WO2005039625A1 - Growth hormone secretagogue receptor agonists - Google Patents

Abstract

The present invention relates to growth hormone secretagogue (GHS) receptor agonists. In particular, the present invention relates to novel therapeutic uses of GHS receptor agonists. The novel applications of GHS receptor agonists relate to thermic control of body temperature which has proven to be beneficial in a number of pathologic conditions.

Description

GROWTH HORMONE SECRETAGOGUE RECEPTOR AGONISTS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to growth hormone secretagogue (GHS) receptor agonists. In particular, the present invention relates to novel therapeutic uses of GHS receptor agonists.

BACKGROUND OF THE INVENTION

Cerebrovascular events leading to ischaemia constitute the third most common cause of deaths in Western societies and it is the most common cause of disability among adults. Patients surviving acute injury to the central nervous system (CNS), whether arising from ischaemia or trauma, are often seriously disabled and constitute a major socio-economic burden.

It has been shown however, that the extent of the tissue damages and hence also the extent of the subsequent disability can be much reduced if the body temperature of the patient is cooled soon after the onset of the ischemic event. So far, it has only been possible to lower the body temperature of a patient by mechanical means, e.g. surface cooling with a cold air flow generator; combination of cooling blankets and cold saline gastric lavage; hypothermic cardiopulmonary bypass; local cortical cooling (Seacost device); endovascular cooling (Innercool Therapies device)

Growth hormone secretagogues and the GHS receptor

Growth hormone release from the anterior pituitary lobe somatotrophs is regulated by the hypothalamic hypophysiotrophic factors GHRH (growth hormone-releasing hormone) and somatostatin. Release of growth hormone from anterior pituitary somatotrophs is also stimulated by synthetic growth hormone secretagogue (GHS)-receptor agonists (an agonist is a drug that activates a receptor). With the cloning of the orphan GHS-receptor from anterior pituitary cells (Howard et al., 1996) it became clear that synthetic ligands - peptides or non-peptides - can be administered to elicit marked growth hormone release from anterior pituitary. However, GHS-receptor is present in many other tissues including the hypothalamus, thyroid gland, heart, lung, kidney, and skeletal muscle and it is distinctly different from the GHRH-receptor, which is primarily expressed by anterior pituitary somatotrophes.

Recently, the endogenous ligand for the GHS-receptor was identified in rat stomach as a 28-amino acid post-translationally modified peptide (Kojima et al., 1999). Human ghrelin is a 28-amino acid modified peptide where a hydroxyl group of the third N-terminal amino acid (a serine) is esterified by n- octanoic acid (Kojima et al., 1999). The peptide was named ghrelin for its growth hormone releasing capabilities. However, its was subsequently discovered that this peptide has an important physiological role as an endogenous stimulator of food intake (Kamegai et al., 2000; Tschόp et al., 2000; Ariyasu et al., 2001 ). Thus, plasma ghrelin levels rise before food intake in ad libitum fed humans as well as animals; this rise coincides with sensation of hunger (Cummings et al., 2001 ).

Both ghrelin and synthetic GHS-receptor agonist analogues have been proposed as useful therapeutic agents for the treatment of wasting, or cachexia (unintended loss of body weight, particularly lean body, or muscle, mass). Cachexia is a common finding in the later stages of many forms of cancer as well as HIV-positive human subjects. This condition is prevalent even in HIV-infected individuals successfully treated with anti- retrovirals. Ghrelin, GHS-receptor agonist analogues and growth hormones facilitate increase in lean body mass, and growth hormone is approved for treating the cachexia associated with HIV disease. Ghrelin also has a modulatory impact on several physiological functions including locomotive activity and overall sympathetic tone (Tang-Christensen et al., 2003). Some of these actions are likely to be induced via GHS- receptors localised within the central nervous system (CNS), as they require administration of ghrelin by the intracerebrovent cular route to elicit maximal inhibition of locomotor activity. Other therapeutic areas that are being investigated for ghrelin would overlap with those pursued with other GHS- receptor agonist analogues (growth hormone deficiency in both children and adults; and in clinical conditions such as congestive heart failure, osteoporosis are potential targets together with improvements of body composition and function in elderly patients).

Ghrelin and derivatives thereof with GHS receptor agonist activity, act on the GHS-receptor. The first class of synthetic ligands acting as stimulators of growth hormone release from isolated pituitary somatotrophs were derived from the hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) (Bowers et al., 1984). The biological response used to screen for biological activity of GHRP-6 and analogues thereof was secretory activity of the anterior pituitary somatotroph. Since then, other potent growth hormone secretagogues such as GHRP-1 , GHRP-2, hexarelin, NN703, and ipamorelin have been synthesised:

GHRP-1 : Ala-His-D-(2')-Nal-Ala-Trp-D-Phe-Lys-NH2 GHRP-2: D-Ala-D-(2')-Nal-Ala-Trp-D-Nal-Lys-NH2 Hexarelin: His-D-MeTrp-Ala-Trp-D-Phe-Lys-NH2 Ipamorelin: H-aminoisobutyric acid-His-D-2Nal-D-Phe-Lys-NH2 NN703: Comp. #41 in Peschke et al. 1999, Eur J ed Chem 34:363-380)

Due to the poor oral bioavailability (<1 %) of the peptide GHS-receptor agonist analogues, considerable medicinal chemistry efforts have been focused towards synthesising non-peptide compounds mimicking the action of GHRP-6 on anterior pituitary cells. Thus, a number of benzolactams (e.g. L-692,429) and spiroindanes (e.g. MK-0677; and other described in WO94/13696) have been found to induce growth hormone release via the GHS-receptor in several mammals including man (Smith et al., 1993 Science 260:1640-1643; Patchett et al. 1995 Proc Natl Acad. Sci, USA 92:7001- 7005). The compound MK-0677 constitutes an excellent example of a small spirodane acting as an agonist at the GHS-receptor. It is more than likely that other GHS receptor agonists will be identified in the future.

The GHS-receptor is a G-protein coupled receptor (GPCR). Effects of GHS- receptor activation include depolarisation of target tissue cell membranes, inhibition of potassium channels, increased inositol triphosphate (IP3) levels, and transient increase in intracellular calcium (Pong et al., Molecular Endocrinology 10:57-61 ; Guan et al., Mol. Brain Res. 48:23-29; McKee et al., 1997 Genomics 46:426-433).

Traditionally, GPCRs have been assumed to exist in a quiescent state unless activated by the binding of an agonist. It is now appreciated that many GPCRs, including the GHS-receptor can exist in a partially activated state in the absence of their endogenous agonists. The basal activity (constitutive activity) can be inhibited by compounds called inverse agonists. Both agonists and inverse agonists interact with a receptor, in that they alone can activate or inactivate receptors. In contrast, classic or neutral antagonists compete against agonists and inverse agonists for access to the receptor, but do not possess the intrinsic ability to inhibit elevated basal or constitutive receptor responses. The GHS-receptor was recently demonstrated to possess significant constitutive, or agonist independent receptor activity (Hoist et al. 2003 Mol. Endocrinol.) Cerebral ischaemia and traumatic brain damage

Any event, which results in cerebral hypoxia including ischaemia, toxicity and acute injury can cause severe damage to tissues of the CNS including neurones of the brain, retina and spinal cord. At present, cerebrovascular events leading to ischaemia constitutes the most common cause of disability among adults. Patients surviving acute injury to the CNS, whether arising from ischaemia or trauma, are often seriously disabled.

There has previously been no effective therapeutic regime for use in treatment of such CNS injuries and no way of limiting the damage that occurs as a result of hypoxia or ischemia. Ischaemia caused by occlusion of any of the branches of the internal carotid artery or of any of the branches of the vertebral arteries have been treated successfully with thrombolytic agents such as Streptase or human recombinant tissue plasminogen activator (Alteplase). However, thrombolytic therapy has several limitations as it must be initiated no later than 3 hours after the debut of symptoms, and cannot be applied to patients unless acute imaging of the injured brain has confirmed a limited lesion size and ruled out haemorrhagic pathology.

Mild hypothermia, in which the temperature of the CNS is kept at 30-34 °C, has been attracting attention as a method for the treatment of ischaemic brain disorders and traumatic brain disorders. Several mechanic cooling methods have been developed to obtain mild hypothermia as therapeutic end point.

Surface cooling can be obtained with a cooling blanket and in combination with cold saline gastric lavage patients can be cooled to a core body temperature of 30-32 °C. Surface cooling can also be obtained using a cold air flow generator (e.g. PolarAir, Augustine Medical, Inc, Eden Prairie, Minnesota, USA). More direct approaches such as extracorporal circulation with cardiac pulmonary bypass device are also efficient means to decrease core body temperature. More recent core body cooling technologies include endovascular cooling by intra-arterially inserted closed loop catheters flushed with cold saline (e.g. Celcius Control Catheter System, Innercool Therapies, Inc, San Diego, USA) and disposable cooling patches applied directly to the cortical surface of brain (Seacoast Technologies, Inc, Portsmouth, NH, USA). However, all of the invasive cooling methods are associated with marked adverse risks for the patients and they are also expensive and inconvenient in use.

Profound hypothermia (body temperature <30 °C) can be fatal. Hirsch and Muller were the first to point out that mild hypothermia is an effective way to counteract and reduce CNS damage in the aftermath of cerebral ischaemia (Hirsch and Muller, 1962). They also emphasised the importance of accurate measurements of body temperature in predicting the protective effect of hypothermia.

The brain protective effects of mild hypothermia has been demonstrated in several rodent models of global cerebral ischaemia and head injury models (Ginsberg et al., 1992; Clifton et al., 1993; Maher and Hachinski, 1993; Miyazawa et al., 1993; Colbourne and Corbett, 1994; Miyazawa and Hossmann, 1994; Weisend and Feeney, 1994). Ongoing randomised clinical intervention trials are currently under way to prove the efficacy of mild hypothermia as a neuroprotective treatment of humans suffering focal cerebral ischaemia. In a retrospective study of patients suffering a cerebrovascular stroke those having the lowest body temperature at admission displayed best survival rates and developed significantly milder neurological deficits compared to stroke patients with elevated body temperature at admission (Olsen et al., 2003). SUMMARY OF THE INVENTION

The present invention lies in the surprising recognition that GHS receptor agonists have a hypothermic effect and are thus extremely useful for treating and limiting tissue damage that occurs as a result of e.g. hypoxia or ischemia.

The present invention thus relates to the use of a GHS receptor agonist for the manufacture of a hypothermic medicament.

Another aspect of the present invention relates to methods of treating ischemia in a mammal, wherein GHS receptor agonist is administered to said mammal before or after the onset of ischemia.

A third aspect of the invention relates to a method of lowering body temperature of a mammal that is undergoing organ transplant surgery.

A fourth aspect of the present invention relates to a method of treating ischemia by co-administration of GHS receptor agonist along with other drugs. Also, mechanically induced lowering of core body temperature might be carried out in combination with the treatments of the present invention.

A final aspect of the invention relates to lowering of body temperature in conditions where the body temperature is higher than 37 °C.

Definitions

"Fever" as used in the present context is characterized as a condition where the body core temperature is adjusted to a temperature above 37 °C. Fever is usually observed in connection with disease, in particular in connection with infection diseases. An elevated body temperature (>37 °C) is frequently observed in conditions such as infection diseases accompanied by fever. An elevated body temperature may also be observed when the body has been exposed to excessive heat and/or the capacity of the body to normalize the elevated temperature is impaired.

As used herein the term "treating" or "treatment" refers to the treatment of a disease in general or to a specific curative treatment as well as the alleviation of the symptoms of pain and suffering. Both prophylactic and curative methods of treating disease are covered by the word therapy.

As used herein the term "growth hormone secretagogue (GHS)-receptor agonist" refers to a chemically heterogeneous selection of compounds all eliciting elevated signalling activity of GHS-receptor activated intracellular pathways. Examples of these compounds include, but are not limited to: GHRP, GHRP-1 , GHRP-2, hexarelin, and ipamorelin and active analogues thereof. Another group of GHS receptor agonists include monoclonal antibodies to the GHS receptor, wherein said antibodies are selected on basis of their GHS receptor agonist activity. It is well known in the art how to produce such antibodies. Also, continuous screening programs will almost inevitably result in the identification of many other types of GHS receptor agonists in the future.

A GHS-receptor "agonist" is defined as a compound giving rise to increased activity of GHS-receptor mediated intracellular signalling events (inositol triphosphate accumulation and accumulation of intracellular Ca⁺⁺). GHS- receptor activation by a GHS-receptor agonist give rise to increased growth hormone release from primary-culture of anterior pituitary cells in a dose- dependent manner without stimulating the release of other pituitary hormones. In vivo, GHS-receptor agonists give rise to increased food intake, increased longitudinal growth, positive energy balance and increased body weight.

A GHS-receptor "antagonist" is defined as a compound that competes with a GHS-receptor agonist or inverse GHS-receptor agonist for binding to the GHS-receptor thereby blocking the action of the GHS-receptor agonist or inverse GHS-receptor agonist on the GHS-receptor. However, a GHS- receptor antagonist (also known as a neutral GHS-receptor agonist) has no effect on constitutive GHS-receptor activity.

"Hypoxia" is defined as lowered oxygen content in any given tissue giving rise to cell death and accompanying functional deficits.

"Ischaemia" is defined as tissue hypoxia due to impaired supply of oxygenated arterial blood to the tissue in question. The terms "hypoxia" and "ischemia" are thus sometimes used interchangeably.

"Arterial thromboembolism" is defined as a vascular lesion giving rise to impaired blood supply, be it partial or complete, to tissues lying distal to the thromboembolic lesion.

"Infarct" is defined as the irreversible tissue damage occurring as a result of partial or complete obliteration of blood supply to a specific tissue area.

"Reperfusion injury" is defined as secondary events (such as alterations in the perfusability and reactivity of the microcirculation), which may aggravate the original ischemic injury at discrete sites of the brain once perfusion is reestablished (be it forced or spontaneously) after arterial occlusion.

"Truncated ghrelin agonists" are defined as size reduced analogues of ghrelin, which are active at the GHS-receptor in a fashion indiscernible to that seen for other GHS-receptor agonists. Thus, truncated ghrelin agonists bind at the receptor and stimulate receptor activity.

"Parenteral administration" is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. For the purpose of this invention, peripheral parenteral routes include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration. Intravenous, intramuscular, and subcutaneous routes of administration of the compounds used in the present invention are preferred. Intravenous and subcutaneous routes of administration of the compounds used in the present invention are highly preferred. For parenteral administration, an active compound used in the present invention is preferably combined with distilled water at an appropriate pH.

"External body cooling devices": this term refers to any device that can assist in cooling down body temperature. Examples include but are not limited to: extracorporal circulation, cooling blankets, cold saline gastric lavage, endovascular cooling catheters, cooling device applied direct to the cortical surface of the brain.

Pharmacological agents, which inhibit the expression or action of proinflammatory cytokines include, but are not limited to, glucocorticoid receptor agonists, non-steroidal anti-inflammatory drugs (NSAID), aspirin, TNFα inhibitors/inactivators, antagonists of COX and COX2

Pharmacological agents, which inhibit cellular adhesion molecules include, but are not limited to, antibodies to vascular adhesion molecules Pharmacological agents, which act to reperfuse or oxygenate tissues include, but are not limited to, recombinant tissue plasminogen activator (rTPA), urokinase, and streptokinase.

Pharmacological agents which assist in temperature normalization include, but are not limited to, acetaminophen, aspirin, and barbiturates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns methods for using Growth hormone secretagogue receptor agonists to reduce neurological damage resulting from cerebral ischaemia or brain injury. In particular, the present invention relates to a novel medical use of Growth hormone secretagogue receptor agonists to induce hypothermia, which is beneficial for post-ischaemic neurological outcome. Such growth hormone secretagogue receptor agonists may also be used as drugs intended for treatment of other medical conditions for which hypothermia is considered an important element of therapy, such as certain types of operations, in particular organ transplant operations and especially in the case of heart transplants.

In a first aspect, the invention relates to the use of a GHS receptor agonist for the manufacture of a hypothermic medicament. The GHS receptor agonist is typically selected from the group consisting of: ghrelin, truncated peptide ghrelin agonists, GHRP, GHRP-1 , GHRP-2, hexarelin, and ipamorelin, monoclonal antibodies with GHS receptor agonist activity, L-692,429, MK- 0677, benzolactams with GHS receptor agonist activity, and spirodanes with GHS receptor activity.

Medicaments according to the invention are useful for treating e.g. ischemic conditions, reperfusion injury, and vascular occlusions such as occlusion of a cerebral vessel and a cardiac vessel. Such medicaments are likewise useful for treating conditions characterized by an elevated body temperature.

In a preferred embodiment, GHS receptor agonist medicaments according to the invention further comprise one or more drugs selected from the group consisting of: steroids, antiedema drugs, thrombolytics, thrombolytic clot solubilizing drugs, polyanions, and anticoagulants. A preferred thrombolytic clot solubilizer is tissue plasminogen activator.

Another aspect of the present invention relates to methods of treating ischemia in a mammal, wherein GHS receptor agonist is administered to said mammal less than 12 hours, preferably less than 10, 8, 6, 5, 4, 3, 2, or 1 hours after the onset of ischemia. Similarly, the GHS receptor agonist might be administered to said mammal before the onset of ischemia. Preferably, body temperature of the mammal receiving GHS receptor agonist is monitored continuously, in order to detect and take appropriate action in case the temperature drops to a critically low level.

In a preferred embodiment, GHS receptor agonist administration is accompanied by additional lowering of the body temperature of said mammal by means of external body cooling devices. GHS receptor agonist administration may take place before the surgery or during the surgery.

A third aspect of the invention relates to a method of lowering body temperature of a mammal that is undergoing organ transplant surgery. GHS receptor agonist is administered to said mammal prior to or during said surgery. A preferred organ transplant surgery procedure is a heart transplant.

A final aspect of the present invention relates to a method of treating ischemia by co-administration of GHS receptor agonist with a compound selected from the following group: nitric oxide synthase inhibitors, antioxidants, sodium channel blockers, potassium channel openers, glycine site agonists, NMDA 2-receptor subtype B antagonists, AMPA (2-amino-3- (methyl-3-hydroxyisoxazol-4-yl)propanoic acid))/kainate receptor antagonists, calcium channel blockers, GABA-A receptor agonists, anti-inflammatory agents, antagonists of cycloxygenase, PPARγ agonists, and drugs which assist in temperature normalization.

Because of their hypothermia inducing capabilities, compositions containing ghrelin, functional analogues hereof, as well as other GHS-receptor agonists (eg. MK-0677, GHRP-6, GHRP-1 , GHRP-2, ipamoraline, NN703) are appropriate means for treatment of neuronal tissue injury that may result from such factors as injury, toxicity, hypoxia or ischaemia. Target tissues include the brain, spinal cord and retina.

The present invention relates to the additional therapeutic benefits that may be gained by treating hypoxic brain injury, stroke, or traumatic brain injury with GHS-receptor agonist induced mild hypothermia alone or in combination with other types of hypothermic agents and other types of compounds. These measures include compounds that protect neurones from toxic insults, inhibit the inflammatory reaction after brain damage and/or promote cerebral reperfusion. In particular, combination therapy with compounds antagonising the neurotoxic actions of cerebral N-methyl-D-aspartate (NMDA) receptor activation should be approached, as activation of this receptor class is the principal cause of neuronal dysfunction and neuronal death that occur after CNS insults. However, other pathological aspects should also be addressed as it is likely that simultaneous inhibition of several pathological mechanisms may provide an unexpected synergistic benefit over and above that which may be achievable with GHS-receptor agonists alone.

Hypoxic damage of neurones occur during ischaemia and/or traumatic injury to the CNS because a number of toxic products are formed, which can further damage neurones injured by the primary insult. This secondary damage may involve brain cells, which were not initially affected by the primary insult. Such toxins include, but are not limited to: nitric oxide (NO); reactive oxygen and nitrogen intermediates, such as superoxide and peroxynitrite; lipid peroxides; TNFα; IL-1 and other interleukins; cytokines and chemokines; cycloxygenase and lipoxygenase derivatives and other fatty acid mediators such as leukotrienes and prostaglandins; and hydrogen ions.

Inhibiting the formation action or accelerating the removal of these toxins may protect brain cells from damage during hypoxic, ischaemic, or traumatic injury. The beneficial effects of inhibiting formation or accelerating the removal of these toxins may be additive or synergistic with benefits of decreasing core body and brain temperature with GHS-receptor agonist treatment. Examples of compounds that inhibit the formation or action of these toxins, or accelerate their removal include, but are not limited to, nitric oxide synthase inhibitors, antioxidants, sodium channel blockers, potassium channel openers, glycine site agonists, NMDA 2-receptor subtype B antagonists, AMPA (2-amino-3-(methyl-3-hydroxyisoxazol-4-yl)propanoic acid))/kainate receptor antagonists, calcium channel blockers, GABA-A receptor agonists, and anti-inflammatory agents (wide infra).

Enhanced cellular activation induced by central nervous system hypoxia leads to excessive cellular depolarisation and loss of ionic homeostasis, which can lead to loss of cellular integrity (i.e. maintenance of membrane potential and resistance). The resulting cellular death is known as necrosis. Before necrosis develops, cell-to-cell interaction as well as cellular interaction with the extracellular matrix. Such interaction with its immediate surroundings is crucial for cellular survival and proper cellular function.

Therapies that conserve cell-to-cell and cell-to-extracellular matrix interactions during hypoxic, ischaemic and/or traumatic brain injury are expected to reduce dysfunction and cell death. The beneficial effects of such cell-to-cell and cell-to-extracellular matrix conserving agent may be additive or synergistic with the benefits of GHS-receptor agonist induced hypothermia. Participating mechanisms to hypoxia induced disruption of cell- to-cell and cell-to-extracellular matrix interaction include, matrix metalloproteases such as MMP-1 through 13. Examples of compounds that inhibit these enzymes include, but are not limited to those referred to in the following patents and patent applications: US 5861510; EP 606046; EP 935963; WO 9834918; WO 9808825; WO 9808815; WO 9803516; WO 9833768.

Brain hypoxia, whether it originates from ischaemia or traumatic CNS injury leads to an inflammatory response mediated by various components of the immune system. Hypoxia, ischaemia or trauma-induced activation of the immune system within the CNS can exacerbate cellular dysfunction and death. Multiple mechanisms are involved in immune system mediated cell death exacerbating hypoxia, iscaemia or trauma-induced CNS injury. Immune competent cells resident to the brain such as microglia are activated following CNS injury. In addition, peripheral immune competent cells are recruited by local tissue damage and migrate into the lesioned brain site and its immediate surroundings. Invading immune competent cells include monocytes/macrophages, neutrophils and T lymphocytes.

The underlying mechanisms governing the recruitment of immune competent cells into the CNS overlap to a large extent with those responsible for recruitment of immune activation in peripheral sites. As a first step in the immune activation, cells and vessels of the injured CNS area commence production of signalling proteins activating immune competent cells in the blood stream, which eventually enter the area in and around the damaged brain tissue. These activated immune cells promote a major part of the deleterious events including release of toxins and disruption of cell-to-cell and cell-to-extracellular matrix interactions occurring in the aftermath of hypoxic, ischaemic or traumatic brain damage.

Therefore, it can be hypothesised that inhibition of recruitment of immune competent cells, their vascular adhesion, cellular activation and formation and release of proteases and toxins in response to CNS hypoxia, ischaemia or trauma will reduce the subsequent cellular death or dysfunctions related to these insults. The beneficial effects of inhibiting hypoxia, ischaemia or trauma-induced CNS injury immune cell recruitment may be additive or synergistic with the benefits of inducing hypothermia with GHS-receptor agonists.

Compounds that inhibit recruitment and activation of immune competent cells include, but are not limited to, antagonist of a variety of cytokine and chemokine receptors. Compounds that inhibit the adhesion of immune competent cells to the endothelial lining of vessels included, but are not limited to, antibodies against a wide variety of cellular adhesion molecules. Compounds that inhibit function of activated immune competent cells include, but are not limited to, antagonists of intracellular enzymes involved in transducing the activation signal into cellular function such as antagonists of cycloxygenase (COX)-1 and COX-2, several ser/thr and tyr kinases and intracellular proteases. Recruitment, adherence, and activation of CNS resident and peripheral immune competent cells can also be inhibited by activation of cellular signalling pathways opposing this activation. Compounds that activate such opposing immune neutralising pathways include, but are not limited to, PPARγ agonists (e.g. troglitazone, rosiglitazone, pioglitazone, balaglitazone)

The growth hormone secretagogues such as the growth hormone releasing peptides GHRP-6 and GHRP-1 are described in US 44111890, and the publications WO 8907110, WO 8907111. BHT920 as well as hexarelin and GHRP-2 are described in WO 9304081.

The family of ghrelin and related truncated ghrelin agonists are described in WO 01/92292.

Synthetic peptides acting as stimulators of growth hormone release via interaction with the GHS-receptor are described in WO 9413696, WO 9411012, and US 6482825

Ghrelin and GHS-receptor agonists can be administered to a subject, by which we refer to as a mammal including for example, a human, a rat, a mouse, a dog, a cat, or a farm animal. Reference to a subject does not necessarily indicate the presence of a disease or disorder. The term subject includes for example mammals being treated to help alleviate a disease or disorder, and mammals being treated prophylactically to retard or prevent the onset of a disease or disorder.

Ghrelin and GHS-receptor agonists can be used to achieve a beneficial hypothermic effect in a subject in treatment of fever, heat-induced hyperthermia, hypoxia induced tissue damage (as seen in cerebral ischaemia, CNS injury, intracerebral haemorrhage, stroke, myocardial infarction, or kidney infarction).

Similarly, grehlin and GHS-receptor agonists can also be used in connection with performing certain types of operations where it is desirable to lower the patients body temperature. Examples of such operations include organ transplants, in particular heart transplants.

Co-administration of drugs The formulation and mode of administration of GHS receptor agonist is preferably compatible with a concomitant administration of drugs conventionally or experimentally used to alleviate symptoms of ischaemic brain diseases. Such therapies include administration of thrombolytic agents such as Streptokinase and human recombinant tissue plasminogen activator (Alteplase, Reteplase) for which the regime has been widely publicised (Wardlaw et al., 2003).

Co-administration of ghrelin analogues with anticoagulants shall also be considered a therapeutically beneficial treatment to follow in patients suffering non-haemorrhagic brain ischaemia due to thromboembolic complications. Examples of anticoagulants include but are not limited to: warfarin, Phenprocoumaron, Clopidogrel, Heparin, Reviparin, Dalteparin, Tinzaparin, Enoxaparin, Antithrombin, Lepirudin, Dipyridamol, Abciximab, Aspirin, Trifiban, and Eptifibatid.

Also, GHS receptor agonists can be co-administrated along with one or more of the following drug types: NR2B subtype selective N-methyl-D-aspartate (NMDA) receptor agonist; sodium channel antagonist, nitric oxide synthase (NOS) inhibitor; glycine site antagonist; potassium channel opener; AMPA/kainate receptor antagonist; a calcium channel antagonist; a GABA-A receptor modulator (e.g. a GABA-A receptor agonist); or anti-inflammatory agent.

Pharmaceutical Compositions

In yet another aspect of the present invention, pharmaceutical compositions of the above GHS receptor agonists are provided. Such pharmaceutical compositions may be compositions for parenteral administration, compositions for oral administration, or compositions for inhalation (pulmonary, nasal or other forms of administrations). Guidance for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences 18th edition, Ed. Gennaro, Mack Publishing, 1990.

In addition, parenteral administration directly into the central nervous system is possible in situations with e.g. traumatic brain damage and open brain surgery. Peripheral, parenteral administration is the preferred route of administration.

Of the non-parenteral routes, the lower respiratory route is preferred for administration of peptides used in the instant invention. Various formulations of peptide compounds for administration to the lower respiratory tract are disclosed in US 5284656 and US 5364838. WO 9619197 discloses aerosol formulations of various peptides suitable for enhancing lower respiratory tract absorption of the compounds used in the instant invention. The oral route of administration is preferred for compounds used in the instant invention.

Pharmaceutical compositions according to the present invention comprising effective amounts of active compounds in the form of peptide, peptide derivatives or non-peptides together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, are comprehended by the invention.

Such compositions may comprise various buffers (e.g. Tris-HCI, acetate, phosphate), pH values and ionic strengths; additives such as detergents and solubilizing agents (e.g. Tween 80, Polysorbate 80), anti-oxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. Thimersol, benzyl alcohol) and bulking substances (e.g. lactose, mannitol, starch, gelatin); incorporation of the material into particulate preparations of polymeric compounds such as polyacetic acid, polyglycolic acid, hyaluronic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. The compositions may be prepared in solutions, tablets, dried powder, such as lyophilised form, etc.

The ghrelin agonist compounds (ghrelin, truncated ghrelin analogues, GHRP- 6; GHRP-1 ; GHRP-2; MK-0677; hexarelin, etc.) may be prepared in the form of pharmaceutically acceptable salts, especially acid-addition salts, including salts of organic acid such as formic acid, fumaric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, and the like. Suitable inorganic acid-addition salts include salts of hydrochloric, hydrobromic, sulphuric and phosphoric acids and the like. Further examples of pharmaceutically acceptable inorganic and organic acid addition salts include the pharmaceutically acceptable salts listed in the Journal of Pharmaceutical Science 1997, 66:2, and which are well known in the art.

Also included in this invention are pharmaceutically acceptable salts and hydrates, which the GHS receptor agonists are able to form.

The acid addition salts may be obtained as the direct products of compound synthesis. In the alternative, the free base may be dissolved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.

The ghrelin agonists intended for use in this patent may form solvates with standard low molecular weight solvents using methods well known in the art.

The ghrelin agonists may be administered in pharmaceutically acceptable acid addition salt form. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms. A pharmaceutical composition for use in accordance with the present invention comprises one or more compounds included in the incomplete list of GHS receptor agonists as active ingredient(s) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent.

Controlled release preparations may be achieved by the use of polymers to complex or absorb the active compound used in the present invention. Extended duration may be obtained by selecting appropriate macromolecules, for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulphate, and by selecting the concentration of macromolecules, as well as the methods of incorporation, in order to prolong release of the active compounds. Another possible method to extend the duration of action by controlled release preparations is to incorporate an active compound used in the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.

Alternatively, instead of incorporating a compound into these polymeric particles, it is possible to entrap a compound used in the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerisation, for example, hydroxymethylcellulose or gelatin- microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions. Such methods are well known in the art and disclosed, e.g. in Remington's Pharmaceutical Sciences 18th edition, Ed. Gennaro, Mack Publishing, 1990.

The pharmaceutical compositions can be sterilised and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring agents and the like, which do not deleteriously react with the active components.

EXAMPLES

Example 1

Evaluation of hypothermic potential of GHS receptor agonists

Male Sprague Dawley rats (250-350 g) are housed individually under standard laboratory conditions with ad libitum access to food and water. Seven days prior to onset of experimentation, rats are equipped with intra- arterial catheters and a temperature telemetry device (Minimitter, Bend, OR, USA) giving online information about core body temperature. A subset of rats are also equipped with an intracerebroventricular cannula placed in the right lateral cerebral ventricle. Data acquisition is mastered electronically using the software provided by MiniMitter Inc.

The hypothermic effects of ghrelin, GHRP-6, and MK-0677 are evaluated by administering the compounds intra-arterially in conscious rats. Applied systemic doses are equivalent to those used to induce growth hormone secretion and to increase feeding in rats, which for native ghrelin is in the range of 0,1 ; 0,5; 1 ; 2; 5; 10; 15; 20; 25; 30; 35; 40; 45; 50; 60; 70; 80; 90; or 100 mg/kg. Body temperature is followed continuously with a datalogger or with other means of measuring temperature.

Intracerebroventricular administration af ghrelin, GHRP-6, and MK-0677 is tested in a subset of rats using doses in the range of: 0,1 ; 0,5; 1 ; 2; 5; 10; 15; 20; 25; 30; 35; 40; 45; or 50 microgram/rat. Body temperature is followed continuously up to 48 hours after administration of a single dose of the active GHS-receptor agonist. Typically, body temperature is measured with a datalogger that records the temperature with an interval of 1 sec, 5 sec, 10 sec, 1 min or 10 min.

Subsequently continuous administration of the pharmacologically active GHS-receptor agonist is tested by attaching the rats to a continuous delivery system (osmotic minipumps, Alzet).

The effect of various ambient temperatures on GHS-receptor agonist induced core body hypothermia are tested. In a subsequent series of experiment, ghrelin, GHRP-6, and MK-0677 is administered to rats housed at various ambient temperatures ranging from 0, 5, 10, 15, 20, 25, and 30 °C.

Subsequently, appropriate doses of GHS receptor agonist can be chosen based on desired body temperature and/or desired ambient temperature.

Example 2

Administration of GHS receptor agonists in ischemic rats

The therapeutic benefits of hyperthermia as protection against neurological damage due to focal CNS ischaemia is obtained with mild hypothermia (30- 35 °C) (Miyazawa et al., 2003). Therefore, hypothermia inducing efficacy of ghrelin and GHS-receptor agonist will be dose titrated to that temperature range.

To elucidate the therapeutic effect of ghrelin and GHS-receptor agonist- induced hypothermia on ischaemia-induced cerebral tissue damage, an arterial occlusion model is used. A modified version of Tamura's method is used to induce transient cerebral ischaemia (Tamura et al., 1981a, b). Male Sprague Dawley rats (250-350 g) are equipped with indwelling arterial femoral catheters and intracerebroventricular cannula seven days before onset of experimentation. On the day of the experiment, the rats are mildly anaesthetised and a small- gauged nylon filament is inserted in the right carotid artery and pushed into an occluding position in the right middle cerebral artery for 30-180 minutes. Rats randomised to the control group receive GHS receptor agonist with or without concomitant exposure to lowered ambient temperature as subsequent post-ischaemic treatment.

The rats, randomised to the pharmacotherapeutic arms of the experiment, receive single injections of hypothermia inducing doses of a ghrelin or GHS- receptor agonists composition up to 24 hours after the ischaemic episode (at 30 minutes, 1 , 2, 6, 12, 24 hours), with or without altered ambient room temperature. Rats in the control group receive either saline water or a composition similar to the GHS receptor agonist composition but of course without GHS receptor agonist.

Example 3

Histologic examination of ischemic rats

Animals are perfused with 4% paraformaldehyde via the ascending aorta. Fixed brains are removed from the skull and cryoprotected in 20% sucrose for at least 24 hours, before 12 micron thick serial coronal sections are cut on a freezing microtome. Mounted sections are counterstained in haematoxylin and eosin and examined on a standard microscope to which a digital camera is fitted.

The tissue damage resulting from transient occlusion of the middle cerebral artery is assessed by histological examination of infarct volume in the right hemisphere of rats killed at various time points after the ischaemic episode

(24 hours, 3, 7 and 21 days). Infarct and penumbra volumes are measured using stereological techniques. Pharmacotherapeutic efficacy is determined as the percentage reduction of infarct volume in ghrelin or GHS-receptor agonist treated rats compared to vehicle injected rats (control).

Example 4

Co-administration of GHS and other putative neuroprotective agents

Once the optimal neuroprotective dose of ghrelin and GHS-receptor agonists have been identified according to the guidance given in Example 1 , these compounds shall preferably be co-administered with other putative neuroprotective agents such as NMDA receptor antagonists of which MK-801 represent an excellent model compound. NMDA receptor antagonist can reduce neuron loss in animal models of cerebral ischaemia and head trauma.

Examples of neuroprotective agents include but are not limited to: NR2B subtype selective N-methyl-D-aspartate (NMDA) receptor agonist; sodium channel antagonist; nitric oxide synthase (NOS) inhibitor; glycine site antagonist; potassium channel opener; AMPA/kainate receptor antagonist; a calcium channel antagonist; GABA-A receptor modulator (e.g. a GABA-A receptor agonist); and anti-inflammatory agents. Specific examples of these groups of compounds are found in: The RBI handbook of receptor classification, 1994 Kebabian, J.W. and Neumeyer, J.L. (eds) ISBN: 0- 9640548-0-9.

The rats are subsequently examined as described in Example 3. References

Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, Suda M, Koh T, Natsui K, Toyooka S, Shirakami G, Usui T, Shimatsu A, Doi K, Hosoda H, Kojima M, Kangawa K, Nakao K (2001 ) Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. 86:4753-4758.

Bowers CY, Momany FA, Reynolds GA, Hong A (1984) On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114:1537-1545.

Clifton GL, Allen S, Barrodale P, Plenger P, Berry J, Koch S, Fletcher J, Hayes RL, Choi SC (1993) A phase II study of moderate hypothermia in severe brain injury. J Neurotrauma 10:263-271 ; discussion 273.

Colbourne F, Corbett D (1994) Delayed and prolonged post-ischemic hypothermia is neuroprotective in the gerbil. Brain Res 654:265-272.

Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS (2001 ) A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50:1714-1719.

Ginsberg MD, Sternau LL, Globus MY, Dietrich WD, Busto R (1992) Therapeutic modulation of brain temperature: relevance to ischemic brain injury. Cerebrovasc Brain Metab Rev 4:189-225.

Hirsch H, Muller HA (1962) Funktionelle und histologische Veran derungen des Kaninchengehims nach kompletter Gehirnischamia. Pflugers Arch 275:227-291. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum Cl, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Van der Ploeg LH, et al. (1996) A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974-977.

Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I (2000) Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression. Endocrinology 141 :4797-4800.

Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-660.

Maher J, Hachinski V (1993) Hypothermia as a potential treatment for cerebral ischemia. Cerebrovasc Brain Metab Rev 5:277-300.

Miyazawa T, Hossmann KA (1994) [Temperature effect on ischemic brain injury]. No To Shinkei 46:29-37.

Miyazawa T, Bonnekoh P, Hossmann KA (1993) Temperature effect on immunostaining of microtubule-associated protein 2 and synaptophysin after 30 minutes of forebrain ischemia in rat. Acta Neuropathol (Berl) 85:526-532.

Miyazawa T, Tamura A, Fukui S, Hossmann KA (2003) Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies. Neural Res 25:457-464.

Olsen TS, Weber UJ, Kammersgaard LP (2003) Therapeutic hypothermia for acute stroke. Lancet Neural 2:410-416. Tamura A, Graham Dl, McCulloch J, Teasdale GM (1981a) Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1 :61-69.

Tamura A, Graham Dl, McCulloch J, Teasdale GM (1981 b) Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1 :53-60.

Tang-Christensen M, Vrang N, Orthmann S, Larsen PJ, Horvarth T, Tschδp M (2003) Ghrelin induces long lasting changes in food intake and locomotor behaviour. Endocrine Society Abstracts: P3-94 497.

Tschδp M, Smiley DL, Heiman ML (2000) Ghrelin induces adiposity in rodents. Nature 407:908-913.

Wardlaw JM, Sandercock PA, Berge E (2003) Thrombolytic therapy with recombinant tissue plasminogen activator for acute ischemic stroke: where do we go from here? A cumulative meta-analysis. Stroke 34:1437-1442.

Weisend MP, Feeney DM (1994) The relationship between traumatic brain injury-induced changes in brain temperature and behavioral and anatomical outcome. J Neurosurg 80:120-132.

Claims

1. Use of a GHS receptor agonist for the manufacture of a hypothermic medicament.

2. Use of a GHS receptor agonist according to claim 1 , wherein the GHS receptor agonist is selected from the group consisting of: ghrelin, truncated peptide ghrelin agonists, GHRP, GHRP-1 , GHRP-2, hexarelin, and ipamorelin, monoclonal antibodies with GHS receptor agonist activity, L- 692,429, MK-0677, benzolactams with GHS receptor agonist activity, and spirodanes with GHS receptor agonist activity.

3. Use of GHS receptor agonists according to claim 1 or 2 for preparation of a medicament for treatment of ischemia.

4. Use of GHS receptor agonists according to claim 1 or 2 for preparation of a medicament for treating reperfusion injury.

5. Use of GHS receptor agonists according to claim 1 or 2 for preparation of a medicament for treatment of vascular occlusion.

6. Use of GHS receptor agonists according to claim 5, wherein the vascular occlusion is selected from the group consisting of: a cerebral vessel and a cardiac vessel.

7. Use of GHS receptor agonists according to claim 1 or 2 for preparation of a medicament for lowering an elevated body temperature.

8. Use of GHS receptor agonist according to claim 1 or 2, said medicament further comprises one or more drugs selected from the group consisting of: steroids, antiedema drugs, thrombolytics, thrombolytic clot solubilizing drugs, polyanions, and anticoagulants.

9. Use of GHS receptor agonist according to claim 8, wherein the thrombolytic clot solubilizing drug comprises tissue plasminogen activator.

10. A method for treating ischemia in a mammal, wherein GHS receptor agonist is administered to said mammal less than 12 hours after the onset of ischemia.

11. A method according to claim 10, wherein GHS receptor agonist is administered to said mammal less than 6 hours after the onset of ischemia.

12. A method of prevention of ischemia induced tissue damage in a mammal, wherein GHS receptor agonist is administered to said mammal before the onset of ischemia.

13. A method according claim 10, wherein GHS receptor agonist is administered to said mammal and wherein the body temperature of said mammal is also lowered by means of external body cooling devices.

14. A method of lowering body temperature of a mammal that is undergoing organ transplant surgery, wherein GHS receptor agonist is administered to said mammal prior to or during said surgery.

15. A method according to claim 14 wherein the organ transplant is a heart transplant.

16. A method according to claim 10 wherein GHS receptor agonist is co- administered with one or more drugs selected from the group consisting of: steroids, antiedema drugs, thrombolytics, thrombolytic clot solubilizing drugs, polyanions, and anticoagulants.

17. A method according to claim 16, wherein the thrombolytic clot solubilizing drug comprises tissue plasminogen activator.

18. A method according to claim 10, wherein a compound selected from the following group is co-administered together with GHS receptor agonist: nitric oxide synthase inhibitors, antioxidants, sodium channel blockers, potassium channel openers, glycine site agonists, NMDA 2-receptor subtype B antagonists, AMPA (2-amino-3-(methyl-3-hydroxyisoxazol-4-yl)propanoic acid))/kainate receptor antagonists, calcium channel blockers, GABA-A receptor agonists, anti-inflammatory agents, antagonists of cycloxygenase, PPARγ agonists, and drugs which assist in temperature normalization.