A lactam moiety based cryoprotectant, compositions thereof and a method of cryopreservation of biological materials

Abstract

The present disclosure provides a liquid cryoprotectant having a lactam moiety structure of wherein the lactam having at least one hydroxyl functional group; wherein Y is an alkylene or alkenylene group comprising 2 to 50 carbon atoms; wherein 2 to 4 carbon atoms reside in the lactam ring between the I group and the II group; Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, rylenes, and combinations thereof, wherein the aforementioned groups may or may not contain oxygen atom(s); and R is selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms. The present disclosure also provides a composition for cryopreservation having a lactam moiety structure.

Description

Docket No.: 4387PCTA LACTAM MOTETY BASED CRYOPROTECTANT, COMPOSITIONS THEREOF AND A METHOD OF CRYOPRESERVATION OF BIOLOGICAL MATERIALSFIELD OF THE INVENTION

[0001] The presently disclosed process(es), procedure(s), method(s), product(s), result(s), and / or concept(s) (collectively referred to hereinafter as the “present application, present disclosure or invention”) relates generally to a liquid cryoprotectant having a lactam moiety.BACKGROUND OF THE INVENTION

[0002] This present invention relates to a liquid cryoprotectant having a lactam moiety, compositions comprising the cryoprotectant having a lactam moiety, and a method of cry opreservation of biological materials.

[0003] Cry opreservation techniques at temperatures at or below 0°C are routinely used for longtime preservation of biological materials such as cells and tissues of animals (including human cells and tissues) and plants. Thompson et al., Cryopreservation and Thawing of Mammalian Cells, December 2014, John Wiley &Sons, Ltd: Chichester. DOI: 10.1002 / 9780470015902. a0002561. pub2. Effective long-term storage of mammalian cells is crucial to the successful application of such cells as clinical and research tools. For example, stem cells can be used for cell transplantation, tissue engineering, and regenerative medicine. Cryopreserved oocytes, sperm, and embryos can be used in assisted reproductive technologies. In transplantation and regeneration medicine, living tissues such as the skin, cornea, pancreatic islets, dental pulp and heart valves need to be cryopreserved.

[0004] It has been proven that the osmotic imbalance and intracellular ice formation could damage cells during the freezing process, Gao et al., Mechanisms of Cryoinjury in Living Cells, ILAR Journal, 2000, 41 (4): 187-196. To avoid this, cryoprotectants (CPAs) are used to preserve the viability of the cells and tissues during freezing. Cryoprotectants are characterized into three main chemical classes: polyols (e.g., diols, glycerol), amides, and sulfoxides. Commonly used cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), and polyethylene glycol (PEG). Furthermore, some additives, such as macromolecules and sugars, can be added to further decrease the damages on cells and tissues during cryopreservation.Docket No.: 4387PCT

[0005] Among these cryoprotectants, DMSO has a high cell permeability, and is the most effective and frequently adopted. However, DMSO is physiologically toxic and known to cause high blood pressure, nausea and vomiting when the cells are transfused to a recipient. Further, the toxicity of DMSO tends to cripple the cells' survival rates and / or functions after the thawed cells are cultured or transfused into a recipient's body.

[0006] Slow freezing and vitrification are the two traditional approaches to the cry opreservation of biological materials. During slow freezing, the cells are cooled to temperatures slightly below their equilibrium freezing point and ice is seeded in the extracellular media. As ice forms in the extracellular solution, there is a progressive increase in the external solute concentration. As a result, the cell dehydrates, the melting point of the cytoplasm lowers and the formation of intracellular ice is avoided. Vitrification is defined by the viscosity of the sample reaching a sufficiently high value to behave like a solid but without crystallization. Principles of Cry opreservation, Methods in Molecular Biology, vol. 368: Cry opreservation and Freeze-Drying Protocols, Second Edition, Humana Press Inc. This glassy state can be induced in most liquids if cooling occurs rapidly. The addition of cryoprotectants can decrease the required high cooling rates.

[0007] The conventional techniques for the cryopreservation of mammalian cells are generally associated with disadvantages that detract from the potential use of the cells in clinical or research settings. Thus, there is a need for improved cryoprotectant, cryopreservation compositions and methods for cry opreserving biological material.

[0008] Hydroxyalkyl pyrrolidones provide an unexpected results and benefit over DMSO because it can permeate cell membranes (intracellular CPA) and can control ice crystal formation extracellularly. The pyrrolidone structure is the essential element for disrupting H-bond formation, thus results a slower and gentler penetration.

[0009] The structure of hydroxyalkyl pyrrolidone will provide value such as increasing living cell survival rate and penetration of CPA to the membrane and control of ice crystal formation. Hydroxyalkyl pyrrolidone is an effective penetrant but less disruptive to lipid bilayers.

[0010] The following patents illustrate suitable cryoprotectants, cryopreservation composition and a method of cryopreservation: U.S. patents U.S. Pat. No. 7,410,798, U.S. Pat. No. 10,791,730,Docket No.: 4387PCTU.S. Patent No. 9,458,424, U.S. Pub. No. 2010 / 0124564 Al , U.S. Pub. No. 2021 / 0315198A1 , U.S. Pub. No. 2009 / 0130756A1 and PCT. Pub. No. 2024 / 002739A1.SUMMARY OF THE INVENTION

[0011] One aspect of the present application is to provide a liquid cryoprotectant having a lactam moiety structure of:wherein the lactam having at least one hydroxyl functional group; wherein Y is an alkylene or alkenylene group comprising 2 to 50 carbon atoms; wherein 2 to 4 carbon atoms reside in thelactam ring between theugroup and thezgroup; Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein the aforementioned groups may or may not contain oxygen atom(s); and R is selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms.

[0012] Another aspect of the present application is to provide a composition for cry opreservation of a biological material, comprising: the liquid cryoprotectant with a lactam moiety, at least one membrane protectant, a base medium, optionally, a media supplement and optionally, a cry opreservation supporting ingredient.

[0013] One more aspect of the present application is to provide a method of cryopreserving biological material comprising cryopreserving biological material with the composition comprising a liquid cryoprotectant having a lactam moiety structure.Docket No.: 4387PCTDETAILED DESCRIPTION OE THE INVENTION

[0014] Before explaining at least one aspect of the disclosed and / or claimed inventive concept(s) in detail, it is to be understood that the disclosed and / or claimed inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components or steps or methodologies set forth in the following description or illustrated in the drawings. The disclosed and / or claimed inventive concept(s) is capable of other aspects or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0015] As utilized in accordance with the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0016] Unless otherwise defined herein, technical terms used in connection with the disclosed and / or claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0017] The singular forms “a”, “an”, and “the” include plural forms unless the context clearly dictates otherwise specified or clearly implied to the contrary by the context in which the reference is made. The term “Comprising” and “Comprises of’ includes the more restrictive claims such as “Consisting essentially of’ and “Consisting of’.

[0018] For purposes of the following detailed description, other than in any operating examples, or where otherwise indicated, numbers that express, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about". The numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties to be obtained in carrying out the invention.

[0019] All percentages, parts, proportions and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore; do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.Docket No.: 4387PCT

[0020] All publications, articles, papers, patents, patent publications, and other references cited herein are hereby incorporated herein in their entirety for all purposes to the extent consistent with the disclosure herein.

[0021] The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more depending on the term to which it is attached.

[0022] In addition, the quantities of 100 / 1000 are not to be considered limiting as lower or higher limits may also produce satisfactory results.

[0023] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0024] The term “each independently selected from the group consisting of’ means when a group appears more than once in a structure, that group may be selected independently each time it appears.

[0025] The term “cryoprotectant / cryopreservanf ’ is a substance used to protect biological tissue, cell or organism from freezing damage.

[0026] Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.

[0027] Primary Cell: The term, “primary cell,” refers to cells that are directly isolated from a subject and which are subsequently propagated.

[0028] In vivo: As used herein, the term “in vivo” refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems,Docket No.: 4387PCT the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0029] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0030] Cry opreservation: As used herein, the term “cryopreservation” or “freezing” generally refers to a method in which cells are frozen to a temperature below 0°C to maintain cellular viability. Cryopreserved cells maintain viability for an extended period of time in the frozen state, such as for 1, 5, 10 or more years in the cryopreserved state. The cryopreserved cells, once thawed, are able to propagate both for in vitro and in vivo applications.

[0031] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and / or a clone.

[0032] According to one of the embodiments, the present invention relates a liquid cryoprotectant having a lactam moiety structure of:wherein the lactam having at least one hydroxyl functional group; wherein Y is an alkylene or alkenylene group comprising 2 to 50 carbon atoms;Docket No.: 4387PCTRH CH wherein 2 to 4 carbon atoms reside in the lactam ring between theugroup and thez' group;Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein the aforementioned groups may or may not contain oxygen atom(s); andR is selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms.

[0033] According to one more embodiment of the present application, the lactam having at least one hydroxyl functional group is selected from the group consisting of:and combinations thereof, whereinQ is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein any of the aforementioned groups may or may not contain oxygen atom(s); and each Ri, R2, R3, R4, and R5 is independently selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms.

[0034] According to another embodiment of the present application, the lactam moiety having at least one hydroxyl functional group is a hydroxyalkyl pyrrolidone having the structure:Docket No.: 4387PCTwherein Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein any of the aforementioned groups may or may not contain oxygen atom(s).

[0035] According to one more embodiment of the present application, the hydroxyalkyl lactam is selected from the group consisting of l-(2-Hydroxyethyl)-2-pyrrolidone, l-(2- Hydroxyethyl)piperidin-2-one, 1 -(2-hydroxyethyl)azepan-2-one, 1 -(2-Hydroxypropyl)-2- pyrrolidone, l-(2-Hydroxypropyl)piperidin-2-one, l-(2-hydroxypropyl)azepan -2-one, l-(2- Hydroxybutyl)-2-pyrrolidone, l-(2-Hydroxybutyl)piperidin-2-one, l-(2-hydroxybutyl)azepan-2- one and combinations thereof.

[0036] According to one more embodiment of the present application, the hydroxyalkyl lactam is l-(2-Hydroxyethyl)-2-pyrrolidone, having a structure:

[0037] According to one more embodiment, the present application discloses a composition for cryopreservation of a biological material, comprising: i) a liquid cryoprotectant having a lactam moiety structure of:wherein the lactam having at least one hydroxyl functional group; wherein Y is an alkylene or alkenylene group comprising 2 to 50 carbon atoms; wherein 2 to 4 carbon atoms reside in theDocket No.: 4387PCTlactam ring between theugroup and thezgroup; Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein the aforementioned groups may or may not contain oxygen atom(s); and R is selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms, ii) at least one membrane protectant, iii) a base medium, iv) optionally, a media supplement and v) optionally, a cryopreservation supporting ingredient.

[0038] According to one more embodiment of the present application, the composition for cryopreservation further comprising a cryoprotectant not having lactam moiety and a hydroxyl functionality.

[0039] According to another embodiment of the present application, the cryoprotectant not having lactam moiety and a hydroxyl functionality is selected from the group consisting of mono, di, oligo or polysaccharides: sucrose, dextrose, methylcellulose, hydroxycellulose, raffinose, mannitol, lactose, glucose, sucrose, maltose, galactose, trehalose, melibiose, melezitose, mannotriose, stachyose, dextran, hydroxy ethyl starch, maltitol and lactitol; and non-saccharides: polyethyleneglycol, dimethylsulfoxide, ethylene glycol, propylene glycol, glycerol, polyvinvyl pyrrolidone and polyethylene oxide.

[0040] According to another embodiment of the present application, the membrane protectant is a protein, a non-protein, or a combination thereof.

[0041] According to another embodiment of the present application, the protein is selected from the group consisting of: casein, albumin, keratin, collagen, atelocollagen, elastin, gelatin, peptones, fibrinogen, fibronectin, egg protein, egg yolk protein, egg white protein, a soy protein, a wheat protein, a corn protein, milk protein, a hydrolysate thereof and a combination thereof.

[0042] According to one more embodiment of the present application, the protein is skim milk or a component thereof, milk powder or a component thereof, or egg yolk or a component thereof.Docket No.: 4387PCT

[0043] According to one embodiment of the present application, the non-protein is selected from the group consisting of: a lipid, a chemically synthesized lipid and a synthetic lipid, phosphatidylglycerol, phosphatidic acid, l, l',2,2'-tetra-acyl-cardiolipin, phosphatidylcholine, phosphatidyl serine, phosphatidylethanolamine, a polyoxyethylene based lipid, arachidonic, linoleic, linolenic, myristic, oleic, palmitic or stearic fatty acids, cholesterol, Pluronic® F-68, polyvinylalcohol, cholesterol and combinations thereof.

[0044] According to one embodiment of the present application, the base medium is a serum- free cell culture medium, an essentially serum free cell culture medium, a serum reduced cell culture medium or water-based medium.

[0045] According to another embodiment of the present application, the base medium is X- VIVO 15 and X-VIVO 20; further comprising a microcarrier and wherein the optional media supplement is selected from the group consisting of MSCGM-CD™ SingleQuots®, FGM-CD™ SingleQuot™, EGM-2 SingleQuot Kit Suppl. & Growth Factors, MesenCult™ Mesenchymal Stem Cell Stimulatory Supplements (Human), and Stem Pro® MSC SFM.

[0046] According to one more embodiment of the present application, the media supplement is serum replacement, complete media, media supplement or cryopreservation media.

[0047] According to one more embodiment of the present application, the cryopreservation supporting ingredient selected from the group consisting of organic salts, inorganic salts, an iron donor, pectin, beta-mercaptoethanol, dithiothreitol, tri s(2-carboxy ethyl) phosphine, dithioerythritol, thioredoxin, dithionite, 2-mercaptoethylamine, dimethyl thiourea, nordihydroguaiaretic acid (NDGA), 2,3-dimercapto-l-propanol, hydroquinone, an amino acid, an amino acid derivative, antioxidant, vitamins, vitamin derivatives, trace elements, butylated hydroxyanisole, butylated hydroxytoluene, dihydrolipoic acid, tetrahydropapaveroline, 2- thiobarbituric acid or taurine, dimercaptosuccinic acid, allopurinol, deferoxamine, melatonin, catalase, glutathione peroxidase, superoxide dismutase, steroids, glutathione and combinations thereof.

[0048] According to one more embodiment, the present application discloses a method of cryopreserving biological material comprising preserving a biological material with the cry opreserve composition at a temperature lower than 0°C.Docket No.: 4387PCT

[0049] According to one more embodiment of the present application, the biological material is a cell, blood, semen, platelets, genetic material, umbilical cord blood, oocytes, embryos, tissue, sugars, biological molecules and combinations thereof.

[0050] According to one more embodiment of the present application, the biological molecule is selected from the group consisting of proteins, nucleic acids or complexes, antibodies, peptides, glycopeptides, cytokines and combinations thereof.

[0051] According to another embodiment of the present application, the biological material can be composed of combinations of these substances or may be living entities such as cells and tissues. Biological materials are isolated from a variety of natural sources - human, animal, or microorganism - and may be produced by biotechnology methods and other cutting-edge technologies. For example, gene-based and cellular biological material, often are at the forefront of biological material research.

[0052] According to one more embodiment of the present application, the tissue is obtained from an mammal organ such as ovarian, cornea, pancreas, dental pulp, heart.

[0053] According to one more embodiment of the present application, the cell is selected from the group consisting of pluripotent stem cells, embryonic stem cells, bone marrow stromal cells, hematopoietic progenitor cells, lymphoid stem cells, myeloid stein cells, T cells, B cells, macrophages, hepatic cells, pancreatic cells, nucleic acids, carcinoma cells and cell lines.

[0054] Further, certain aspects of the present application are illustrated in detail by way of the following examples. The examples are given herein for illustration of the application and are not intended to be limiting thereof.TOXICOLOGY STUDY

[0055] Toxicology Study of l-(2-Hydroxyethyl)-2-pyrrolidone:

[0056] 1) Toxicokinetics:

[0057] Toxicokinetic studies were conducted for hydroxyethylpyrrolidone (HEP), the relevant HEP properties and data from toxicity studies indicating systemic bioavailability is taken together to assess the general toxicokinetics of HEP.

[0058] The results of the acute oral and the repeated dose oral toxicity study with reproduction / developmental toxicity screening test indicate absorption of HEP by the oral route.Docket No.: 4387PCTSecondly, HEP’s high water solubility, low Log Pow and low molecular weight suggests that HEP may be readily absorbed by the gastrointestinal and respiratory tract.

[0059] The log Pow of -E03 and the high-water solubility suggest that the substance may not be easily taken up by the stratum comeum. This is further substantiated by the lack of systemic effects observed in the acute dermal toxicity study performed with HEP.

[0060] 2) Acute Toxicity

[0061] i) Oral Toxicity:

[0062] In an acute oral toxicity study, HEP demonstrated an LD50 of 17.0 ml / kg body weight in rats. Five groups of albino rats, consisting of three males and two females of the Sherman- Vi star strain, were set aside and observed for a period of one week to assure normalcy. The animals were then starved for 24 hours. Doses at levels of 10.0, 12.6, 16.0, 20.0, and 25.1 ml / kg were determined for each animal and administered directly into the stomach by means of a syringe and stomach tube. Following this, the animals were allowed food and water ad libitum during a fourteen-day observation period. HEP, as tested in rats, had an acute oral LD50 of 17.0 ml / kg.

[0063] In another acute oral toxicity study, HEP demonstrated an LD50 of greater than 14.3 g / kg body weight in rats. Male Wistar rats, 200 to 300 g, were fasted for approximately 18 hours prior to administration of HEP. It was given orally by intubation (5.0, 7.12, 10.14, and 14.43 g / kg). Following treatment, the animals were returned to their cages and food and water were freely available. The rats were observed for signs of toxicity and pharmacological effects for 14 days following treatment. Animals which died during the 14-day observation period were necropsied. The oral LD50 was determined to be greater than 14.3 g / kg body weight.

[0064] ii) Dermal Toxicity:

[0065] In an acute dermal toxicity study (OECD 402), HEP demonstrated an LD50 of > 2000 mg / kg body weight in rabbits. Species / Strain: Rabbit / New Zealand White. Vehicle: HEP administered as supplied. Type of dressing: Occlusive. Signs of Local Toxicity: Dermal reactions were slight to well-defined on day 1 but were absent on days 7 and 14. Signs of Systemic Toxicity: There were no abnormal systemic signs noted in 9 / 10 animals. One male exhibited red staining of the nose / mouth area and an apparent cataract in the right eye on day 5. This was considered to result from a self-inflicted injury unrelated to the test substance. Body weight gains were normal at all weighing periods. Effects in Organs: Necropsy did not reveal any treatment related changes.Docket No.: 4387PCT

[0066] iii) Inhalation Toxicity:

[0067] In an acute inhalation toxicity, LC50 was determined to be > 0.008 mg / L air in rats (highest attainable concentration). Route of administration: inhalation. Type of inhalation exposure: whole body. Vehicle: air. Exposure apparatus: the atmosphere was saturated with vapors of the volatile components of the test substance (at 20°C). Vapors were generated by bubbling 200 L / h air through a substance column of about 5 cm above a fritted glass disc in a glass cylinder. Duration of exposure: 8 h. Concentrations: highest attainable concentration when the test substance is evaporated at 20°C: 0.008 mg / L. Number of animals per sex per dose: 12. Duration of observation period following administration: 7 days. Mortality: No mortality was observed. Clinical signs: No abnormalities detected. Gross pathology: No abnormalities detected.

[0068] 2) Irritation / Sensitization:

[0069] i) Skin Irritation:

[0070] HEP was non-irritating [Primary Irritation Index (PII) = 0.0] to the skin of rabbits. The backs of six rabbits were closely clipped; the skin on one side of the spinal column was abraded, and the skin on the other side remained intact. A 0.5 ml amount of HEP was applied under occlusive patches to both sides of each rabbit. After 24 hours, the patches were removed. The reactions were noted at 24 and 72 hours. PII was calculated as 0.0. HEP produced no erythema or oedema in any of the animals. It was concluded that HEP was non-irritating to rabbit skin under conditions of this test.

[0071] ii) Skin Sensitization

[0072] Guinea Pig “Buehler” Sensitization Study: HEP was evaluated for skin sensitization potential using the Buehler test on Hartley derived albino guinea pigs. HEP did not cause contact dermal sensitization in the test animals. HEP was evaluated for skin sensitization potential using the Buehler test on 20 albino guinea pigs. The animals were shaved over the left flank and divided into a positive control group (10) and a test group (10). Each received an application of 0.5 ml HEP under an occlusive bandage, with 0.15% l-chloro-2,4-dinitrobenzene in 80% ethanol being used as the positive control. After six hours, the bandage was removed, and the area wiped clean. Applications were made one per week at rotating sites for three weeks. The sites were scored, using the Draize scale, at 24 and 48 hours following each application. The skin sensitizationDocket No.: 4387PCT potential of HEP and positive control material was determined by statistically comparing erythema scores obtained 24 and 28 hours after the first sensitizing dose with those obtained after the challenge application. A statistically significant difference was obtained in the positive control group (mean erythema score increased from 0.8 to 1.6) but not in the test article group. In conclusion, there was no evidence of reaction(s) indicative of skin sensitization to HEP under the conditions of the test.

[0073] Mouse Local Lymph Node Assay (LLNA): Three groups each of five female mice were treated with different concentrations (25%, 50% and 100%) of HEP by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal, and immediately weighed using an analytical balance. Both ears were punched at the apical area and the punches were immediately weighed pooled per animal. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. The suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a [3-scintillation counter. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. The stimulation index of the Positive Control group (25% hexyl cinnamic aldehyde, CAS No 101-86-0) was 8.08. The group (SI) values for HEP were 1.14, 1.22 and 1.47 for the 25%, 50% and 100% group, respectively. With SI values <3.0, HEP is not a dermal sensitizer in LLNA.

[0074] Human Repeat Insult Patch Testing (HRIPT): To determine the irritation and / or sensitization potential, HEP was evaluated in HRIPT. A total of 53 subjects (12 male and 41 female), ranging in age from 18 to 63 years completed HRIPT. The quantity of HEP per test patch was approximately 0.2 ml. HEP was placed onto a 2 cm square of Webril™ (Kendall) non-woven cotton fabric affixed to 1 " Dermicel (Johnson & Johnson) cloth tape. The patch was applied to the inner aspect of the arm or the back of each subject and covered with overlapping strips of Blenderm (3M) tape to form occlusion. The subjects removed the patch 24 hours after application. Twenty-Docket No.: 4387PCT four-hour rest periods followed the Tuesday and Thursday removals, and 48-hour rest periods followed the Saturday removal. The site was scored by a trained examiner just prior to the next patch application. This procedure was repeated until 10 applications of the test material had been made. Responses were scored according to the following scale: 0 = no reaction, ± = minimal erythema, 1 = erythema, 2 = erythema and edema, 3 = erythema and moderate edema with papules, 4 = erythema, marked edema, substantial vesiculation. If a subject developed a positive reaction of at least a 2 during the induction phase, the patch was applied to a fresh site for the next application. If a 2-reaction occurred in the new site, no further applications were made. However, the subjects were challenged with the test material. Ten to twenty-one days after application of the last induction patch, the challenge patch was applied to the original contact site and to a fresh, adjacent site. The sites were scored at 24 and 48 hours after application. The panelists were asked to report to the laboratory any delayed reaction which might have occurred after the last reading of the challenge. A total of 53 subject completed HRIPT. With the exception of but a few subjects, the panel was remarkably clear of any reactions during the induction and challenge phases. One subject, no. 5, exhibited reactions starting at the 4th induction patch. At the challenge, the subject exhibited delayed reactions (at 48h reading) at both the original and virgin sites. A second subject had no reactions during the induction and minimum reactions (±) at the challenge. It was decided to rechallenge both of these subjects two to three weeks after the original challenge to determine if sensitization had taken place. Subject no. 5 had a delayed reaction at a virgin site indicating that sensitization did occur. Subject no. 19 had a minimal reaction at 48 hours at the original site. It would be difficult to conclude that this subject was sensitized. In view of the test conditions and the paucity of reactions during the entire test it was concluded that HEP could not be considered a contact sensitizer except in unusual cases of atopic individuals.

[0075] iii) Eye Irritation:

[0076] HEP was minimally irritating to rabbits eyes. A 0. 1 ml amount of HEP was instilled into one eye of each of six rabbits. The other eye served as a control. Ocular reactions were observed and recorded at 24,48 and 72 hours. Only conjunctival effects were observed, and these cleared in all animals by the second day. On the basis of these data, it was concluded that HEP is minimally irritating to rabbit eyes under these test conditions.

[0077] Repeated Dose Toxicity / Reproductive and Developmental Toxicity:Docket No.: 4387PCT

[0078] There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods. Several male and female animals of dose group 3 (1000 mg / kg bw / day) showed salivation after treatment during premating, mating, gestation, and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. One sperm positive high-dose female (1000 mg / kg bw / day) and four sperm positive mid-dose females (300 mg / kg bw / day) did not become pregnant. Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 100, 300 and 1000 mg / kg bw / day) was comparable to the concurrent control group during the entire study period. Male and female animals of all dose groups (1000, 300 and 100 mg / kg bw / day) did not show any abnormalities. No treatment-related changes among hematological parameters were observed. In males of test group 3 (1000 mg / kg bw / day) absolute and relative basophil counts were increased. However, mean values were within historical control ranges (absolute basophil counts 0.00-0.07 Giga / L; relative basophil counts 0.0- 1.1 %). This were the only changed clinical pathology parameters in these individuals. Therefore, these alterations were regarded as incidental and not treatment related. No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 1 and 2 (100 and 300 mg / kg bw / day), urea levels were higher compared to controls. However, the parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment related. No Observed Adverse Effect Level (NOAEL) was determined to be 1 000 mg / kg bw / day based on no effect observed at the highest dose tested.

[0079] Genotoxicity / Mutagenicity

[0080] Bacterial Reverse Mutation “AMES” Assay

[0081] Technical grade HEP was tested against Salmonella typhimurium strains TA98, TAI 00, TA1535, TA1537 and TA1538. The assays were conducted with and without metabolic activation (S-9) in a culture medium which contains insufficient histidine to allow the strains to proliferate.Docket No.: 4387PCTIf the tester strain organisms are mutated by the sample chemical, they gain the ability to grow with insufficient histidine. After incubation for 48 hours at 37°C, these mutant colonies were counted. Results are reported as (average) total number of revertant per plate and the standard error of the mean is calculated. Technical grade HEP gave a positive response in both TA100 and TAI 535 tester strains with and without activation when tested in the concentration range of 0.32 - 200 ml / plate. The TAI 00 strain demonstrated toxicity as evidenced by sparse lawn, at the highest concentration of 200 ml / plate and increased revertant only at the next concentration of 40 ml / plate. The TAI 535 strain demonstrated activity with dose-response. Background revertant were reached at the lowest concentration of 0.32 ml / plate.

[0082] A purified sample demonstrated no mutagenic response either with or without metabolic activation under the same test conditions resulting in the conclusion that the activity is due to an impurity.

[0083] Chinese Hamster Ovary / HGPRT Gene Mutation Assay:

[0084] The cytotoxic effect of technical grade HEP on CHO cells was further assessed in a clonal assay. With this more stringent test of viability which requires single cells to form colonies after treatment with technical grade HEP was found to be nontoxic. Results of the CHO / HGPRT assay show that all concentrations of technical grade HEP tested, from 125 - 1,000 mg / ml, uniformly induced a higher mutation frequency than the control. However, no dose-dependent effect was observed. Since technical grade HEP was not cytotoxic, the mean number of mutant colonies per plate between the treated cultures and the control can be compared. When tabulated as such, cultures treated with technical grade HEP showed a 2-3-fold increase in mutants per plate over the untreated control. This increase, with the exception of the 500 mg / ml concentration which had a high standard deviation, was found to be statistically significant (p<0.05). The positive control, EMS, induced over a 90-fold increase in the number of mutants compared to the spontaneous mutation. This assay resulted in a suggestive to weakly positive conclusion for the technical grade material. No dose-response was seen in the test concentration range of 250 - 1000 mg / ml and the mutation frequency showed only a 2 - 3 times elevation over control. By contrast, the positive control (EMS) showed a 93-fold increase.

[0085] Chinese Hamster Ovary / Sister Chromatid Exchange (SCE):

[0086] An initial cytotoxicity study was carried out to determine the appropriate dose range for use in the cytogenetic assays. The results show that technical grade HEP was not cytotoxic to massDocket No.: 4387PCT cultures of Chinese hamster ovary (CHO) cells from 0.01 - 250 mg / ml under conditions of the assay. Since technical grade HEP was not toxic, the highest concentration used in the cytogenetic assays was 1,000 mg / ml. This assay resulted in a slight elevation in the number of SCE's at the two highest concentrations of technical grade material tested (500 and 1000 mg / ml). Although these increases were statistically significant (t-test), they were considered to be biologically irrelevant in that the magnitude of the response was only a 10% increase. For comparison, the positive control (EMS) gave a >3-fold increase.

[0087] Chinese Hamster Ovarv / Chromosome Aberration Cytogenetic Assay:

[0088] The results of the chromosome aberration assay are shown in from one hundred metaphases examined, three chromosome aberrations were found in CHO cells treated with 125 mg / ml of technical grade HEP. This number was calculated to be statistically significant (p<0.05) over the zero aberration of the control. However, no aberrations were found in cells treated with higher concentrations of the chemical. Since it has often been observed one or two aberrations in control cultures historically, the aberrations scored in this one particular concentration of technical grade HEP are considered not to be biologically significant. The positive control, EMS, induced a significant number of chromosome aberrations within an acceptable range established from historical data. This assay was negative for the concentrations of technical grade HEP (250 - 1000 mg / ml).

[0089] Balb / C 313 Neoplastic Transformation Assay:

[0090] The neoplastic transformation assay was conducted using concentrations varying from 2 - 250 mg / ml. The test compound, technical grade HEP, produced a negative response in the in vitro transformation assay. The positive control, 3-methylcholanthrene, induced a mean of 3.00 Type III foci / plate, which is within the normal range of transformation induced by this carcinogenic arylaromatic hydrocarbon. The results of the cytotoxicity assay conducted in parallel with the transformation assay show higher surviving fractions of cells treated with technical grade HEP than the control. The lower cell count in the control may result from an incomplete trypsinization of cells from the plates in this particular experiment. This assay was negative for the concentration of technical grade material (2 - 250 mg / ml). It was therefore concluded that technical grade HEP contains a potential mutagenic contaminant, as demonstrated by the positive Ames assay, but the contaminant was found to be so weak or present in such low concentration as to be inactive in in vitro mammalian cell assays. Purified HEP was not active.Docket No.: 4387PCT

[0091] Biodegradation and Bioaccumulation:

[0092] Two ready biodegradability studies (OECD 301A and 301F) (BASF AG 01 / 0109 / 26 / 1, 2002) demonstrate the ready biodeagradability of HEP.

[0093] The log Kow was calculated to be below 3, indicating that accumulation of HEP in organisms is not to be expected.

[0094] Ecotoxicology:

[0095] Effects of HEP towards fish, algae and daphnia were found in the same range providing a LC50 (96h) >120 mg / L, an EC50 (48h) >100 mg / L and an ErCso (72h) >100 mg / L, respectively. Thus, HEP is not considered to have harmful effects for aquatic organisms.

[0001] The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration it is believed that one skilled in the art can, given the preceding description, utilize the present invention to its fullest extent. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

Claims

1. Docket No.: 4387PCTWhat is claimed is:

1. A liquid cryoprotectant having a lactam moiety structure of:wherein the lactam having at least one hydroxyl functional group; wherein Y is an alkylene or alkenylene group comprising 2 to 50 carbon atoms;Ru wherein 2 to 4 carbon atoms reside in the lactam ring between the ° group and thegroup;Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein the aforementioned groups may or may not contain oxygen atom(s); andR is selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms.

2. The liquid cryoprotectant according to claim 1, wherein the lactam having at least one hydroxyl functional group is selected from the group consisting of:and combinations thereof, whereinDocket No.: 4387PCTQ is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein any of the aforementioned groups may or may not contain oxygen atom(s); and each Ri, R2, R3, R4, and R5 is independently selected from the group consisting of hydrogen and functionalized and vunfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups, wherein the functionalized and unfunctionalized alkyl, cycloalkyl, alkenyl, and aryl groups may or may not contain heteroatoms.

3. The liquid cryoprotectant according to claim 1, wherein the lactam moiety having at least one hydroxyl functional group is a hydroxyalkyl pyrrolidone having the structure:wherein Q is selected from the group consisting of functionalized and unfunctionalized alkylenes of linear, branched, or cyclic structure, arylenes, and combinations thereof, wherein any of the aforementioned groups may or may not contain oxygen atom(s).

4. The liquid cryoprotectant according to claim 1, wherein the hydroxyalkyl lactam is selected from the group consisting of: l-(2-Hydroxyethyl)-2-pyrrolidone, l-(2- Hydroxyethyl)piperidin-2-one, 1 -(2-hydroxyethyl)azepan-2-one, 1 -(2-Hydroxypropyl)-2- pyrrolidone, l-(2-Hydroxypropyl)piperidin-2-one, 1 -(2-hydroxypropyl)azepan -2-one, l-(2-Hydroxybutyl)-2-pyrrolidone, l-(2-Hydroxybutyl)piperidin-2-one, l-(2-hydroxybutyl)azepan-2- one and combinations thereof.

5. The liquid cryoprotectant according to claim 1, wherein the hydroxyalkyl lactam is l-(2-Hydroxyethyl)-2-pyrrolidone, having a structure:

6. A composition for cryopreservation of a biological material, comprising:Docket No.: 4387PCT vi) the liquid cryoprotectant with a lactam moiety according to claim 1, vii) at least one membrane protectant, viii) a base medium, ix) optionally, a media supplement and x) optionally, a cryopreservation supporting ingredient.

7. The composition for cryopreservation of a biological material according to claim 6, further comprising a cryoprotectant not having lactam moiety and a hydroxyl functionality.

8. The composition for cry opreservation of a biological material according to claim 6, wherein the cryoprotectant not having lactam moiety and a hydroxyl functionality is selected from the group consisting of: mono, di, oligo or polysaccharides: sucrose, dextrose, methyl cellulose, hydroxycellulose, raffinose, mannitol, lactose, glucose, sucrose, maltose, galactose, trehalose, melibiose, melezitose, mannotriose, stachyose, dextran, hydroxy ethyl starch, maltitol and lactitol; and non-saccharides: polyethyleneglycol, dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyvinvyl pyrrolidone and polyethylene oxide.

9. The composition for cry opreservation of a biological material according to claim 6, wherein the membrane protectant is a protein, a non-protein, or a combination thereof.

10. The composition for cry opreservation of a biological material according to claim 9, wherein the protein is selected from the group consisting of: casein, albumin, keratin, collagen, atelocollagen, elastin, gelatin, peptones, fibrinogen, fibronectin, egg protein, egg yolk protein, egg white protein, a soy protein, a wheat protein, a com protein, milk protein, a hydrolysate thereof and a combination thereof.

11. The composition for cry opreservation of a biological material according to claim 9, wherein the protein is skim milk or a component thereof, milk powder or a component thereof, or egg yolk or a component thereof.

12. The composition for cryopreservation of a biological material according to claim 9, wherein the non-protein is selected from the group consisting of: a lipid, a chemicallyDocket No.: 4387PCT synthesized lipid and a synthetic lipid, phosphatidylglycerol, phosphatidic acid, 1 , l',2,2'-tetra- acyl-cardiolipin, phosphatidylcholine, phosphatidyl serine, phosphatidylethanolamine, a polyoxyethylene based lipid, arachidonic, linoleic, linolenic, myristic, oleic, palmitic or stearic fatty acids, cholesterol, Pluronic® F-68, polyvinylalcohol, cholesterol and combinations thereof.

13. The composition for cry opreservation of a biological material according to claim 6, wherein the base medium is a serum-free cell culture medium, an essentially serum free cell culture medium, a serum reduced cell culture medium or water-based medium.

14. The composition for cryopreservation of a biological material according to claim 8, wherein the base medium is X-VIVO 15 and X-VIVO 20; further comprising a microcarrier and wherein the optional media supplement is selected from the group consisting of MSCGM-CD™ SingleQuots®, FGM-CD™ SingleQuot™, EGM-2 SingleQuot Kit Suppl. & Growth Factors, MesenCult™ Mesenchymal Stem Cell Stimulatory Supplements (Human), and Stem Pro® MSC SFM.

15. The composition for cry opreservation of a biological material according to claim 6, wherein the media supplement is serum replacement, complete media, media supplement or cry opreservation media.

16. The composition for cry opreservation of a biological material according to claim 6, wherein the one or more cryopreservation supporting ingredient selected from the group consisting of organic salts, inorganic salts, an iron donor, pectin, beta-mercaptoethanol, dithiothreitol, Tris(2-carboxyethyl) phosphine, dithioerythritol, thioredoxin, dithionite, 2- mercaptoethylamine, dimethyl thiourea, nordihydroguaiaretic acid (NDGA), 2, 3 -dimercapto- 1- propanol, hydroquinone, an amino acid, an amino acid derivative, antioxidant, vitamins, vitamin derivatives, trace elements, butylated hydroxyanisole, butylated hydroxytoluene, dihydrolipoic acid, tetrahydropapaveroline, 2-thiobarbituric acid or taurine, dimercaptosuccinic acid, allopurinol, deferoxamine, melatonin, catalase, glutathione peroxidase, superoxide dismutase, steroids, glutathione and combinations thereof.

17. A method of cryopreserving biological material comprising cryopreserving biological material with the composition of claim 6.Docket No.: 4387PCT18. The method of claim 17, wherein the biological material is a cell, blood, semen, platelets, genetic material, umbilical cord blood, oocytes, embryos, tissue, sugars, biological molecules and combinations thereof.

18. The method of claim 18, wherein the biological molecule is selected from the group consisting of proteins, nucleic acids or complexes, antibodies, peptides, glycopeptides, cytokines and combinations thereof.

20. The method of claim 19, wherein the cell is selected from the group consisting of pluripotent stem cells, embryonic stem cells, bone marrow stromal cells, hematopoietic progenitor cells, lymphoid stem cells, myeloid stein cells, T cells, B cells, macrophages, hepatic cells, pancreatic cells, nucleic acids, carcinoma cells and cell lines.

Images