CN1204232A - Composition for organ cryopreservation and treatment of viral and bacterial infections - Google Patents

Abstract

The invention provides a method for the cryopreservation of organs and the treatment of viral and/or microbial infections, the substance or composition comprising an active agent selected from the group consisting of amides of the general formula R3-CO-NR1R2, in which: R1 and R2 are independently selected from the group including H, Me, halomethyl, saturated and unsaturated C2-C3 alkyl groups, saturated and unsaturated halogenated C2-C3 alkyl groups, hydroxylated alkyl groups; or R1 and R2 are together selected from (CH2)n, wherein n = 4 or 5, or (CH2)2O(CH2)2; and R3 is selected from H, Me and saturated an unsaturated C2-C3 alkyl groups.

Description

Compositions for cryopreservation of organs and treatment of viral and bacterial infections

The present invention particularly relates to substances or compositions for cryopreservation of organs and treatment of viral and/or microbial infections, substances or compositions for cryopreservation of organs, substances or compositions for treatment of viral and/or microbial infections, Method for manufacturing cryopreservation reagent for cryopreservation of organs, method for manufacturing medicine or preparation for treating virus and/or microbial infection, application of cryopreservation reagent in cryopreservation of organs, medicine or preparation for treating virus and/or microbial infection Use in microbial infections, use of cryopreservation reagents in the production of cryopreservation reagents, use of substances or compositions in the manufacture of medicaments or preparations for the treatment of viral and/or microbial infections, methods of cryopreservation of organs, thawing Methods of organ, methods of treatment of viral and/or microbial infections, dosage forms and vaccines.

According to one aspect of the present invention, there is provided a substance or composition for cryopreservation of organs and treatment of viral and/or microbial infections, containing an active agent selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ Substances or compositions of which:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

At least one of _R1 and _R2 may be a methyl group.

Alternatively, at least one of R ₁ and R ₂ may be a fluorinated C ₁ -C ₃ alkyl group.

The active agent can be selected from the group consisting of formamide, methylformamide, dimethylformamide, acetamide, methylacetamide, dimethylacetamide, diethylacetamide, isopropylacetamide, diisopropyl Acetamide, N-acetylpiperidine, N-(β-hydroxyethyl)acetamide, N,N-bis(β-hydroxyethyl)acetamide, N-acetylmorpholine, acrylamide, propionamide, N-fluoromethyl-N-methyl-formamide and mixtures of any two or more thereof.

In a particular embodiment, the active agent may be _{dimethylformamide C3H7NO} (DMF). In another specific embodiment, the active agent may be N-fluoromethyl-N-methyl-formamide, HCON( _CH3 )( _CH2F ).

DMF is generally used as a polar agent and is readily absorbed through the skin, through lung or oral exposure. The absorption rate of liquid DMF through the skin reaches 9.4 mg per square centimeter per hour. DMF is rapidly metabolized, the main biotransformation site is the liver, and most of it is excreted in urine. The major metabolites in rats, rats, hamsters and humans are N-(hydroxymethyl)-N-methylformamide (HMMF), N-(hydroxymethyl)-formamide (HMF) and N- Acetyl-S-(N-methyl-carbamoyl)cysteine (AMMC). Unaltered DMF is excreted in the urine as a small portion of the dose. The limited data available indicated that significant amounts of the dose were left unexcreted and/or excreted as unidentified compounds.

DMF has low acute dermal, oral and inhalation toxicity. It is considered a mild to moderate skin and eye irritant and penetrates the skin readily. No indication for skin sensitization properties.

In several 90-day studies, a NOAEL (No Observed Toxic Effect Level) could not be established. In a 28-day inhalation study in dogs, no effect was found at 63 mg/m3. In another study in dogs, reversible cardiovascular effects were found at 60 mg/m3.

DMF is teratogenic and possibly embryo-lethal. The NOAEL for developmental effects reached a total of 44 mg/kg bw in the oral study and 150 mg/m3 in the inhalation study in rabbits.

A particular feature of the present invention is that by pre-cooling the organ prior to exposure to DMF, the toxicity of DMF is substantially reduced or substantially eliminated during hypothermia. As an example, proteins are denatured in 30% DMF solutions in water or physiological solutions at room temperature. However, at 8°C, according to the method of the present invention, at a DMF concentration level of 30% and a temperature below 0°C, organs are not substantially affected, and DMF concentrations of 40% do not affect organs either.

As described in the examples below, during cryogenic procedures, organs are generally unaffected if a 10% DMF solution is introduced at a temperature between 8-4°C, and organs and solutions are generally unaffected if a 25% to 30% DMF solution is introduced. The temperature should be below 4°C.

During low-temperature storage, the substance or composition of the present invention functions simultaneously as a low-temperature storage agent and a virus- and/or bacteria-inhibiting agent. So, if a virus or bacteria infects an organ, cryopreservation of the organ will simultaneously destroy the viral and/or microbial infection.

For the purposes of this specification, the word "organ" should be interpreted broadly to include heart, liver, skin, tissue, cornea, bone, glands and heart valves.

The present invention provides cryopreservation agents (CPA) and methods of treating mammalian or other tissues, organs or whole bodies with CPA, deep freezing, and storing substantially irregularly until thawed when needed. Typically, the storage life of non-frozen donor organs is about 4 hours, after which time cellular destruction begins, making transcontinental transfer of donor organs difficult or impossible. As will become clear below, the procedure of the present invention is uncomplicated, by immersion in liquid nitrogen after CPA treatment, the freezing temperature rate of -10°C/sec is readily tolerated by the organs. So transcontinental transfer of donor organs and establishment of donor organ banks or storage of cadavers, limbs and organs for medical applications becomes feasible.

Therefore, according to another aspect of the present invention, there is provided a substance or composition for low-temperature storage of organs, a substance or composition containing a low-temperature agent selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ ,in:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl groups, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups group, a hydroxylated alkyl group; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4, or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

At least one of _R1 and _R2 may be a methyl group.

Alternatively, at least one of R ₁ and R ₂ may be a fluorinated C ₁ -C ₃ alkyl group.

Amides may be as previously described herein.

In one particular embodiment, the cryogenic reagent can be dimethylformamide, and in another particular embodiment, the cryogenic reagent can be N-fluoromethyl-N-methylformamide.

Low temperature reagents can be mixed with sodium chloride, EDTA-disodium, sodium bicarbonate, potassium chloride, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride, glucose, Hespes-sodium hydroxide and any two of them Physiological solution combination of one or more components of a mixture.

Specifically, the physiological solution may be Tyrode's solution (referred to herein as Tyrode), University of Wisconsin solution (ViaSpan), Krebs Henseleit, or any other suitable physiological solution.

The present invention extends to _a substance or composition for use in the treatment of viral and/or microbial infections, a substance or composition comprising an active therapeutic agent selected from the group comprising amides of the general formula _R3 -CO- _NR1R2 , wherein:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups; saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

In a particular embodiment, the amide may be dimethylformamide. In another specific embodiment, the amide may be N-fluoromethyl-N-methylformamide.

The viral infection may be a retrograde viral infection, ie acquired human immunodeficiency (HIV-1) virus infection. However, the compounds, substances or compositions of the present invention can be used in the treatment of other viral and/or microbial infections such as German measles, skin acne and random infections associated with immune system diseases such as HIV-1. In particular, the substances or compositions according to the invention may be used in the treatment of viral infections, wherein the virus has a protective coat.

Therefore, the viral infection may be selected from acquired human immunodeficiency virus infection (HIV-1).

A substance or composition may include at least one physiologically acceptable excipient or carrier. The excipient or carrier can be colloidal silicon dioxide.

According to another aspect of the present invention, there is provided a method for manufacturing a cryopreservation agent for cryopreservation of organs, comprising mixing a cryopreservation component selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ and a physiological solution A method of combining steps in which:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ )n, where n=4 or 5, or (CH) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

The physiological solution may be as described herein above.

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

According to another aspect of the present invention, there is provided a method for the manufacture of a medicament or formulation for the treatment of viral and/or microbial infections, comprising combining an active therapeutic agent selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ The step of combining the agent with at least one physiologically acceptable excipient or carrier, wherein

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups,

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

The invention extends to the use of cryopreservation agents comprising cryopreservation components selected from the group comprising amides of the general formula _R3 -CO- _NR1R2 for cryopreservation of organs, wherein _:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

Freeze storage reagents may include freeze storage components and physiological solutions.

R ₁ , R ₂ and R ₃ can be as described herein.

Amides can be as described herein.

The invention further extends to the use of a medicament or preparation comprising an active therapeutic agent selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ for the treatment of viral and/or microbial infections, wherein

R ₁ and R ₂ are each selected from the group consisting of H, Me, halogenated methyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkyl groups a group of groups; or

R ₁ and R ₂ together are selected from (CH ₂ )n, where n=4 or 5, or (CH ₂ )O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

R ₁ , R ₂ and R ₃ can be as described herein.

A medicament or formulation may include at least one physiologically acceptable excipient or carrier.

A physiologically acceptable excipient or carrier may be colloidal silicon dioxide.

Amides can be as described herein.

The present invention extends to the use of freeze storage ingredients selected from the group comprising amides of the general formula _R3 -CO- _NR1R2 in the _manufacture of freeze storage reagents, wherein:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

R ₁ , R ₂ and R ₃ may be as previously described herein.

Amides may be as described previously herein.

_The present invention further extends to the use of a substance or composition in the manufacture of a medicament or preparation for the treatment of viral and/or microbial infections, comprising an activity selected from the group comprising amides of the general formula _R3 -CO- _NR1R2 Substances or compositions of therapeutic agents, wherein

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, haloalkyl a group of groups; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

Typically, the method involves initially perfusing the organ with a physiological solution, such as Tyrode [trade name], and then, in a stepwise or continuous manner, perfusing the organ with a physiological solution containing sequentially increasing concentrations of cryopreserved components while reducing the temperature of the organ.

By initially freezing organs followed by snap freezing in liquid nitrogen and storing organs at liquid nitrogen temperatures; by freezing organs in liquid nitrogen and storing in a freezer; by planning freezing and storing in liquid nitrogen; or in a freezer In the freezing and storage plan, the temperature can be lowered.

The method therefore includes the previous steps of perfusing the organ with a physiological solution and freezing the organ to a temperature between 8 and 5° C., then perfusing the organ with a cryopreservation reagent while sequentially increasing the concentration of the cryopreservation component in the cryopreservation reagent and freezing the organ. organs to storage temperature.

Concentrations of frozen storage ingredients can be increased sequentially from 10% to 40%.

Typical storage temperatures are in the sub-zero temperature range (liquid nitrogen temperature) ranging from about -50°C to -196°C.

Specifically, the storage temperature may be between about -80°C and -196°C. This temperature can be achieved using liquid CFC (chlorofluorocarbon) or liquid nitrogen cryogens.

The invention extends to a method of thawing an organ that has been perfused with a cryopreservation agent and frozen to storage temperature, the method comprising the steps of:

Exposure of the organ to _a cryopreservation agent comprising a cryopreservation component selected from the group comprising amides of the general formula _R3 -CO- _NR1R2 , wherein:

R ₁ and R ₂ are each selected from the group consisting of H, Me, halomethyl, saturated and unsaturated C ₂ -C ₃ alkyl groups, saturated and unsaturated halogenated C ₂ -C ₃ alkyl groups, hydroxylated alkanes groups of radicals; or

R ₁ and R ₂ together are selected from (CH ₂ ) _n , where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups,

until the organ has reached the predetermined temperature above the storage temperature;

Perfusing an organ with a cryopreserved reagent containing a cryopreserved component while sequentially decreasing the concentration of the cryopreserved component in the cryopreserved reagent and increasing the temperature of the organ;

Perfuse the organ with physiological solution.

The predetermined temperature is about -4°C to 4°C.

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

The present invention further extends to a method of treating viral and/or microbial infections comprising administering to an individual having a viral and/or microbial infection an active compound selected from the group comprising amides of the general formula R ₃ -CO-NR ₁ R ₂ A medicament or formulation of a physiologically effective dose of a therapeutic agent, wherein

R _{and R} are each selected from the group comprising H, Me, halomethyl, saturated and unsaturated halogenated _C2 - _C3 alkyl groups, hydroxylated alkyl groups; or

R ₁ and R ₂ together are selected from (CH ₂ ) n, where n=4 or 5, or (CH ₂ ) ₂ O(CH ₂ ) ₂ ; and

R ₃ is selected from H, Me and saturated and unsaturated C ₂ -C ₃ alkyl groups.

The medicament or formulation may comprise at least one physiologically acceptable excipient or carrier as previously described herein.

R ₁ , R ₂ and R ₃ may be as described herein above.

Amides can be as described herein above.

The dosage may be selected so as to provide a blood concentration of the active therapeutic agent in the blood of the individual to be treated of about 2-200 ppm, preferably about 50-100 ppm. It has been found that a peak plasma concentration of DMF of 50-100 ppm increases the CD4 T-cell count in patients with HIV-1 from 312/ml to over 1000/ml within 3 weeks of starting treatment.

Doses may be administered transdermally, ie, transfer of the active therapeutic agent through the skin, such as by application of a skin patch or cotton wool pad that has been impregnated with the active therapeutic agent or a composition containing the active therapeutic agent. The concentration of active therapeutic agent in the compositions for soaking skin patches or cotton wool pads is about 10-100%.

The dosage may however be administered by a method selected from oral dosage, inhalation, by suppository, intravenous and a combination of any two or more thereof. Usually intravenous introduction will be performed at a controlled rate over a period of time.

The present invention therefore extends to dosage forms for the treatment of viral and/or microbial infections comprising topical application means such as skin plasters or pads which absorb or absorb an effective amount of the active substance as described herein above. therapeutic agent.

Topical means may comprise a body of synthetic polymeric material such as TEFLON (trade mark) into which the active therapeutic agent is absorbed or absorbed. Typically, the dosage form will consist of DMF mixed with colloidal silicon dioxide to form a gel for poultice soaking. The plaster may be Virodene PO58 plaster.

The invention extends to vaccines prepared from antibodies harvested from the body of a human infected with the virus and then treated with the methods of the invention.

The virus may be a retrovirus such as HIV-1.

The invention will now be described, by way of example, with reference to the accompanying drawings and examples, in which

Figure 1 shows the temperature change in the aortic cannula during immersion of the rat heart in liquid nitrogen;

Figure 2 is a diagram showing the cannulation procedure and the location of thermocouples in the heart;

Figure 3 shows the HIV-1 viral load (polymerase chain reaction [PCR]) as a function of time of treatment of AIDS patients with Virodene PO58 (DMF) plaster;

Figure 4 shows temperature as a function of time for whole rat heart perfusion, cryopreservation, freezing, thawing and reperfusion according to the method of the present invention.

Figure 5 shows the temperature as a thermocouple and the time of the transient response;

Figure 6 shows the plasma DMF concentration as a function of time during the first set of human toxicity tests;

Figure 7 shows the plasma DMF concentration as a function of time during the second set of human toxicity tests;

Figure 8 is a micrograph of a heart valve magnified 22,500 times from a frozen/thawed rat heart after a cryopreservation process according to the present invention;

Figure 9 is a micrograph of a heart valve magnified 36,250 times from a frozen/thawed rat heart after a cryopreservation process according to the present invention; and

Fig. 10 is a micrograph of a heart valve magnified 28500 times of a frozen/thawed rat heart after a cryopreservation process according to the present invention.

Example 1

low temperature storage

Materials and methods

Model: Isolated perfused rat heart

Twelve BD9 rats each weighing 250-350 grams were utilized. Rats were anesthetized with ethylamylbarbiton sodium according to accepted protocols.

Langendorff Instruments

Two standard Langendorff systems connected to two water baths were utilized, one at cryogenic temperature for freezing the organs to approximately 4-5°C and the other maintained at 37°C. Hearts were perfused with Tyrode-DMF solution using a cold Langendorff system (designated LD2) and in the absence of DMF, ie pure Tyrode, with a warm Langendorff system (designated LD1).

Perfusion composition per liter of solution (Tyrode)

NaCl 137.6mM

Kcl 5.4mM

MgCl ₂ 1.0mM

CaCl ₂ 1.8mM

Glucose 5.0mM

Hespes-NaOH 11.6mM

The pH value of the solution is 7.45

DMF characteristics

(BASF Industrial Chemistry, Dimethylformamide Technical Print M5351e, October 1989)

N,N-Dimethylformamide (DMF) is a cell differentiation agent with the following properties

Molecular formula: C ₃ H ₇ ON

Relative molar mass: 73,095 g/mol

Freezing point: -61℃

DMF is a colorless, high-boiling, mobile, polar, hygroscopic liquid with a faint characteristic odor. As an inert solvent with a high dielectric constant, DMF is a good solvent, soluble in water and liquids and has a low gas pressure at 23°C. Contact of DMF with strong oxidizing agents, aluminum alkyls and halogenated chemicals can cause a fire or explosion.

Store DMF in airtight containers below -5°C. The perfusate/CPA(OV) solution was mixed quantitatively when needed and not stored for later use.

temperature measurement

As shown in Figure 2, temperature was measured continuously by a small (0.5 mm diameter) T-type thermocouple positioned within the aortic cannula. This location was chosen as the best unity between proximity to the heart and minimal interference with heart motion. Thermocouples can precisely follow temperature changes at a rate of more than 45°C/sec. Temperature was sampled once per second during the experiment using a PC-30 12 bit A/D card (Eagle Electronics) with a custom amplifier (incorporating cold spot temperature compensation). The temperature measurement system was calibrated with a commercial Fluke 52K-model thermocouple thermometer. Measurements in the range -100°C to +100°C proved accurate to within 2% of the true temperature. During snap freezing in liquid nitrogen, the heart was submerged in liquid nitrogen just above the aorta (see Figure 2). Temperature measurements during cryopreservation experiments were performed in the left ventricle and in the aortic duct as mentioned above. A heart submerged in liquid nitrogen reached a temperature of -196°C within 20 seconds, compared to -75°C at the aortic tip. The freezing rate of the left ventricle is about 10°C/sec during the rapid freezing process, and it is 7.5°C/sec at the aortic tip.

Perfusate/CPA(OV) mix used:

Tyrode 90% and DMF 10% called (OV1) solution

Tyrode 80% and DMF 20% called (OV2) solution

Tyrode 75% and DMF 25% called (OV25) solution

Tyrode 70% and DMF 30% called (OV3) solution

All percentages are volume/volume (v/v) percentages.

technology

Hot/cold (37°C/5°C) LD1 and cold (5°C) LD2 Langendorff systems were prepared as described above. All perfused rat hearts were frozen at LD1 using a single pure Tyrode's solution and using this system. In LD2 pure Tyrode and an OV mixture of storage protectants were used only at cold temperatures (5°C).

Keep DMF at -5 °C in a sealed container in a freezer storage area. Make 10%, 20%, 25%, and 30% DMF solutions in Tyrode's solution immediately before use.

In the first set of experiments, rats were anesthetized, their hearts excised, immersed in cold Tyrode's solution, cannulated through the left ventricle, connected to a hot/cold Langendorff system (LD1) containing pure Tyrode's solution at 37°C and perfused for 8 minutes to fix the shrinkage (Figure 4-note a), then the temperature of the system was lowered to 5°C and maintained at this temperature for 10 minutes (Figure 4-note b-c).

The abdomen was transected with Mayo scissors just below the rib cage to expose the diaphragm, and then the diaphragm was excised at the abdominal and lateral margins to expose the heart and lungs. At this point, the lungs are no longer functioning and the ischemic clock kicks in. The mediastinum between the heart and the rib cage was then excised and the heart was gently lifted and excised with diaphragm scissors. The heart is then placed in a cold Tyrode beaker and submerged. The heart is then lifted by cutting the edge of the aorta, closing the aorta to the cannula with the aid of cotton thread, and trimming the edge of the suture.

The heart was then separated from the LD1 system and connected to the LD2 system (Figure 4—notes c–d). Perfusion of hearts from the LD2 system was started at 5°C with pure Tyrode, and then the perfusion solution was changed to 50 ml of OV1 solution (LD2) (Figure 4—note d). Perfuse the heart for 10 min until the OV1 solution is exhausted. System LD2 was then filled with 30 mL of OV1 solution and perfused continuously for 8 min. System LD2 was then filled with 30 ml of OV3 solution, and perfusion was stopped after 8 min before OV3 was depleted. The heart was then covered with a thin layer of cotton wool and immersed in OV3 solution at 0 °C. The heart was then completely submerged in liquid nitrogen until the temperature of the left ventricle reached -196 °C. Freeze for 20 seconds at a freezing rate of -10°C/second. Hearts were maintained at a temperature of -196°C for a period of time ranging from 10 seconds to 45 minutes before reheating.

The heart was then thawed by placing it in a 100 ml glass containing 70 ml of OV3 at 0°C for 12 minutes (Figure 4, annotations g-i). Thereafter, the left ventricular temperature ranged from +4°C to -4°C. On the cold still connected Langendorff system (LD2), perfusion was started for another 2 minutes with 20 ml OV3. The LD2 system was then filled with 20 mL of OV2 solution and perfusion continued for 2 minutes, followed by 30 mL of OV1 solution for 4 minutes, and then 100 mL of pure Tyrode for 12 minutes. The total reperfusion time was 20 minutes. The heart was then removed, reconnected to the cold/warm Langendorff system LD1 (Fig. 4-note j-k), perfusion was started at a temperature of 5°C with pure Tyrode solution (Fig. 4-note k) and the system was reheated to a temperature of 37°C (Fig. 4 - Notes k-m) until the left ventricular temperature is 37°C. The heart begins to contract within 3 to 6 minutes when the temperature reaches 14 to 30°C. The beating heart was kept in a warmed Langendorff system for 30 minutes, after which the experiment was terminated.

In the second set of experiments, OV1 and OV25 solutions were used. In this set of experiments, OV25 replaced OV2 and OV3.

Embrittlement of the heart usually occurs if freezing in liquid nitrogen is initiated above 8°C. The cotton-wool wrap slows down the drop in temperature by creating an insulating pouch around the heart.

All solutions were oxidized by pre-blowing with a 95% oxygen/5% _CO2 gas mixture. Diffusion of DMF in the cells was fast enough that no osmotic swelling was observed. Air bubbles in the perfusate entering the heart must be avoided as they can cause infarction causing ischemia and interfering with the addition and removal of CPA. Large bubbles are lethal. The pressure head of the perfusate is preferably 90-100 cm. A pressure head of 37.5 cm resulted in improper perfusion.

Transfer electron microscopy of the subendocardia and subcardia was performed on all hearts at the completion of the experiment (see Figures 8-10).

The experiment as described above was repeated on 100 rat hearts.

result

As shown in Figure 1-note b, it can be seen that rapid freezing can be achieved with a temperature cooling rate of -75°C/sec. The heart was submerged in liquid nitrogen to fix the temperature of the aortic cannula tip at -75°C. The associated temperature inside the heart is -196°C. After the heart was removed from the liquid nitrogen and reheated, the temperature rapidly reached -7°C (see Figure I—note c).

In this series of experiments, hearts that had initially contracted 3 to 6 minutes after hyperperfusion with the Langendorff system were restarted at a myocardial temperature range of 14 to 30 °C. Watch for an active, rhythmic beating heart for at least 30 minutes. Coronary flow returns to precool speed and heart rate returns to precool value. No damage was visible to the naked eye. There was no visible damage to the intracellular structures observed with an electron microscope at magnifications of 22500, 28500 and 36250 (Figs. 8-10).

In the case of cryopreservation of the liver, it is important to arrest liver function prior to the introduction of CPA, since a functioning liver will break down DMF into toxic compounds (AMCC). DMF is hepatotoxic. Liver function ceases at about 8°C and the liver must be frozen to a minimum of 4°C before being introduced into DMF. The whole body must be kept at or below 4°C before introducing the DMF solution. To freeze the whole body, the body is placed in a heart-lung machine, the blood is replaced with a pure physiological solution, and frozen to 4°C. CPA (OV solution) is introduced and circulated throughout the body until all cells are saturated with CPA. The body was then covered with a pad imbibed with CPA solution and frozen in liquid nitrogen. When reheating, a minimum temperature of 4°C must be maintained until all the CPA solution is flushed from the body cells. The body is then reheated with pure physiological solution up to 11°C before the blood is introduced into the body.

discuss

Past attempts to store solid organs by cryopreservation at temperatures up to -196°C while reacquiring a functional state after the freezing process have not been successful (Wang T, Banker MC, Clayden N, Hicks GL Jr, Layne JR Jr, Mammalian heart explant Frozen storage of body VI. The effect of thawing speed on functional recovery, "Cryobiology" 29, (470-477), 1992; Wang T, Connery CP, Batty PR, adult mammalian heart Cryopreservation, Cryobiology 29 (171-176), 1991; Wicomb WN, Hill DJ, Collins GM, 24-hour ice storage of rabbit hearts, Journal of Heart and Lung Transplantation, 13:5, (891-894) , 1994. To the best of the applicant's knowledge, there have been no successful experiments in which solid organs were frozen at temperatures below -80°C and subsequent macroscopic and microscopic evaluations showed no visible organ damage.

Intracellular damage caused by crystallization, toxicity and osmotic stress. The Journal of Cryogenics has well documented these issues. Previously reported successful cryopreservation was limited to single cells and sperm. These procedures often use slow or planned freezing in which the absolute volume of the structures is very small (Rall WF, Fahy GM, Cryopreservation of rat embryos at -196°C by vitrification, Nature, 313, ( 573-575), 1985). In single cells, high temperature cooling rates and freezing are assumed to have less effect because volume changes have less damaging effects on single cells than on intact organs.

When submerged in liquid nitrogen, like the electrolyte-water solution, 25% DMF and Tyrode's solutions do not exhibit volume expansion. So the inventors used this concentration in some rat heart experiments.

The cooling rates to the temperatures encountered in the rat heart experiments did not have any deleterious effect, and no intracellular damage occurred.

For successful cryopreservation of whole organs, media and processes with the following properties are required:

1. The medium must quickly replace the water in the cells;

2. The medium must reach all cells in the organ simultaneously (Burrows FA, Bissonette B, Ways to block blood flow velocity to the brain when profound hypothermia and low cardiopulmonary flow or circulation are exploited in neonatal and infant cardiac surgery, "Can Anaesth ", 40:4, (298-307), 1993);

3. The medium must change the properties of the mixture of water and medium so that no or little volume change occurs upon freezing;

4. The medium must be nontoxic to cells and tissues;

5. Dielectric properties must be maintained over a wide temperature range;

6. The medium must have a low viscosity at freezing temperatures, usually close to water;

7. A medium that is liquid at sub-zero temperatures.

The inventors have found that dimethylformamide (DMF) meets most of the requirements mentioned above.

The rat heart was used as a model since it is easy to evaluate in experimental work. The manner in which function can be tested in the rat heart in experimental models is well established, so comparative information is available. Interest initially focused on the function of the heart following cryopreservation techniques. However, electron microscopy of the tissue was performed in all cases in order to investigate the possibility of superstructural changes.

Electron microscopy showed no visible damage to intracellular structures, despite a very careful study of the signals of cellular damage.

The heart recovered particularly well in the experiment after encountering extreme temperature cooling rates and very low endpoint temperatures. Macroscopically no damage can be seen, as indicated by the systolic movement, and the function is good.

The present invention opens up the potential for long-term storage of solid organs and tissue structures and the establishment of organ banks. Example 2

HIV treatment

Such as the phase 3 clinical trial of Virodene PO58 skin plaster on HIV-1 positive patients, and the toxicity study on humans. At the same time, the inventors also conducted in vitro studies on DMF on cell cultures.

DMF (Virodene PO58) is used to treat patients infected with HIV-1 by applying a skin patch soaked in DMF gel to the patient's body. Two skin patches are applied to different parts of the patient's body such as the forearm. Each skin patch contained 7.064 grams of gel containing DMF (92.5% m/m) and colloidal silicon dioxide (7.5% m/m). The role of the gel is to prevent the liquid DMF from leaking out of the plaster. Since DMF evaporates quickly, make these plasters no more than 12 hours before application. The desired DMF level in the patient's blood is 100 ppm. For a patient weighing approximately 60 kg, an amount of approximately 14 grams is required to produce a level of 100 ppm over a 12 hour period. N-acetyl-cysteine-glutathione and/or essential phospholipids are administered to the patient as a hepatic booster injection at a dose of 250 mg to 300 mg per day. Alternatively, glutamate can be administered to patients as a hepatic booster injection (oral or intravenous). According to Marz and Nohova, the surface area required to absorb this dose is approximately 127.2 square centimeters. To obtain a blood level of 100ppm, approximately 1.272 grams of DMF must be absorbed per hour, so approximately 7.064 grams of DMF are required per patch to deliver the required dose, with a surface area of 6.36 square centimeters (per patch) . The absorption rate of DMF is 9.4 mg/cm2/hour. In theory, this treatment would deliver 125-135ppm, but due to evaporation of the DMF, 100ppm was obtained. Absorbency varies from patient to patient according to various factors such as skin type and skin thickness. In order to obtain the desired DMF level in the patient, the plasma DMF concentration was measured for each patient and the treatment was gradually adjusted according to the DMF level of each patient (see Figures 6 and 7, for example, various plasma DMF concentrations in the same treated patients level).

So about 7.064 grams of DMF and silica gel was loaded per post. Use each patch for a 12-hour period, one patch per week for a 12-week cycle, or two patches per week for a 6-week cycle.

In some patients within three weeks (see Figure 3), blood tests with as few as three treatments showed an increase in CD4 T-cell counts from 350 to 1,000 and a rapid decrease in PCR (polymerase chain reaction) from 120,000 ( viral load) to 500/ml. The PCR test was performed with a Roche amplicor HIV detector. A viral load of <500/ml plasma was considered undetectable.

Some treated patients had severe acne or exhibited symptoms of German measles prior to treatment. When treated with DMF so that the DMF level in the patient's blood was 50-100 ppm, German measles symptoms and severe acne cleared or disappeared within 7 days.

Before treatment with DMF, conduct a comprehensive basic clinical and psychological evaluation of the patient. The assessment provides the patient's basic biochemical and hematological data. Simultaneously detailed viral serological tests (HIV-1) are carried out in order to determine the viral count of the patient's whole body, these tests are carried out on a weekly or per treatment basis.

During the treatment period, the concentration of DMF in the patient's blood is determined every hour. Intravenous thin lines were introduced each morning for blood sampling and kept open by infusing normal saline at 20 ml/hr, while daily monitoring of the active metabolite AMCC originating from DMF (eg, via 4-hour urine samples) was performed. Subsequent application of DMF was adjusted according to measured changes in blood levels of DMF concentration resulting from changes in absorption variability and full hematology and biochemical profiles were performed daily to detect any changes in liver function. Daily clinical and psychological assessments were performed concurrently.

Daily viral serology to determine the daily total body viral count and to detect improvement in the patient's immune status (CD4 T-helper cells) and predictors was performed concurrently. Serological work is based on p24 antigen and quantitative PCR or alternatively by other methods. Weekly specific measurements of CD4 count and β-2-macroglobulin are also performed to monitor the patient's immune status and predict improvement of factors. In order to shorten treatment to maximize clinical effect and minimize side effects, all clinical and laboratory data are loaded into a centralized data system to simplify rapid response to any changes in hazards.

test includes

a) Serum: S-Na, SK, S-Cl, S-CO ₂ , S-urea, Surate, S-Creat,

S-Ca, S-Ca, S-Mg, S-Phos, Total Serum, S-Conjd;

b) Erythrocytes: HB Det, Erythrocytes, Haematocrit, MCV, MCH,

MCHC, RDW;

c) Protein electrophoresis: ST-protein, S-albumin, S-total Glob, S-αGlob,

S-α2 Glob, S-βGlob, S-γGlob;

d) Differentiation number of white blood cells: number of white blood cells, absolute value of Neutro, absolute value of Lypho,

Mono absolute value, Eosino absolute value, Baso absolute value;

e) Liver enzymes: S-Alk, Phos, S-γGT, S-Alt(SGPT), S-AST(SGOT),

S-LD;

f) Virology: Cell markers, PCR, β2-microglobulin, P24 antigen, C-reaction

sex protein, and CK-MB concentration;

g) blood analysis for DMF levels;

h) Urinalysis for AMCC levels.

DMF appears to act as one of a reverse transcriptase inhibitor and a protease inhibitor. In vitro experiments performed indicated that it appears to be the soluble properties of DMF that solubilizes virus particles such as coats.

Claims (51)

1. the material or the composition that are used for the treatment of the cryopreservation of organ and virus and/or infected by microbes contain to be selected from and comprise general formula R ₃-CO-NR ₁R ₂The material or the composition of activating agent of group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

2. material according to claim 1 or composition, wherein R at least ₁And R ₂In one be methyl group.

3. material according to claim 1 or composition, wherein R at least ₁And R ₂In one be to fluoridize C ₁-C ₃Alkyl group.

4. material according to claim 3 or composition, wherein activating agent is N-fluorinated methyl-N-methyl-formamide.

5. according to claim 1 or described material of claim 2 or composition, wherein activating agent is a dimethyl formamide.

6. the material or the composition that are used for the cryopreservation organ contain to be selected from and comprise general formula R ₃-CO-NR ₁R ₂The material or the composition of active low temperature reagent of group, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me and halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

7. material according to claim 6 or composition, wherein R at least ₁And R ₂In one be methyl group.

8. material according to claim 6 or composition, wherein R at least ₁And R ₂In one be to fluoridize C ₁-C ₃Alkyl group.

9. material according to claim 8 or composition, wherein active low temperature reagent are N-fluorinated methyl-N-methyl-formamides.

10. according to claim 6 or described material of claim 7 or composition, wherein active low temperature reagent is dimethyl formamide.

11. any one described material or composition of comprising according to claim 5 to 10, wherein low temperature reagent contains sodium chloride with comprising being selected from, the EDTA-disodium, sodium bicarbonate, potassium chloride, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride, the physiological solution of the composition of the group of glucose and Hespes-sodium hydroxide and wherein any two or more mixture.

12. be used for the treatment of the material or the composition of virus and/or infected by microbes, contain to be selected from and comprise general formula R ₃-CO-NR ₁R ₂The material or the composition of active therapeutic agent of group, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

13. material according to claim 12 or composition, wherein virus infections is selected from acquired human immunodeficiency virus infection (HIV-1).

14. according to claim 12 or 13 described material or compositions, R at least wherein ₁And R ₂In one be methyl group.

15. according to claim 12 or 13 described material or compositions, R at least wherein ₁And R ₂In one be to fluoridize C ₁-C ₃Alkyl group.

16. material according to claim 15 or composition, wherein active therapeutic agent is N-fluorinated methyl-N-methyl-formamide.

17. according to any described material or composition that claim 12 to 14 comprises, wherein active therapeutic agent is a dimethyl formamide.

18., can accept excipients or carrier comprising at least a physiology according to any described material or the composition that claim 12 to 17 comprises.

19. material according to claim 18 or composition, wherein physiology can be accepted excipients or carrier is a colloidal silicon dioxide.

20. make the cryopreservation compositions and methods that is used for the cryopreservation organ, comprise comprising general formula R with being selected from ₃-CO-NR ₁R ₂The cryopreservation composition of group of acid amides and the method for the step that physiology solution merges, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

21. make the method for the medicament or the preparation that are used for the treatment of virus and/or infected by microbes, comprise comprising general formula R with being selected from ₃-CO-NR ₁R ₂The active therapeutic agent of group of acid amides and at least a physiology method that can accept the step that excipients or carrier merge, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

22. contain to be selected from and comprise general formula R ₃-CO-NR ₁R ₂The application of cryopreservation reagent in the cryopreservation organ of cryopreservation composition of group, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

23. contain to be selected from and comprise general formula R ₃-CO-NR ₁R ₂The medicament of group active therapeutic agent or the application of preparation in treatment virus and/or infected by microbes of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

24. the application of cryopreservation composition in making cryopreservation reagent, this composition is selected from and comprises general formula R ₃-CO-NR ₁R ₂The group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

25. material or composition are used for the treatment of the medicament of virus and/or infected by microbes or the application in the preparation in manufacturing, this material or composition comprise being selected from and contain general formula R ₃-CO-NR ₁R ₂The active therapeutic agent of group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, the group of halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

26. the method for cryopreservation organ, this method comprise that this cryopreservation composition is selected from and contains general formula R with the cryopreservation reagent perfused organ's of containing the cryopreservation composition step ₃-CO-NR ₁R ₂The group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, the group of halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group; With

Perfused organ's temperature is reduced to storage temperature, stores temporarily at this temperature to major general's organ.

27. method according to claim 26, comprise the front with physiological solution perfused organ and freezing organ to 5 step to 8 ℃, use cryopreservation reagent perfused organ then, increase the concentration of cryopreservation composition in the cryopreservation reagent gradually and organ is cooled to storage temperature simultaneously.

28. method according to claim 27, wherein the concentration of cryopreservation composition is increased to 25% to 40% gradually.

29. according to claim 27 or the described method of claim 28, wherein the cryopreservation temperature is between-4 ℃ and-196 ℃.

Pour into and be cooled to the method for the organ of cryopreservation temperature with cryopreservation reagent 30. thaw, the method comprising the steps of: organ is contacted with the cryopreservation reagent that contains the cryopreservation composition, and this cryopreservation composition is selected from general formula R ₃-CO-NR ₁R ₂The group of acid amides, wherein;

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group

Reach predetermined temperature on the cryopreservation temperature up to organ;

With the cryopreservation reagent perfused organ of containing the cryopreservation composition, the while reduces the concentration of cryopreservation composition in the cryopreservation reagent gradually and increases the temperature of organ; With

Use the physiological solution perfused organ.

31. the method for treatment virus and/or infected by microbes, the method comprising the steps of: the individuality of infective virus and/or microorganism is used contained being selected from of physiology effective dose and contain general formula R ₃-CO-NR ₁R ₂The medicament or the preparation of active therapeutic agent of group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

32. method according to claim 31, wherein selected dosage provide the haemoconcentration of activating agent in the blood of the individuality to be treated that is about 50-200ppm.

33. method according to claim 32, wherein selected dosage provides the haemoconcentration of 50-500ppm.

34. according to any described method that claim 31 to 32 comprises, wherein dosage passes through subcutaneous administration.

35. any described method that comprises according to claim 31 to 34, wherein dosage is by being selected from oral dose, sucking, by suppository, and the method administration of intravenous administration and wherein any two or more associating.

36. be used for the dosage form of virus and/or infected by microbes treatment, comprise the instrument of topical application, absorb on it or be absorbed being selected from of effective dose and contain general formula R ₃-CO-NR ₁R ₂The active therapeutic agent of group of acid amides, wherein:

R ₁And R ₂Be selected from separately and comprise H, Me, halogenated methyl, saturated and unsaturated C ₂-C ₃Alkyl group, saturated and unsaturated halogenation C ₂-C ₃Alkyl group, the group of hydroxylating alkyl group; Or

R ₁And R ₂Be selected from (CH together ₂) n, wherein n=4 or 5, or (CH ₂) ₂O (CH ₂) ₂With

R ₃Be selected from H, Me and saturated and unsaturated C ₂-C ₃Alkyl group.

37. the vaccine of the Antibody Preparation of gathering in the crops the health of the people after any described method that has also comprised according to claim 31 to 35 with the virus infections people is treated.

38. basically as described earlier in this article and the material or the composition that are used for cryopreservation organ and treatment virus and/or infected by microbes of illustrating.

39. basically as described earlier in this article and the material or the composition of the cryopreservation that is used for organ of illustrating.

40. basically as described earlier in this article and the material that is used for the treatment of virus and/or infected by microbes or the composition of illustrating.

41. basically as described earlier in this article and the cryopreservation compositions and methods of the manufacturing of illustrating.

42. basically as described earlier in this article and the manufacturing medicament of illustrating or the method for preparation.

43. basically as described earlier in this article and the application of cryopreservation reagent in the cryopreservation of organ of illustrating.

44. the concrete application in virus and/or infected by microbes treatment with medicament of illustrating or preparation as described earlier in this article basically.

45. basically as described earlier in this article and the cryopreservation composition of the illustrating application in making cryopreservation reagent.

46. the application in making medicament or preparation with material of illustrating or explanation as described earlier in this article basically.

47. basically as described earlier in this article and the method for the cryopreservation organ of illustrating.

48. basically as described earlier in this article and the method for the organ that thaws of illustrating.

49. basically as described earlier in this article and the treatment virus illustrated and/or the method for infected by microbes.

50. basically as described earlier in this article and the dosage form of illustrating.

51. as described earlier in this article and illustrate concrete vaccine.

Images