WO2009071098A2

WO2009071098A2 - Use non-human mammal capable of perspiration for determining the effect of hypothermia inducing drugs in humans

Info

Publication number: WO2009071098A2
Application number: PCT/DK2008/050296
Authority: WO
Inventors: Jacob Gotfredsen; Uno Jakob Weber; Malene Weis
Original assignee: Neurokey A/S
Priority date: 2007-12-05
Filing date: 2008-12-05
Publication date: 2009-06-11
Also published as: WO2009071098A3

Abstract

The present invention relates to a method for determining the effect of a compound for inducing hypothermia in a human being by use of non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied.

Description

Use non-human mammal capable of perspiration for determining the effect of hypothermia inducing drugs in humans

Field of invention

Background of invention

The effect of use of therapeutic hypothermia is internationally acknowledged in relation to the treatment and/or prevention of ischemia.

Ischemia is the lack of oxygenated blood flow to various body parts and organs. Cerebral ischemia is an ischemic condition where the brain or parts of the brain do not receive enough blood flow to maintain normal neurological function. Cerebral ischemia can be the result of various serious diseases such as stroke and cardiac arrest, or the result of arterial obstruction such as strangulation. Severe or prolonged cerebral ischemia will result in unconsciousness, brain damage or death.

The neuroprotective efficacy of induced hypothermia following or during ischemia of the brain is evident in experimental animal models of stroke [1 -7]. In humans, two trials conducted in cardiac arrest patients have shown improved neurological outcome of inducing hypothermia [8;9]. The therapeutic hypothermia did not increase the complication rate in these two trials and the use of induced hypothermia in comatose survivors of cardiac arrest is now recommended internationally [10].

Preferably the regulation of the core temperature of the body is induced by a pharmaceutical composition comprising a compound capable of inducing hypothermia. Therefore, there is a need for identification of new and improved compounds capable of inducing hypothermia. In consequence hereof animal models for studying the hypothermic effect of such compounds are needed. The present animal models are predominantly concerned with the study of small (when compared to the human body) animals, such as rats and mice [1 - 7], i.e. in animals having a body weight of between 3Og and 30Og.

Summary of invention

The present inventors have found that a method for determining the effect of an compound for inducing hypothermia in a human being by administration of the same compound to a non-human mammal is more reliable if the body weight of the non- human mammal is comparable to that of a human being and if further the mecha- nism for thermoregulation for the non-human mammal is comparable to that of a human, i.e. if the non-human mammal is capable of perspiration, in particular where the perspiration is through glands on the skin.

Accordingly, the present invention relates to a method for determining the effect of a compound for inducing hypothermia in a human being, said method comprising the steps of: a) providing a non-human mammal capable of perspiration, b) administering to said non-human mammal an amount of said compound, c) measuring the temperature of said non-human mammal at least once, d) based on the measurement of the temperature in step c) determining whether the agent is capable of inducing hypothermia in an human.

In a second embodiment the present invention relates to a method for determining the effect of treatments and drugs on human being, said treatments or drugs having a hypothermic effect in humans, said method comprising the steps of a) providing a non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied, b) obtaining a hypothermic effect on the non-human mammal by i. mechanical hypothermia therapy, and/or ii. providing a drug c) observe the effect of the temperature regulation on the non-human mammal d) based on c) determine the effect on human beings. The mechanical hypothermia therapy may either be surface hypothermia therapy or invasive hypothermia therapy, as defined: Surface hypothermia therapy, such as:

• Applying ice, cold water, or other liquid or firm elements to the skin, mucous membranes, or in proximity hereof

• Dressing patients in thermo-vests, thermo-hats, or other garments capable of inducing hypothermia

• Using mist-, flow-, or other tents in order to induce hypothermia Invasive hypothermia therapy, such as: • Intravenous administration of cold fluids (<37°C), e.g. saline

• Insertion of cooling agents such as metallic coils, tubes, or other foreign bodies in blood vessels, body cavities or in other bodily locations

In a third embodiment non-human mammal is of the family Bovidiae, such as the subfamily Bovinae, the genus Bos, in particular the species B. Taurus as it has a body weight comparable to that of the human being for which the effect is to be studied. In a particularly preferred embodiment the non-human mammal is a calf. In a fourth embodiment the non-human mammal is of the family Equidae, in particular the genus Equus, such as the species E. caballus. In a fifth embodiment the non-human mammal is the off-spring of any of the said non-human mammals.

Further the non-human mammal may be a primate.

In a sixth embodiment the non-human mammal of any of the previous embodiments has a weight that corresponds to the weight of a human body, i.e. is in the range of 2-150 kg, such as 5-100 kg, such as 10-90 kg, in particular 50-90 kg, or 60-90 kg, or 70-90 kg, such as 75-85 kg.

The present invention discloses a method for determining the effect of a compound for inducing hypothermia in a human being, said compound being for the prepara- tion of a medicament capable of inducing hypothermia in an individual, in particular where the medicament is for the treatment of ischemia in an individual in need thereof. It is also an aspect of the present invention to provide a method for assaying the effect of a medicament comprising a compound capable of inducing hypothermia in an individual.

Definitions

Compound: Chemical or biological material useful for the treatment of a disease or for obtaining a therapeutic effect.

Hypothermia: Lowering of the body temperature below normal level.

Ischemia: Restriction in blood supply with resultant dysfunction or damage of tissue.

Ischemic tissue damage: Tissue damage due to ischemia.

Off-spring is the product of reproduction, a new non-human mammal produced by one or more parents.

Perspiration (also known as sweating or transpiration) is the production of a fluid consisting primarily of water that is excreted by the sweat glands in the skin of mammals. Perspiration is a means of thermoregulation.

Pharmaceutical composition, drug or medicament refers to any chemical or biological material, compound, or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Some drugs are sold in an inactive form that is converted in vivo into a metabolite with pharmaceutical activity. For purposes of the present invention, the terms "pharmaceutical composition" and "medicament" encompass both the inactive drug and the active metabolite.

Thermoregulation is the ability of an organism to keep its body temperature within certain boundaries. Detailed description of the invention

The study of medical induction of hypothermia in animals having a body weight that differs significantly from the body weight of human beings is bound to less reliable compared to studies in animals with body weights similar to humans because the volume that has to be reduced in temperature when inducing hypothermia is significantly different in small and large animals. Accordingly, animals with a body weight comparable to the weight of the human body are preferred over smaller animals such as rats and mice. In human beings perspiration, where water is excreted by the sweat glands, is an important means for thermoregulation. The inventors of the present invention have realised that not only body weight but also mechanism of thermoregulation may be of importance when a non-human mammal is to be used in a method for determining the effect of a compound for inducing hypothermia in a human being. Most animals usually used for studying the effect of compounds for use in the production of medicaments do not perspirate to the same extend as human beings. For example dogs have only few sweat glands and accomplish their temperature regulation by panting which evaporates the moist lining of the oral cavity. It is believed that this will lead to a different thermoregulation profile as compared to the profile for human beings and the effectiveness of compounds having hypothermic effect may therefore be either over- or underestimated if e.g. dogs are used for determining the effect. Therefore, in a particularly preferred embodiment the non- human mammal is capable of perspiration in a manner similar to that of human beings. In particular calves are preferred because they both have a body weight comparable to the one of human beings and they are capable of perspiration in a way that is similar the one of human beings.

Ischemia

Ischemia is the reduction or abolition of blood supply to a tissue. The associated deficiency of oxygen and nutrients may lead to cell death (necrosis) in areas of the affected tissue. The damage induced by the lack of oxygenated blood in the brain occurs in two stages. First cellular metabolism is arrested due to lack of oxygen and some cells and tissue will die within minutes as a consequence hereof. Secondly a cascade of processes such as apoptosis are initiated and continue up to 12 hours after the event that initially induced the ischemic state has been abolished. The tis- sue damaged by the second cascade can be crucial and cause greater harm to the individual than the primary damage happening within the first minutes of ischemia.

Ischemia may occur under various circumstances; of special relevance to the pre- sent invention are the circumstances relating to cardiovascular diseases, asphyxia and traumatic brain injuries.

Cardiovascular diseases

Cardiovascular disease is the most common cause of death and of physical as well as mental impairment in the developed world. A similar development is seen in the rest of the world as it emulates the lifestyle of the Western hemisphere with its fatty diets, lack of exercise and increasing average lifespan.

The main causes of death and disability among cardiovascular diseases are myo- cardial infarction, acute coronary syndrome, cardiac arrest and stroke, but many less common cardiovascular diseases may be equally detrimental to the individual affected. These less common diseases include among others arterial aneurism, subarachnoid haemorrhage, arteriosclerosis, angina pectoris, hypertension, hypercholesterolemia, cardiac arrhythmia, cardiomegaly, cardiomyopathy, heart valve regur- gitation and heart valve stenosis.

Each of the cardiovascular diseases mentioned, as well as others not mentioned, may cause ischemia of organs. This ischemia, whether of the brain, heart or other organs, may lead to death or impairment if not treated rapidly.

It is an object of the present invention to provide a method for determining the effect of a compound for inducing hypothermia in a human being, said compound being useful for the production of a medicament for the treatment or prophylaxis of an individual suffering from or at risk of suffering from of ischemia such as due to cardio- vascular diseases such as, but not limited to: myocardial infarction, acute coronary syndrome, cardiac arrest, stroke, arterial aneurism, subarachnoid haemorrhage, arteriosclerosis, angina pectoris, hypertension, hypercholesterolemia, cardiac arrhythmia, cardiomegaly, cardiomyopathy, heart valve regurgitation and heart valve stenosis. The said method comprises the steps of a) providing a non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied, b) administering to said non-human mammal an amount of said com- pound, c) measuring the temperature of said non-human mammal at least once, d) based on the measurement of the temperature in step c) determining whether the agent is capable of inducing hypothermia in an human.

Particularly mammals having a body weight comparable to that of human beings, i.e. in the range of 50-90 kg, or 60-90 kg, or 70-90 kg, such as 75-85 kg are preferred. The mammal may be a calf.

Asphyxia

Asphyxia (suffocation) is a common cause of death and of physical as well as men- tal impairment in perinatals, neonatals or newborns, children and adults of all ages.

Asphyxia can be divided into perinatal asphyxia and non-perinatal asphyxia. Perinatal asphyxia is the medical condition resulting from deprivation of oxygen to an infant long enough to cause apparent harm; the infant may at the time of oxygen depriva- tion still reside in the mother's uterus and/or birth canal, it may occur during the process of delivery or may occur immediately after delivery.

Non-perinatal asphyxia is a condition of severely deficient supply of oxygen to the body that arises from being unable to breathe normally. Common causes hereof include drowning, strangulation and exposure to toxic gasses. Asphyxia causes generalized hypoxia, which primarily affects the tissues and organs most sensitive to hypoxia first, such as the brain, hence resulting in cerebral hypoxia. The absence of effective remedial action will very rapidly lead to unconsciousness, brain damage and death.

It is an object of the present invention to provide a method for determining the effect of a compound for inducing hypothermia in a human being, said compound being useful for the production of a medicament for the treatment or prophylaxis of an individual suffering from or at risk of suffering from of ischemia due to asphyxia such as: perinatal asphyxia and/or non-perinatal asphyxia. The said method comprises the steps of a) providing a non-human mammal capable of perspiration, b) administering to said non-human mammal an amount of said compound, c) measuring the temperature of said non-human mammal at least once, d) based on the measurement of the temperature in step c) determining whether the agent is capable of inducing hypothermia in an human.

In a particular embodiment, where determining the effect of an agent for inducing hypothermia in a human being under perinatal conditions, the compound is adminis- tered to the off-spring while it resides within the uterus and/or birthcanal.

In another embodiment the present invention provides a method for determining the effect of a compound for inducing hypothermia in a human being, said compound being useful for the production of a medicament for the treatment or prophylaxis of an individual suffering from or at risk of suffering from elevated body temperature, e.g due to an infection, ischemic damage, fever, in particular hyperpyrexia, heat- or sunstroke or the like.

In a still further embodiment the present invention provides a method for determining the effect of a compound for inducing hypothermia in a human being, said compound being useful for the production of a medicament for induction of hypothermia in an individual in need of surgery prior to or during the surgery, for example in the case of organ transplants.

Any of the embodiments above may also be applied to the situation where a combination of two or more compounds, such as a combination of three, four, five, six, seven, eight, nine or ten compounds, are used. In a particular embodiment the further compound is selected from the group of: analgesics, opioids, GABAs and adrenergic antagonists. Hypothermia

Hypothermia is the lowering of the core temperature of the body below normal level. Normal body temperature in an adult human measured rectally over 24 hours is 37 degree Celsius +/- 0.6 degree Celsius and is thus variable between individuals, and over time within the individual. Hypothermia as a medical condition is usually defined as the effects seen on the body once the core temperature drops below 35 degree Celsius. It may become critical, if the body temperature falls below 32 °C. In the present application hypothermia is defined as the lowering of the core body temperature below normal levels. This implies that any temperature below the normal core body temperature of the specific individual with its natural variations at the given point in time of the day, or period, herein is defined as being hypothermic. In particular, hypothermia is a temperature below 35.5 °C, such as below 35 °C, such as below 34.5 ⁰C, such as below 34.0 °C or below 33.0 °C.

Body temperature may be measured by a variety of means by mercury, electronic or plastic strip thermometers on different areas of the body such as the forehead, mouth, armpit, ear or rectum. It is presently understood, that the temperature referred to in the present application is the core body temperature, and that some of the above methods of measurement will indicate a different temperature than the core temperature.

It is of importance, that induction of hypothermia in an individual can follow a predictable course and be responsive to the dose in which the compound capable of inducing hypothermia is administered. An aspect of the method of the present inven- tion is therefore that it may be used to study the effect of treatments and drugs on human beings, said treatments or drugs having a thermoregulating effect in human beings. Accordingly, the present invention covers a method for determining the effect of treatments and drugs on a human being, said treatments or drugs having a temperature regulating effect in humans, said method comprising the steps of a) providing a non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied, b) obtaining a thermoregulating effect, preferably a hypothermic effect, on the non-human mammal by 1 ) mechanical means, such as cooling or heating, and/or 2) providing a drug c) observe the effect of the temperature regulation on the non-human mammal d) based on c) determine the effect on human beings.

It is thus an object of the present invention to provide a method for determining the effect of a compound for the production of a medicament for the induction of hypothermia in an individual, wherein the compound is capable of inducing hypothermia to any range of temperatures between 37 and 31 degree Celsius, such as between 36.5 and 31.5 degree Celsius, such as between 36 and 32 degree Celsius, such as between 35.5 and 32.5 degree Celsius, such as between 35 and

33 degree Celsius, such as between 34.5 and 33.5 degree Celsius. The ranges may furthermore be between 37 and 34 degree Celsius, such as between 36.5 and 34.5 degrees, such as 36 and 35 degrees, alternatively between 34 and 31 degree, such as between 33.5 and 31.5 degree, or between 34 and 32 degree, such as 33 and 32 degree Celsius, alternatively between 36 and 33 degree or 35 and 32 degree

Celsius. Preferably, the compound of the present is capable of inducing hypothermia in the range of between 36 to 32 degree Celsius, and more preferably between 35 and 33 degree Celsius.

Preferred compound

The present method for determining the effect of a compound for inducing hypothermia in a human being is applicable to any compound. Various useful compounds can be found in PA 2007 01742, PA 2008 01 105, PA 2007 01743, PA 2008 00716, PA 2008 01337, PA 2007 01744, PA 2008 01 104, PA 2007 01745 and PA 2008 00498 as well as in WO 2007/140786, WO 2008/040360 and WO 2008/040361.

Medicament The method for determining the effect of a compound for inducing hypothermia in a human being by any of the herein described compounds may be performed by preparing, producing and thus providing a medicament or pharmaceutical composition comprising at least one of said compounds. In a further embodiment the method of the present invention may be used to evaluate the effectiveness of a compound for inducing hypothermia in a human being in combination with at least one further active ingredient, such as active ingredients selected from the group of: cannabinoids, neurotensins, analgesics, opiods, GABAs and adrenergic antagonists.

Pharmaceutical composition

Whilst it is possible to apply the method of the present invention to a compound per se (i.e. as a raw chemical) it is also possible to apply the method to a fully formu- lated pharmaceutical composition.

The compounds to be tested by the method of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compounds of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, bind- ers, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

The compounds to be tested by the method of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.

The compound for which the effect for inducing hypothermia in a human being is to be tested by the method of the present invent may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Administration

Various ways of administering the compound for inducing hypothermia in the method of the present invention are contemplated and reflect the main routes of drug delivery to human beings.

The main routes of drug delivery in the treatment of human beings with compounds/drugs for inducing hypothermia are intravenous, oral, and topical, as will be described below. Other drug-administration methods, such as subcutaneous injection or via inhalation, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The mucosal membrane to which the pharmaceutical preparation of the invention is administered may be any mucosal membrane of the individual to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, mouth or rectum.

Compounds applied in the method of the invention may be administered parenterally, that is by intravenous, intramuscular, intraspinal, subcutaneous, intraarterial, intracardiac, intraosseous, intradermal, intrathecal, transdermal, transmucosal, inhalational, epidural, sublingual, intravitreal, intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. The compounds may also be administered by inhalation, that is by intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.

The compounds to be applied in the method of the present invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially.

A preferred embodiment of the present invention is the administration by injection, suppository, oral administration, sublingual tablet or spray, cutaneous administration, or inhalation. More preferably the administration form is by injection, wherein the injection is intravenous, intramuscular, intraspinal, intracerebral, intraperitoneal, subcutaneous, a bolus or a continuous administration.

A highly preferred embodiment of the present invention is the intraperitoneal administration.

The most preferable mode of administration is by intravenous injection.

Dosage

The dose administered to the non-human mammal in the method of the present invention should be an "effective amount" or an amount necessary to achieve an "effective level" in the mammal.

Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on inter-individual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the non-human mammal that corresponds to a concentration of a compound according to the invention.

Dosage regime and Duration of treatment

The method of the present invention may be used to calculate the correct amount of medicament to be administered in any suitable dosage regime, suitable as to the potency of the compound / drug, the target temperature to be reached, the speed of action of the compound, the metabolic stability of the compound, the duration of the treatment and how often the medicament optimally is to be administered.

It is within the scope of the method of the invention that the compound to be investigated is to be administered at intervals of 30 minutes to 24 hours, such as intervals of 1 to 23 hours, 2 to 22 hours, 3 to 20 hours, 4 to 18 hours, 5 to 16 hours, 6 to 14 hours, 7 to 12 hours or 8 to 10 hours. Preferably, the administration occurs at intervals of 1 to 6 hours, such as 2 to 5 hours, 3 to 4 hours.

In one embodiment, the compounds to be tested in the method of the present invention are administered at a dosage of between 10 μg to 80 mg pr kg body mass either sequentially or consecutively.

In one embodiment, for determining the effect of a compound for inducing hypo- thermia in a human being by the method of the present invention, the treatment with compounds is from 6 to 72 hours.

Examples

Example 1 - Calf study model

In order to evaluate a sought after hypothermic effect of a treatment by test compounds according to the invention, the treatment is tested in the calf study model. The calf model is used because the body weight of the calves is comparable to the body weight of humans.

Study subjects

The evaluation is carried out on male "sortbroget malkeko/Holstein" calves with a body weight of 75-85 kilo or 70-90 kilo. The calves are positioned in individual booths; they are not sedated; they are fed twice a day (milk at 8:00 A.M. and hay/food pills at an unspecified hour, depending on the effects of the compounds examined); and they are subjected to a day cycle consisting of 16 hours of light followed by 8 hours of dark. The ambient temperature is 20°C+/-2°C.

Drug administration/device-based therapy The compounds investigated are administered i.v. (in the jugular vein) and/or i.p. and/or s.c. as bolus injections and consists of 1 solitary injection, alternatively 2-8 repeated injections within a timeframe of 12-24 hours from the initial injection.

Generally 4 different doses plus vehicle are tested producing varying degrees of hypothermic responses. Hypothermic effect

The primary effect evaluated is hypothermia. Temperature is measured using a temperature probe (ADInstruments) that is surgically positioned in vena cava crani- alis one week prior to commencement of the study. The probe is connected to a Powerlab ensuring the required read-outs.

Temperature is measured 100 times/second and compounded every 15 minutes from 1 hour prior to drug administration to 24 hours subsequent to administration of the test compounds.

Other effects

Blood pressure and heart rate is measured 100 times/second and compounded every 15 minutes from 1 hour prior to drug administration to 24 hours after the com- pounds have been administered. Data is collected using a Powerlab.

Example 2 - Bovine study model

In order to evaluate an effective hypothermic dose of a test compound, the compound is tested in the bovine study model. The efficacy of a compound tested in the bovine model may be correlated with the efficacy of other compounds tested in the same bovine study model. Calves in the weight target range from 65 to 95 kilo are of primary interest. Calves of higher weight are not be comparable to overweight/obese humans since their weight gain does not arise from increased amount of fatty tissue, but on the contrary from a larger muscle percentage.

Study subjects

Fifteen male Danish Holstein calves are moved to an intensive research barn. The calves are kept separated form other cattle throughout the trial. The insulated barn has 4 separate rooms (sections) each with 4 pens and no windows. A 16 hour light and 8 hour dark program is imposed. Each pen is equipped with a rubber mattress and further bedded with sawdust. Pens are cleaned daily and new sawdust added. Calves are loose housed, but each day around milk-feeding calves are tied for 1 hour to get accustomed to the experimental sampling situation, where calves has to be tied due to the fitted cords from the probes and catheters (see below). Rooms are automatically heated and ventilated and temperatures are set at 20⁹C +/- 2°C. Real time recordings of room temperature and humidity are performed. The calves are kept in the intensive barn for 29 days before the experimental sampling periods are initiated. At an age of app. 50 days (body weight 75 to 85 kg) calves are assigned to the sections of four and injected with test compound. Body temperature, arterial blood pressure, and heart rate are monitored 60 min prior to injection and during 24 h following injection.

During the pre-trial period all calves received surgery. Calves are surgically implanted with permanent indwelling catheters in the right jugular vein and right carotid artery under total anesthesia prior to the experimental sampling periods.

Four of the calves are used to test the sampling procedure, blood pressure probes, equipment and the recording of data without receiving test compound or vehicle.

All calves stay healthy during the trial and none of the calves receive veterinary treatments during the trial except for the preplanned treatments with antibiotics and analgesics connected with surgical procedures.

The calves are fed milk-replacer restrictively (4 kg of milk-replacer with 123 g dry matter/kg supplied once daily) and have ad libitum access to artificially-dried hay and starter concentrate during the trial. During the 29-day pre-trial period, the daily gain is on average 666 gram/day. Calves growing at this level of daily gain will usually be considered as well performing calves.

On the sampling days, 8 calves are fitted with at temperature probe (MLT 1401 ; ADInstruments) inserted into the jugular vein via the permanent catheter. The arterial catheter is connected to a pressure transducer and filled with saline containing 100 IU sodium heparin. Data are recorded using Powerlab (Chart 5 version 5.5.4, ADInstruments) at 100 Hz. All pressure probes are individually tested and calibrated against a 2-m water column.

The test compound iss injected into the left-side jugular vein via a temporary catheter implanted at least one day before injection. Data analyzed within sampling period

All calves complete the planned injections and recordings with no loss of data apart from shorter periods of time (minutes) where temperature probes accidentally are disconnected or repositioned. Pressure transducers are flushed when needed and pressure data is only missing or invalid for shorter periods before these flushings and during the flushing procedure.

The recorded data are averaged in 15-min intervals prior to data analysis. Data are sampled from exactly 60 min before injection of test compound and until 23 hours after injection. Data recorded during the 60 min prior to injection are defined as baseline.

Variables describing temperature, blood pressure and heart rate are considered as repeated measures and analyzed by using the autoregressive order 1 structure in the mixed model procedure of SAS (SAS Institute, 2001 ). The model includes the effects of block (calves housed in the same room), treatment and time as well as the interaction between treatment and time. Orthogonal polynomial contrasts are used to estimate the linear, quadratic, and cubic effects of treatment.

Variables describing number of hours where the body temperature deviated from baseline are constructed within calf and period. Variables are constructed for +0.5, +1.0, +2.0, +2.5, -0.5, -1 .0, -2.0, and -2.5°C.

A variable describing time when body temperature returns to or exceed baseline after injection is calculated as the minimum value for time fulfilling the conditions: time > 0.25 h post injection & (15 min average temperature - baseline temperature) > 0.

Variables with only one observation within period and calf are analyzed using a model including the effects of block and treatment. Orthogonal polynomial contrasts are used to estimate the linear, quadratic, and cubic effects of treatment.

Means are given as least squares means ± residual standard error of the mean. Allocation of animals

Three test days (TestDay 1 , TestDay 2 and TestDay 3; each of the test days are separated by three days without testing) are planned involving testing on 16 calves. Eight calves are tested each test day. The calves to be used at the first two days have not been used previously in the study, while all the calves to be used at Day 3 have been used either 4 or 8 days earlier. At TestDay 3, seven out of eight calves have received the test compound and one out of eight have gotten placebo at either TestDay 1 or TestDay 2. Test days: TestDay 1 is designed at dose finding. TestDay 2 is designed at dose finding. TestDay 3 is designed to test the effect of repetitive doses.

Drug formulation: The test compound may be formulated in several ways according to standard procedures known in the art (e.g. Remington: The Science and Practice of Pharmacy [1 1])-

Hypothermic effect The primary effect evaluated is hypothermia. Temperature is measured using a temperature probe that is surgically positioned in a femoral artery two weeks prior to commencement of the study. The probe is connected to telemetry equipment (e.g. implanted telemetry from Data Sciences International) ensuring the required readouts.

Temperature is measured every 15 minutes from 1 hour prior to drug administration to 12 hours after drug administration, and every 30 minutes subsequently until 24 hours after drug administration. Temperature measurement is conducted via a permanent femoral artery temperature probe (telemetry).

The minimum temperature as well as a graph of the temperature at each point of measurement is recorded for each dose of compound or compound mixture. Other effects

Blood pressure, heart rate and ECG is registered every 15 minutes from 1 hour prior to drug administration to 12 hours after drug administration, and every 30 minutes subsequently until 24 hours after drug administration.

Example 3 - Extended Bovine study #1

Study subjects

The evaluation is carried out on 80 Holstein bull calves purchased for the study that is conducted over 8 study days. At an age of app. 50 days (body weight 75 to 85 kg) calves are assigned to the sections and injected with test compound. Body temperature, arterial blood pressure, and heart rate are monitored 60 min prior to injection and during 24 h following injection. Each calf receives experimental injection one time. See the above for further specifications.

Each study week consists of two study days. For each study week 20 calves are purchased. These calves will be at the test facilities for approximately 2 weeks before the operation period. Two calves serve as a buffer in case of sickness or unexpected high or low weight gain in the first two weeks after the animals arrive at test facilities. The remaining 18 calves are operated and 16 are ultimately included in the study. The surplus of two operated calves is an extra measure in order to deal with sickness and unexpected high or low weight gain in the two-week period from operation to the study day.

Calf study outline

The study days are allocated to the examination of the following compounds and purposes:

The TEST-I at day 3 is used to investigate continuous doses. The calves are given the indicated doses until a stable temperature is reached; the reached temperature is then kept stable by administering an appropriate amount of the test compound. The calves remain stable at the reached temperature for the duration of the experiment.

Administration

Day 1 -5: Each calf receives a single intravenous bolus injection of 2 ml administered over 7-8 minutes followed by saline flush of 5 ml. Day 6-7: Each calf will receive an initial intravenous bolus injection of 2 ml administered over 7-8 minutes, followed by one or several similar intravenous injections for up to 12 hours after the initial injection.

Pharmacokinetics / Pharmacodynamics (PK/PD)

Blood samples will be taken before and after injection of compounds in order to examine PK/PD. The specifics depend on a meeting with Xendo (www.xendo.dk) where the topic will be a tailor-made pharmacokinetic/pharmacodynamic model for the calf study.

Drug administration

The compound investigated are administered i.v. as bolus injections and may consist of 1 solitary injection, alternatively 2-4 repeated injections within a timeframe of 24 hours from the initial injection, alternatively as a continuous administration.

The primary effect evaluated is hypothermia. Temperature is measured using a temperature probe that is surgically positioned in a femoral artery two weeks prior to commencement of the study. The probe is connected to telemetry equipment (e.g. implanted telemetry from Data Sciences International) ensuring the required readouts.

The minimum temperature as well as a graph of the temperature at each point of measurement is recorded for each dose of compound or compound mixture.

Other effects

References

[1 ] Busto R, Dietrich WD, Globus MY, Valdes I, Scheinberg P, Ginsberg MD: Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 1987; 7(6):729-738.

[2] Barone FC, Feuerstein GZ, White RF: Brain cooling during transient focal ischemia provides complete neuroprotection. Neurosci Biobehav Rev 1997; 21 (1 ):31 -44.

[3] Onesti ST, Baker CJ, Sun PP, Solomon RA: Transient hypothermia reduces focal ischemic brain injury in the rat. Neurosurgery 1991 ; 29(3):369-373.

[4] Ooboshi H, lbayashi S, Takano K, Sadoshima S, Kondo A, Uchimura H, Fujishima M: Hypothermia inhibits ischemia-induced efflux of amino acids and neuronal damage in the hippocampus of aged rats. Brain Res 2000; 884(1 ~2):23- 30.

[5] Colbourne F, Corbett D, Zhao Z, Yang J, Buchan AM: Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model. J Cereb Blood Flow Metab 2000; 20(12):1702-1708.

[6] Kawai N, Okauchi M, Morisaki K, Nagao S: Effects of delayed intraischemic and postischemic hypothermia on a focal model of transient cerebral ischemia in rats. Stroke 2000; 31 (8):1982-1989.

[7] Maier CM, Ahern K, Cheng ML, Lee JE, Yenari MA, Steinberg GK: Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: effects on neurologic outcome, infarct size, apoptosis, and inflammation. Stroke 1998; 29(10):2171 -2180.

[8] Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest: N Engl J Med 2002; 346(8) :549-556.

[9] Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, Smith K: Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002; 346(8) :557-563.

[10] Bernard S: Therapeutic hypothermia after cardiac arrest: now a standard of care. Crit Care Med 2006; 34(3):923-924.

[1 1 ] Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

Claims

1. A method for determining the effect of a compound for inducing hypothermia in a human being, said method comprising the steps of: e) providing a non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied, f) administering to said non-human mammal an amount of said compound, g) measuring the temperature of said non-human mammal at least once, h) based on the measurement of the temperature in step c) determining whether the agent is capable of inducing hypothermia in an human.

2. A method for determining the effect of treatments and drugs on human being, said treatments or drugs having a hypothermic effect in humans, said method comprising the steps of e) providing a non-human mammal capable of perspiration, said mammal preferably having a body weight comparable to that of the human being for which the effect is to be studied, f) obtaining a hypothermic effect on the non-human mammal by iii. mechanical means, such as cooling, and/or iv. providing a drug g) observe the effect of the temperature regulation on the non-human mammal h) based on c) determine the effect on human beings.

3. The method of claim 1 -2 wherein the non-human mammal is a primate.

4. The method of any of claims 1 -2, wherein the non-human mammal is of the fa- miliy Bovidae, such as the subfamily Bovinae, the genus Bos, in particular the species B. Taurus.

5. The method of any of claims 1 -2, wherein the non-human mammal is of the family Equidae, in particular the genus Equus, such as the species E. caballus.

6. The method of claim 1 or 2 wherein the non-human mammal is an off-spring of the non-human mammal of any of claims 3-5.

7. The method of claim 1 or 2 wherein the weight of the non-human mammal is in the range of 2-150 kg, such as 5-100 kg, such as 10-90 kg, in particular 50-90 kg, or 60-90 kg, or 70-90 kg, such as 75-85 kg.

8. The method of any of claims 1 -2, 4 and 6-7 wherein the non-human mammal is a calf.

9. The method of any of claims 1 -8 wherein the compound is administered injec- tion, suppository, oral administration, sublingual tablet or spray, cutaneous administration, or inhalation, more preferably the administration form is by injection, wherein the injection is intravenous, intramuscular, intraspinal, intracerebral, intraperitoneal, subcutaneous, a bolus or a continuous administration or even more preferably by intraperitoneal administration.

10. The method of any of claims 1 -9 wherein the compound for inducing hypothermia in a human being is administered with at least one other compound, wherein the compounds may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially.

1 1 . The method of any of claims 1 -10 wherein the core body temperature is measured by a by mercury, electronic or plastic strip thermometers on different areas of the body such as the forehead, mouth, ear or rectum.