be essentially positive, negative, neutral, or polar. At a pH lower than their pI, they carry a net positive charge. Above their pI, they carry a net negative charge. Thus, by changing the pH, the properties of zwitterionic amino acids can be changed.

[0067] Alternatively, complex formation is carried out using cation-π interactions. Some experiments were conducted to find the best pairs to stabilize the formulation while maintaining homeostasis in vivo and reducing AKI, neurotoxicity, and ototoxicity. Among the various quality-by-design (QBD) tests performed, l-arginine as a cationic amino acid was selected for complex formation.

[0068] According to another embodiment, L-arginine is preferred over lysine and histidine. Formulations without L-lysine were proven to have failed in stabilization or be toxic. Furthermore, note that the Pka 3 and pI values of L-arginine are the highest among the three amino acids, and arginine can provide the highest competitive inhibition due to its highest charge.

Table 1

[0069] According to yet another preferred embodiment, both l-arginine and l-lysine provide competitive binding with megalin, but l-arginine is preferred. Since arginine is also involved in NO production and needs to be regulated, achieving competitive binding while optimizing NO secretion was another challenge skillfully managed by the present invention. Optimization of amino acid concentration is important, as slight variations disrupt the homeostasis necessary to achieve the perfect balance for reducing toxicity.

[0070] According to a preferred embodiment, the ratio of said arginine to said agent is from 0.1:1 to 3:1.

[0071] According to a preferred embodiment for producing a supramolecular cationic complex, the polymer is selected from the group of natural polysaccharides as a scaffold base. The reason for selecting polysaccharides is that they provide a continuous energy source. The selection of polysaccharides was based on charge neutrality. Therefore, the selection of natural polysaccharides played an important role in the decision-making.

[0072] According to another embodiment, the natural polysaccharide is selected from the group comprising dextran, poly(sialic acid), pullulan, dextrin, hyaluronic acid, chitosan, and heparin. Other natural polysaccharide agents that can be used alternatively are guar gum, gum arabic, tragacanth gum, pine gum, karaya gum, locust bean gum, agar, alginate, carrageenan, pectin, starch, c-starch, xanthan gum, succinoglucan, acrylic acid graft copolymer, and the like.

[0073] According to yet another preferred embodiment of the present invention, the polymer selected for SMCC formation is ordinary low molecular weight dextran with charge neutrality, which has a linear backbone of α-linked d-glucopyranosyl repeating units. Low molecular weight dextran particularly refers to dextran with a molecular weight of less than 60 KDa. In the prior art, the selected dextran had a high degree of polydispersity of 10 7 ~10 8It has a high molecular weight in the range of kDa, is suitable for conjugation, and increases the circulation time. These high molecular weight dextrans and their derivatives have been used in drug administration by covalent bond formation in most prior arts. Instead of adopting a predefined approach, in the present invention, low molecular weight dextran is selected without any chemical modification, which provides a neutral scaffold surface for capturing cationic complexes.

[0074] The main determinants of proper organ function are the microcirculation for performing organ function and the proper supply and utilization of oxygen at the cellular level. Due to the very complex structure of the renal microvasculature, the need to meet high energy demands, and the fact that the kidney is border ischemic, the kidney becomes a very vulnerable organ to hypoxic injury in AKI. In the normal steady state, the supply of oxygen (O2) to the kidney tissue is well regulated. However, in the state of illness or sepsis, due to the dysfunction of the renal microvasculature, the delicate balance between oxygen supply and demand is disrupted.

[0075] This dysfunction is mainly due to the interaction of oxygen processing, nitric oxide metabolism, and radical formation in the kidney. The oxygen requirement of the kidney is mainly determined by ATP production.

[0076] Therefore, the selection of polysaccharide as a polymeric scaffold is such that the formed complex is confined to the scaffold of neutral polysaccharide without any chemical bond, any conjugation, covalent bond, or micelle formulation, as a continuous energy source, and for capturing polybasic / cationic drug entities and cationic amino acids to form supramolecular cationic complexes, and the supramolecular cationic complexes are formed by changing the pH of basic amino acids by zwitterionicity or by cation-π interaction.

[0077] Here, it is important to state that since the formation of SMCC is based on similar charge interactions by changing the pH of arginine due to the zwitterionic nature of arginine or by performing cation-π interactions, the ratio of arginine to cationic agents has played an important role. Since arginine binds competitively to drugs with receptors, the ratio of arginine to polymyxin (more toxic than aminoglycoside and with a higher Pka value) is higher than the ratio of arginine required for aminoglycoside. The ratio of L-arginine to polymyxin drug is between 3:1 and 0.5:1, while the ratio of L-arginine to aminoglycoside drug is between 0.1:1 and 1:1.

[0078] Another critically important issue that is only managed when addressing the reduction of the multi-organ toxicity of polybasic drugs is microcirculation dysfunction. This can significantly limit the ability of the circulation to provide adequate oxygen to fuel oxidative phosphorylation for ATP production and can directly impair the function of the Na / K ATPase pump.

[0079] However, inflammation and oxidative stress can also greatly alter the delicate balance between oxygen supply and consumption in the brain and kidneys. Furthermore, disruption of the homeostasis between reactive oxygen species (ROS) and nitric oxide (NO) caused by inflammation in the nerves and kidneys can contribute to neurotoxicity and nephrotoxicity.

[0080] Some drugs with known neurotoxicity and nephrotoxicity are used in today's medical practice because antibacterial resistance increases when other drugs do not produce results. The continuous supply of energy for ATP production, along with microcirculation management, is creatively managed by selecting unmodified low molecular weight dextran as a polysaccharide as a scaffold for SMCC.

[0081] According to a preferred embodiment of the present invention, the novel feature of the present invention is low molecular weight plain dextran. Specifically, dextran 40 kDa (hereinafter referred to as D40 or dextran 40) is used. This is also non-conjugated and does not form micelles. Without chemical modification in its neutral form for physically capturing polybasic drug entities and cationic amino acids to form a supramolecular cationic complex, when administered to a subject in need, it reduces multi-organ toxicity by managing multiple mechanisms that would otherwise cause toxicity simultaneously.

[0082] It is important to state here that the main drawback of conjugation is that the half-life of the drug becomes longer and the toxicity further increases. Dextran 40 further provides excellent stability by cryoprotection at temperatures from -45°C to 60°C. D40 has been observed to not only function as an energy source required for ATP production but also contribute to the improvement of blood flow and microcirculation.

[0083] Other forms of low molecular weight dextran are highly toxic, and it has been proven through experiments that D40 is selected as the safest neutral polysaccharide. Using D40 at a specific ratio of 100% or less of the drug component avoids its harmful effects and toxicity such as intracellular accumulation. It is important to emphasize that ordinary dextran is used in the present invention without chemical modification, without conjugation, or without covalent bond formation.

[0084] According to another embodiment, dextran 40 improved microcirculation flow by two mechanisms: by reducing blood viscosity through blood dilution and by inhibiting erythrocyte aggregation. Dextran 40 also functions as an inhibitor of T lymphocyte adhesion to endothelial cells (EC). Dextran 40 inhibits the constitutive and cytokine-induced binding of T cells to EC by selectively interfering with the clustering of adhesion molecules on T cells. This process is thought to play an important role in inducing extravasation of leukocytes to the periphery during inflammation.

[0085] According to yet another embodiment, the addition of one or more sugar-based compounds is optional and is at the discretion of one skilled in the art to improve the crystal structure and stability of the formulation according to the type of treatment for which the final formulation is selected.

[0086] In addition to nephrotoxicity, polybasic agents are also associated with ototoxicity. It is well established that polybasic agents such as gentamicin, amikacin, apramycin, plazomycin, polymyxin B, and polymyxin E accumulate in the epithelial cells of the cochlea of the inner ear via megalin. Once inside the cell, the agents accumulate in lysosomes and the endoplasmic reticulum, where they bind to calreticulin and increase the level of misfolded proteins within the cell.

[0087] After further accumulation, the polybasic agent is released into the cytosol, causing oxidative stress and apoptosis. Therefore, to reduce multi-organ toxicity, the formed SMCC complex exhibits antioxidant properties that reduce ROS and avoid apoptosis. D40 has been experimentally proven to form supramolecular cationic complexes (SMCCs) with amino acids and polybasic agents and exhibit these properties.

[0088] According to one of the most preferred embodiments, the supramolecular cationic complex is formed by physical interactions including electrostatic interactions without any conjugation, covalent bonding, or micellar formulation. (Figures 1-5) Clearly distinguish between normal drug-induced toxicity and the reduction of SMCC formulation-based toxicity.

[0089] The resuscitation of the damaged kidney requires an integrated correction of the homeostasis between oxygen and reactive oxygen and nitrogen species. To demonstrate the effectiveness of selective polysaccharides, particularly dextran, in the restoration of microcirculatory oxygenation, in parallel with the improvement of oxidative stress and the source of the ATP requirements of the Na / K ATP pump that functions to protect renal function after septic AKI, several experimental therapies have been conducted.

[0090] Dextran 40 is the only dextran that exhibits immunomodulatory functions and reduces (by about 40%) the release of nitric oxide by cells, both in the absence and presence of lipopolysaccharide (LPS). Furthermore, dextran 40 is more potent (70%) than other dextrans in suppressing lipid peroxidation. These indicate dextran with a weight of 40 kDa, which is considered ideal for use as an antioxidant and immunomodulatory agent in the present invention.

[0091] Despite many beneficial effects discovered so far, ensuring a complete understanding of the relative advantages and potential adverse effects of L-arginine on human / animal metabolism poses another challenge in the present invention.

[0092] The main adverse effects of L-arginine include the acceleration of the onset and / or growth of certain malignancies. Therefore, safe dosage optimization to achieve homeostasis is one of the important inventive steps of the present invention. This includes, but is not limited to, the essential components of the present invention required to regulate the NO levels produced by L-arginine. Arginine plays an important role in competitive inhibition with megalin.

[0093] Therefore, the selection of specific weight and molar ratios that have been proven to be less toxic when combined with other components is carefully studied through a series of experiments. The innovative first arginine appears to be charged like a polybasic drug, but its charge changes with pH changes so that it can form complexes. Therefore, concentration optimization becomes an essential part of the inventive step of the present invention.

[0094] Yet another challenge to overcome is the significant adverse effects of dextran, including anaphylaxis, volume overload, pulmonary edema, cerebral edema, or platelet dysfunction, hypotension, shock, and cardiac arrest. The rare but serious complication of the dextran osmotic pressure effect is acute renal failure.

[0095] Therefore, another important advancement of the present invention is the selection of the correct molecular weight of dextran, and then, using the optimization of the dextran concentration, not only provides the energy source necessary to prevent AKI and multiple organ toxicity, but also improves microcirculation, controls NO production, and achieves its antioxidant effect.

[0096] According to a preferred embodiment of the present invention, the ratio of arginine to D40 is between 0.25:1 and 7.5:1.

[0097] According to yet another embodiment, the ratio of the cationic complex to the polymer is from 1:0.05 to 1:0.5 in SMCC.

[0098] According to yet another important embodiment, maintaining the charge molecular weight balance is essential for SMCC to achieve homeostasis in the body and provide the stability of the composition and the formulations made therefrom for at least 24 months (end of shelf life).

[0099] Therefore, based on the Flory-Huggins theory of the entropy term and the Debye-Hückel theory of the electrical / electrostatic interaction term, when σ3r ≧ 0.53 (σ is the charge density / charge per unit area, r is the polymer molecular weight), the conditions for complexation are satisfied, which is ≧ 53 in the present invention, and it is possible to prove complexation.

[0100] According to yet another one of the most important embodiments of the present invention, it is the ratio of each component in the complex. The ratio is very important for achieving homeostasis after administration to the target in need. After a series of experiments to check the reduction of animal toxicity and after verifying the best formulation for achieving the maximum antioxidant effect, the ratio of each component of SMCC was optimized.

[0101] According to a preferred embodiment, in the formed SMCC, the ratio of the cationic drug to the cationic amino acid to the low molecular weight dextran is from 1:0.1:0.1 to 1:3:1.

[0102] According to yet another preferred embodiment, in the formed SMCC, the ratio of the cationic agent to the cationic amino acid to low molecular weight dextran is from 1:0.1:0.2 to 1:3:0.75.

[0103] According to one of the most preferred embodiments of the present invention, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is polymyxin B and polymyxin E or their pharmaceutical salts, which electrostatically bind to the cationic amino acid L-arginine using cation-π interactions while capturing the cationic complex formed with low molecular weight dextran D40.

[0104] According to yet another one of the most preferred embodiments of the present invention, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is amikacin and apramycin or their pharmaceutical salts, which electrostatically bind to the cationic amino acid L-arginine due to the pH change of arginine and simultaneously capture the cationic complex formed with low molecular weight dextran D40.

[0105] According to yet another most preferred embodiment of the present invention, for the early recognition of acute kidney injury, it is carried out by using biomarkers such as kidney injury molecule 1 (KIM-1) and cystatin C. KIM-1 is highly upregulated in proximal tubular cells after kidney injury. The serum level of cystatin C is a more powerful predictor of renal outcome and cardiovascular toxicity risk than creatinine level. Figures 2 and 3 show that when each control and F57 were administered amikacin 400 mg / kg * 3 times a day, apramycin 500 mg / kg * 3 times a day, each control and F175, polymyxin 7.5 mg / kg * 3 times, each control and F30, colistin 12 mg / kg * 3 times a day, all control groups damaged the kidneys, and a negative trend of the graph was observed with Kim-1 and cystatin-C, while the formulation of the present invention showed a positive bar trend, clearly indicating the least kidney damage.

[0106] According to one of the most preferred embodiments of the present invention, various formulations of the polybasic agent are optimized for maintaining homeostasis after administration in order to reduce multi-organ toxicity. Figures 4 and 5 represent a comparison with a reference agent. In the experiment, several test formulations were administered to different groups of rats together with a reference control agent (commercially available reference product) for 2 days at each TID administration. Samples were collected from each group before and after administration. The biochemical parameters BUN and creatinine were measured for renal function tests in plasma / serum. F-30 of polymyxin B, F-108 of colistin, F-57 of amikacin, and F-175 of apramycin had little or no effect on the serum biochemical levels that were found to be significantly elevated in commercially available formulations at the same administration level, and it was found that the control groups of all polybasic agents showed a significantly higher renal impairment.

[0107] According to yet another embodiment of the present invention, the addition of either lysine / histidine with arginine increased the toxicity. A slight deviation in the charge neutralization potential results in higher toxicity compared to the best ratio optimized by the present invention. From a series of experiments, the inventors concluded that the reduction in toxicity is significantly less than that optimized in the present invention in any other combination or ratio, or in replacing arginine with other cationic amino acids.

[0108] According to another preferred embodiment, the ratio of the agent to the polymer is from 1:0.1 to 1:1 in SMCC. Removal of any component from the complex results in increased toxicity, especially removal of D40 or reduction by more than one-tenth of the drug concentration, which likely results in even higher toxicity than the original drug, because at concentrations below that, the balance of nitric oxide cannot be maintained, sufficient antioxidant action cannot be obtained, and the resulting toxicity of the drug product is further enhanced by the toxicity of high arginine. Experiments were conducted to test the nitrotyrosine levels in the blood of rats comparing the control with the selected formulation of the present invention. An increase in the increase in nitrotyrosine values was observed in the control group indicating nitrosative stress, which was significantly reduced in the compositions of the present invention and the formulations made therefrom.

[0109] According to yet another preferred embodiment of the present invention, a concentration of D40 above the drug product does not enable the complex to be realized during formulation, and the complex so formed is not stable. Replacing D40 with dextran 20KDa or dextran 60KDa resulted in very high mortality. Repeated experiments have proven that it was the safest for reducing toxins among all D40s.

[0110] According to yet another one of the most important embodiments of the present invention, the supramolecular complex is formed by changing the arginine pH due to the zwitterionic nature of arginine to enable the formation of a cationic complex.

[0111] According to another important embodiment of the present invention, alternatively, the supramolecular complex is formed by cation-π interactions.

[0112] According to yet another embodiment of the present invention, the ratio of the cationic amino acid arginine to the polymer varies depending on the nature of the bond. In the case of the polymyxin drug with cationic pi interaction, the ratio of arginine to D40 in the polymyxin polybasic drug composition and the formulations made therefrom is between 7.5:1 and 2.5:1.

[0113] According to yet another preferred embodiment of the aminoglycoside, wherein the arginine charge changes by varying the pH due to the zwitterionic nature of the aminoglycoside before binding to the agent, in such a case, the ratio of arginine to D40 is between 0.25:1 and 1:1. Increasing arginine beyond the amount of dextran in such formulations dramatically increases nitrosation and oxidative stress and results in higher toxicity.

[0114] According to another embodiment, the complex is administered to a subject in need thereof by a parenteral route, and the subject is preferably a mammal.

[0115] According to a preferred embodiment, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is polymyxin B or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine and low molecular weight dextran D40 at a ratio of agent:cationic amino acid:dextran of 1:1.4:0.2 to 1:2.5:0.5.

[0116] According to another preferred embodiment, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is polymyxin E or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine and low molecular weight dextran D40 which provides a scaffold for physical capture, and the ratio of agent:cationic amino acid:dextran is 1:0.5:0.1 to 1:2:0.25.

[0117] According to yet another preferred embodiment, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is amikacin or a pharmaceutically acceptable salt thereof, which is electrostatically combined with L-arginine and D40 at a ratio of agent:cationic amino acid:dextran of 1:0.2:0.3 to 1:0.5:0.75.

[0118] According to another preferred embodiment, the polybasic / cationic agent used to prepare the SMCC composition and the formulations made therefrom is apramycin or a pharmaceutically acceptable salt thereof, which is combined with L-arginine and D40 at a ratio of agent:cationic amino acid:dextran of 1:0.1:0.2 to 1:0.5:0.75.

[0119] According to another important embodiment of the present invention, the supramolecular cationic complex maintains homeostasis when administered parenterally to mammals and reduces multi-organ toxicity without disturbing the pharmacokinetics of the individual agents. The composition is formulated as a liquid or lyophilized formulation.

[0120] The following examples are provided to illustrate the present invention. However, it should be understood that the present invention should not be limited to the specific conditions or details described in these examples.

[0121] Example 1 - Optimization Study of SMCC Formulations

Table 2-1

Table 2-2

[0122] Example - 2 Comparative Study of Plasma Oxidative Stress Markers in a Rat Model Using a Reference Agent and the Formulation of the Present Invention

Table 3-1

Table 3-2

[0123] According to the above research, the antioxidant capacity of each formulation is evaluated using the sera of rats used in the nephrotoxicity evaluation study. Changes in antioxidant parameters such as catalase activity, SOD (superoxide dismutase) activity, and ferric ion reducing antioxidant power (FRAP) in serum were evaluated using well-established procedures and compared with the reference drug group. After TID administration of each drug (reference drug / commercial formulation), the results of % change were observed to be negative, indicating an increase in oxidative stress. The formulation group of the present invention for each drug tested showed a significant decrease in oxidative stress represented by a positive percentage change, indicating the antioxidant capacity of the drug and thereby reducing toxicity. The test for nitrotyrosine showed that nitrosative stress increased with the administration of all drug control groups and decreased dramatically to less than 1 or to a negative value in the formulation of the present invention that shows the role of homeostasis maintained by the components of this composition.

[0124] Comparative study of antioxidants between the control drug group in Example 3 and the formulations with and without arginine and dextran

Table 4-1

Table 4-2

[0125] According to the above research, in vitro tests were conducted to examine the antioxidant ability of each formulation of the present invention, and comparisons with reference products were made in the presence or absence of arginine and dextran. The test parameters included the TEAC (Total Equivalent Antioxidant Capacity) assay, superoxide anion radical scavenging, hydrogen peroxide radical scavenging, and reducing power assay. As a result, F-30, F-108, F-57, and F-175 showed significantly higher scavenging activities compared to the control. When comparing formulations without arginine, aminoglycosides still showed higher antioxidant ability than the control. When comparing formulations without dextran, the polymyxin group showed significantly higher antioxidant ability than the control, indicating that the final formulations with specific ratios of each excipient had the highest antioxidant ability. From this experiment, it was revealed that in the absence of arginine or dextran, free radicals increased significantly, and each component played a major role in maintaining homeostasis.

[0126] (Main features of the invention) The present invention provides a novel composition and formulation of a polybasic drug for reducing multi-organ toxicity.

[0127] The present invention provides a supramolecular cationic complex composition and formulation of similarly charged molecules without any chemical cross-linking or covalent bond formation.

[0128] The present invention provides concentration optimization of each compound at a predefined ratio to achieve the best possible effects and homeostasis after administration of a drug that minimizes or reduces toxicity.

[0129] The present invention provides a composition for the formation of a supramolecular cationic complex involving a very selective selection of cationic compounds and polymers that target multiple mechanisms simultaneously, resulting in almost complete equilibrium in vivo.

Claims

**Claim 1** A composition comprising a polybasic / cationic agent that reduces multi-organ toxicity in mammals by forming a supramolecular cationic complex without chemical cross-linking and covalent bonding, wherein the supramolecular cationic complex (a) the polybasic / cationic agent selected from the group of aminoglycoside or polymyxin antibiotics, (b) a cationic compound selected from the group of ethoxylated amines, quaternary ammonium compounds, amino acids, wherein the amino acid is selected from L-arginine, L-lysine, histidine, a cationic compound, (c) a polymer for the scaffold base, and the polymer is low molecular weight dextran without chemical modification, the supramolecular cationic complex is formed by cationic electrostatic interaction, the weight ratio of the polybasic / cationic agent: the cationic compound: the low molecular weight dextran is from 1:0.1:0.1 to 1:3:1, (d) the supramolecular cationic complex is administered to a subject in need by a parenteral route, a composition. **Claim 2** The composition according to claim 1, wherein the cationic compound is L-arginine. **Claim 3** The composition according to claim 1, wherein the low molecular weight dextran is dextran 40 kDa. **Claim 4** The supramolecular cationic complex is formed by electrostatic interaction without any conjugation, covalent bonding or micelle formation, the weight ratio of the polybasic / cationic agent to the polymer is from 1:0.1 to 1:1, the weight ratio of the cationic compound to the polybasic / cationic agent is from 0.1:1 to 3:1, The composition according to claim 1, wherein the supramolecular cationic complex maintains homeostasis and reduces multi-organ toxicity when administered parenterally to a mammal. **Claim 5** The polybasic / cationic agent is polymyxin B or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine together with dextran 40 kDa that provides a scaffold for physical capture, The weight ratio of the polybasic / cationic agent: the cationic amino acid L-arginine: the dextran 40 kDa is from 1:1.4:0.2 to 1:2.5:0.5, the composition according to claim 1. **Claim 6** The polybasic / cationic agent is polymyxin E or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine together with dextran 40 kDa that provides a scaffold for physical capture. The composition according to claim 1, wherein the weight ratio of the polybasic / cationic agent: the cationic amino acid L-arginine: the dextran 40 kDa is from 1:0.5:0.1 to 1:2:0.

25.

7. The polybasic / cationic agent is amikacin or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine together with dextran 40 kDa that provides a scaffold for physical capture. The composition according to claim 1, wherein the weight ratio of the polybasic / cationic agent: the cationic amino acid L-arginine: the dextran 40 kDa is from 1:0.2:0.3 to 1:0.5:0.

75.

8. The polybasic / cationic agent is apramycin or a pharmaceutically acceptable salt thereof, which is electrostatically combined with the cationic amino acid L-arginine together with dextran 40 kDa that provides a scaffold for physical capture. The composition according to claim 1, wherein the weight ratio of the polybasic / cationic agent: the cationic amino acid L-arginine: the dextran 40 kDa is from 1:0.1:0.2 to 1:0.5:0.75.

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