CASPASE- 2 INHIBITORS

FIELD OF THE INVENTION

The invention is in the field of medicinal biology and chemistry and relates to novel compounds, pharmaceutical compositions and use thereof, which inhibit pro-apoptotic caspase-2 to prevent and/or treat diseases and injuries where caspase-2 activity is implicated.

BACKGROUND OF THE INVENTION

Neuronal cell death occurs during embryogenesis to remove excess of neurons to ensure appropriate pre- and post-synaptic connections and to allow formation of a functional adult brain. Besides post-mitotic death related to normal ageing, environmental or genetic mutational factors may induce neuronal death in the adult human during acute injuries (for instance, hypoxia- ischemia, stroke, spinal cord injury, trauma) or chronic neurodegenerative diseases. Cell death associated with these disorders may occur by three distinct mechanisms, exhibiting morphological and biochemical features of necrosis, autophagy or apoptosis. Both physiological and pathological neuronal deaths are often associated with defective apoptosis regulation and signalling pathways that lead to this active cell suicide mechanism may be divided in cysteinyl aspartate-specific protease (caspase) -dependent versus caspase- independent pathways in mammalian cells.

Neuronal apoptosis is an active cell suicide mechanism that can be divided into sequential phases, including initiation, decision, execution and degradation. This cascade of events is driven by the activation of specific machinery that involves both the activation of cysteine-dependent aspartate- specific proteases (caspases) and the mitochondrion which may act as a decisive (or amplifier) regulatory organelle. Indeed, mitochondrial alterations include loss of mitochondrial inner membrane electrochemical gradient and release of apoptogenic factors such as cytochrome c Smac/Diablo and Apoptosis Inducing Factor. Once released from mitochondria, these effectors trigger caspase-dependent and/or caspase- independent cytoplasmic and nuclear dismantling. Hence, mitochondrial factors combined with caspases contribute to the degradation phase of apoptosis, resulting in cell shrinkage, nuclear condensation, emission of apoptotic bodies and appearance of "eat- me" signals such as phosphatidyl-serines translocation to the outer leaflet of the plasma membrane before phagocytosis.

So far, several caspases have been identified in humans. In particular, there are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases (e.g., caspase-2, caspase-8, caspase-9, caspase-10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g., caspase-2, caspase-6, caspase -7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process.

Several caspase inhibitors have been disclosed in the art. Compounds able to inhibit caspase-2 activity have been reported in WO 2004/103389, WO 2005/105829 and WO 2006/056487.

In particular, the pentapeptide 5-(2,6-Difluoro-phenoxy)-3(R,S)-{2(S)- [2(S)-(3-methoxycarbonyl-2(S)-{3-methyl-2(S)-[(quinoline-2-c arbonyl)- amino]butyrylamino } -propionylamino)-3 -methyl-butyrylamino] - propionylamino}-4-oxo-pentanoic acid methyl ester is a selective caspase-2 inhibitor, disclosed in WO 2005/105829, currently under development for the treatment of neonatal brain injury.

The compound has the formula reported below

It has also been quoted in the art as Quinoline-2-carbonyl-(¾j-Val-(¾ ⁾ - Asp(OMe)-(¾j-Val-(¾j-Ala-( ?,S Asp(OMe)-CH ₂ OC ₆ H ₃ (2,6-F ₂ ) or TRP 601.

However, there is still a need for caspase-2 inhibitors with improved properties in particular in terms of pharmacokinetics and ADME (adsorpion, distribution, metabolism and excretion) properties.

In particular, it would be highly advantageous to provide more efficacious caspase-2 inhibitors for use for the treatment of neonatal brain ischemia which is a major cause of neurodevelopmental damage in pre-terms infants and remains a major unmet medical need.

SUMMARY OF THE INVENTION

The present invention is directed to a caspase-2 inhibitor of general formula (I)

(I)

wherein: n is 0 or 1;

A represents N, N(H),O, S, or N-O (i.e. N-oxide)

... adjacent to the group A can be a single bond when A is O or S or a single or double bond when A is N(H) or N, respectively;

X, the same or different from each other, is independently selected from halogen atoms, preferably fluorine;

Xi represents H or a halogen atom, and

R _l5 the same or different from each other, is independently selected from H and a linear or branched (C1-C4) alkyl group, preferably from H, methyl and ethyl.

In a second aspect, the invention provides pharmaceutical compositions comprising a compound of general formula (I) as active ingredient, and, optionally, one or more pharmaceutically acceptable excipients.

In a third aspect, the invention provides a compound of general formula (I) for use as a medicament.

In a fourth aspect the invention provides a compound of general formula (I) for use for the prevention or treatment of a disease where caspase-2 activity is implicated, such as neonatal brain injury.

In a fifth aspect the invention provides the use of a compound of general formula (I) in the preparation of a medicament for the prevention or treatment of a disease where caspase-2 activity is implicated, such as neonatal brain injury.

In a sixth aspect the invention provides a method for the prophylaxis or treatment of a disease where caspase-2 activity is implicated, such as neonatal brain injury, said method comprising the administration of a therapeutically effective amount of a compound of general formula (I).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as it is commonly understood by one of skill in the art to which this subject matter belongs.

The term "halogen atoms" includes fluorine, chlorine, bromine and iodine.

The expression "linear or branched alkyl" refers to straight and branched chained alkyl groups wherein the number of constituent carbon atoms is in the range 1 to 4. Particular examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl and t-butyl.

For "dosage", it is meant the unitary amount of drug to be administered.

"An effective amount of a compound for treating a particular disease" is an amount that is sufficient to ameliorate, or in some manner reduce, the symptoms associated with the disease.

The term "high level of chemical purity" refers to a compound wherein the total amount of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC), is less than 5%, advantageously less than 2.5%, preferably less than 1.0, more preferably less than 0.5% w/w.

The term "prevention" means an approach for reducing the risk of onset of a disease.

The term "treatment" means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, decrease in the extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term can also mean prolonging survival as compared to expected survival if not receiving treatment. BRIEF DESCRIPTION OF THE DRAWING

Figure - In vitro activity of TRP 701 versus other compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a caspase-2 inhibitor of general formula (I)

When A is N and is a double bond, the invention also encompasses the corresponding N-oxides on the quinoline ring.

In a preferred embodiment of the invention, A is N and ^ is a double bond so forming a quinoline ring.

More preferably, the caspase-2 inhibitor is a compound of general formula (II) in which n = 1

(II)

wherein each of X, i and Ri have the meanings defined above.

Moreover, the invention encompasses pharmaceutically acceptable salts and/or solvates thereof.

Pharmaceutically acceptable salts include those in which acidic functions, when present, are reacted with an appropriate base to form, e. g. sodium, potassium, calcium, magnesium, ammonium and choline salts.

When A is N and . . . is a double bond, pharmaceutically acceptable salts also include those obtained by reacting the nitrogen of the quinoline ring, functioning as a base, with a strong inorganic or organic acid to form, for example, salts of hydrochloric acid and trifluoromethanesulfonic acid.

The compounds of general formula (I) are derivatives of the amino acid sequence Valinyl-Aspartyl-Vanilyl-Alanyl-Aspartyl and contain asymmetric centers having either the absolute configuration R corresponding to L or the S configuration corresponding to the D series.

The invention includes the optical stereoisomers and mixtures thereof.

Preferably, the asymmetric centers of the amino acid residues Valinyl and Alanyl have the absolute configuration (S) while that of the Aspartyl residue could be (S) or (R,S).

A preferred group of compounds of general formula (II) is that wherein Ri is H, each of X is F, and i is H.

Accordingly, in one of the preferred embodiments of the invention, the caspase-2 inhibitor is the compound 2-quinolinylcarbonyl-(S)-Valinyl-(S)- Aspartyl-(S)-Valinyl-(S)-Alanyl-(R,S)-Aspartyl 2,3,5,6-tetrafluorophenyl ester.

Another preferred group of compounds of general formula (II) is that wherein Ri is methyl, each of X is F, and Xi is H.

Accordingly, in one of the preferred embodiments of the invention, the caspase-2 inhibitor is the compound 5-(2,3,5,6-tetrafluoro-phenoxy)-3(R,S)- {2(S)-[2(S)-(3-methoxycarbonyl-2(S)-{3-methyl-2(S)-[(quinoli ne-2- carbonyl)-amino]-butyrylamino}-propionylamino)-3-methyl-buty rylamino]- propionylamino}-4-oxo-pentanoic acid methyl ester, also quoted hereinafter with the internal code TRP 701.

Said compound can also be called 2-quinolinylcarbonyl-(S)-Valinyl-

(S)-Aspartyl (methyl ester)-(S)-Valinyl-(S)-Alanyl-(R,S)-Aspartyl (methyl ester) 2,3,5,6-tetrafluorophenyl ester or quinoline-2-carbonyl-(¾ ⁾ -Val-(¾ ⁾ - Asp(OMe)-(^-Val-(¾j-Ala-( ?,Sj-Asp(OMe)-CH ₂ OC ₆ H(2,3,5,6-F ₄ ). Another preferred group of compounds of general formula (II) is that wherein is ethyl, each of X is F, and i is H.

Accordingly, in one of the preferred embodiments of the invention, the caspase-2 inhibitor is the compound 2-quinolinylcarbonyl-(S)-Valinyl-(S)- Aspartyl (ethyl ester)-(S)-Valinyl-(S)-Alanyl-(R,S)-Aspartyl (ethyl ester) 2,3,5,6-tetrafluorophenyl ester.

Unless otherwise indicated, the present invention includes all of those compounds wherein the ring bearing group A is substituted, in any suitable free position, with the residual portion of the molecule trough the amido linkage as represented in formula (I).

The compounds of the invention, being very lipophilic, may readily cross the blood-brain barrier and diffuse into the brain. Moreover, the addition of more halogen atoms, in particular fluorine, on the terminal phenoxy group, make the resulting compounds endowed with a more rapid onset of action in comparison to compounds with less halogen atoms such as TRP 601. Without being limited by the theory, it was afterwards hypothesized that the supplementary halogen atoms on the aromatic cycle may even enhance the electron delocalisation of the relevant compound which, in turn, makes it more prone to rapid interaction with the active thiol (SH) group of the active site in the pocket of caspase-2.

The compounds of general formula (I) may be prepared by known methods. Some of the processes which can be used are reported in Schemes 1, 2 and 3, wherein A, X, Xi and Ri have the above reported meanings.

The conditions and the reagents to be utilised are disclosed in WO 2005/105829, whose teaching regarding the synthesis is incorporated herein by reference.

However said synthetic pathways should not be viewed as limiting the scope of the synthetic methods available for the preparation of the compounds of the invention.

The schemes refer to the preparation of the compounds of general formula (I) wherein n = 1 and _^ is a double bond,_but they can be applied as well for the preparation of the compounds wherein n = 0 and _^ is a single bond, using the suitable intermediates accordingly.

The starting materials and the reagents are known or, if not commercially available per se, can be readily prepared according to known methods.

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Advantageously, the compounds of general formula (I) are utilised with a high level of chemical purity for the preparation of pharmaceutical compositions, for administration in any convenient way.

Suitable dosages of the compounds of the invention may easily be established by the attending physician and will depend on the type of patient, age, nature of the disease and on the mode of drug delivery. Dosages of the order of about 0.01 mg to about 5 mg per kilogram of body weight may be useful, preferably of about 0.1 and 3 mg/kg, more preferably of about 0.5 to about 2 mg/kg.

Pharmaceutical compositions may be prepared by admixing a compound of general formula (I) in a suitable dosage and one or more pharmaceutically acceptable excipients. Depending on the nature of the medical disease or condition to be treated and the type of patient, the pharmaceutical compositions may be formulated to be delivered by any suitable route, including oral, intravenous, parenteral, inhalation, intranasal, topical, subcutaneous, intramuscular, rectal, intraperitoneal, intracerebroventricular, intrahippocampal or other intracerebral delivery, intracerebral implantation of instrumentation for mechanical delivery such as of Gelfoam ^® impregnated with a compound of the invention. Suitable dosage forms include all those known to the skilled person, such as tablets, capsules, powders, sustained release formulations, ointments, gels, creams, suppositories, eye drops, transdermal patches, syrups, solutions, suspensions, aerosols, solutions for nebulizers, nasal sprays etc.

Suitable known excipients, include carriers, diluents, wetting agents, emulsifying agents, binders, coatings, fillers, glidants, lubricants, disintegrants, preservatives, surfactants, pH buffering substances and the like. Examples of excipients are provided in the Handbook of Pharmaceutical Excipients, 5 ^th ed. (2006), Ed. Rowe et al., Pharmaceutical Press. In preferred embodiments the compositions are formulated for delivery by intravenous, subcutaneous, intraperitoneal or intracerebral routes.

In some embodiments of the invention, the compositions may be formulated in form of liposomal solutions or microsuspensions. In other embodiments may be formulated in form of aqueous solutions, optionally pH- buffered.

The solvent wherein the compound of general formula (I) should be dissolved may consists of only water or of a mixture of water and a co- solvent, miscible with water, selected from the group consisting of ethanol, propylene glycol, polyethylene glycol, polypropylene glycol, and glycerol or mixtures thereof.

In a preferred embodiment, the composition is formulated in form of aqueous solution, optionally pH-buffered comprising a compound of general formula (II) wherein Ri is H, each of X is F, and Xi is H, or a salt thereof at a dosage comprised between 0.02 and 0.25 mg/kg, more preferably between 0.05 and 0.2 mg/kg.

The compositions may also comprise, if required, one or more other therapeutic agents, preferably those currently used in the treatment of neonatal diseases.

The compounds of general formula (I) may be used for prophylactic purposes or for the treatment of a wide range of diseases involving caspase-2 activity.

For example, said compounds are advantageously useful for preventing, reducing and treating pathologies characterized by cell death, particularly in hypoxic -ischemic (H-I) brain damages and stroke-like situations brain injuries: for example, global or focal, transient or permanent, adult or neonatal H-I (ischemia with or without hypoxia/hypoglycaemia) with origin at cerebral or heart level, with or without reperfusion, or MCAO (Middle Cerebral Artery Occlusion).

Preferably, the compounds of the invention are utilized for the treatment of neonatal brain injury that encompasses Perinatal Arterial Stroke (PAS), Perinatal Hypoxic-Ischaemic Encephalopathy (HIE), and Periventricular Leucomalacia (white matter injury) in premature babies, more preferably for the treatment of Perinatal Hypoxic-Ischaemic Encephalopathy.

The compounds of the invention may also be useful for:

preventing and/or treating apoptosis during chronic degenerative diseases e.g. neurodegenerative disease including Alzheimer's disease, Huntington's' disease, Parkinson's' disease, Multiple sclerosis, amyotrophic lateral sclerosis, spinobulbar atrophy, prion disease, dementia, or

preventing and/or treating retinal pericyte apoptosis, retinal neurons apoptosis glaucoma, retinal damages resulting from local ischemia, diabetic retinopathy, or - preventing and/or treating epilepsy, or

- preventing and/or treating apoptosis during spinal cord injury, or to prevent and/or treat apoptosis resulting from traumatic brain injury, retinal ischemia or

preventing and/or treating apoptosis during pathological situations of focal cerebral ischemia or - providing cerebroprotective effect, or - preventing and/or treating cytotoxic T cell and natural killer cell- mediated apoptosis associated with autoimmune disease and transplant rejection, or

preventing and/or treating cell death of cardiac cells including heart failure, cardiomyopathy, viral infection or bacterial infection of heart, myocardial ischemia, myocardial infarct, and myocardial ischemia, coronary artery by-pass graft, or

preventing and/or treating mitochondrial drug toxicity e. g. as a result of chemotherapy or HIV therapy, or preventing and/or treating cell death during viral infection or bacterial infection, or

preventing and/or treating inflammation or inflammatory diseases, inflammatory bowel disease; sepsis and septic shock, or

- preventing cell death from follicle to ovocyte stages, from ovocyte to mature egg stages and sperm (for example, methods of freezing and transplanting ovarian tissue, artificial fecundation), or

preserving fertility in women and men after chemotherapy, or preserving fertility in females and males animals, or to prevent and/or treat, macular degenerescence and glaucoma, or

preventing and/or treat acute hepatitis, chronic active hepatitis, hepatitis-B, and hepatitis-C, or

preventing and/or treating hair loss, and said hair loss due-to male-pattern baldness, radiation, chemotherapy or emotional stress, or

- treating or ameliorating skin damage (due to exposure to high level of radiation, heat, burns, chemicals, sun, and autoimmune diseases), or preventing cell death of bone marrow cells in myelodysplasia syndromes (MDS), or - preventing and/or treating pancreatitis, or

preventing and/or treating respiratory syndrome, or

- preventing and/or treating osteoarthritis, rheumatoid arthritis, psoriasis, glomerulonephritis, atherosclerosis, and graft versus host disease, or preventing and/or treating disease states associated with an increase of apoptosis.

The invention is further illustrated by the following Examples.

EXAMPLES

Example 1 - In vitro activity

The in vitro caspase-2 inhibition activity of 2-quinolinylcarbonyl-(S)- Valinyl-(S)-Aspartyl (methyl ester)-(S)-Valinyl-(S)-Alanyl-(R,S)-Aspartyl (methyl ester) 2,3,5,6-tetrafluorophenyl ester (TRP 701) in comparison to other compounds was evaluated according to the following protocol.

Human recombinant caspase-2 (25-50U;, BIOMOL, Plymouth, Pennsylvania, USA) were pre-incubated 30 min with inhibitors (0.001- 2 μΜ) in final 100 μΐ final assay buffer (50 mM HEPES, pH 7.4, lOOmM NaCI, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol) and then mixed with 200 μΜ of the fluorogenic caspase substrates (BIOMOL) Ac-VDVAD-AMC. An IC ₅₀ value corresponding to the concentration inhibiting 50% of caspase activity was determined from the dose-response sigmoid curve using steady- state fluorescence approach. The cleavage of AMC-based substrate by human recombinant caspase-2 was measured after 2h at 37°C on a fluorescence microplate reader by monitoring emission at 510 nm upon excitation at 405 nm.

The compounds used for comparative purposes were:

TRP 601 (Quinoline-2-carbonyl-(¾ -Val-(^-Asp(OMe)-(^-Val-(^-

Ala-(7?,S)-Asp(OMe)-CH ₂ OC ₆ H ₃ (2,6-F ₂ )) identified with the code MZ77027; rac-TRP 601, i.e. racemic TRP 601 identified with the code BS 03140; M3 which is the compound 2-quinolinylcarbonyl-S-Valine, an inactive compound structurally related to TRP601 ; and

TRP 600 (Quinoline-2-carbonyl-(¾ -Val-(^-Asp(OMe)-(^-Val-(^-

Ma-(R,S)-Ma ((R,Sj-CH ₂ OC ₆ H ₃ (2,6-F ₂ )), another inactive compound structurally related to TRP601.

The dose response sigmoid curve is reported in Figure. Curves are mean of three independent runs.

TRP 701 turned out to be a selective caspase-2 inhibitor. It also turned out to be able of inhibiting caspase-2 activity with an IC ₅₀ similar to that of TRP 601, i.e. of around 60-90 nM. Example 2 - Impact on in vivo caspase-2 kinetics and brain tissue preservation upon pathological condition

The activation kinetics of caspase-2 as well as efficacy based on histology parameters is evaluated though a convenient animal model, i.e., where caspase-2 is early active. Such model allows to compare caspase-2 inhibitory potency and protective effect of 2-quinolinylcarbonyl-(S)-Valinyl- (S)-Aspartyl (methyl ester)-(S)-Valinyl-(S)-Alanyl-(R,S)-Aspartyl (methyl ester) 2,3,4,5,6-pentafluorophenyl ester (TRP 701) versus TRP601.

This model of neonatal stroke (cerebral ischemia with reperfusion) gives rise to an ipsilateral penumbra progression and leads to cortical injury (infarction) at 48 h. This may in turn develop a cavity. This experimental model exhibits a real reperfusion step as noted in clinical syndrome and is relevant in term of brain maturation and blood-brain-barrier with the term human newborn. The pattern of lesion is very similar to that found in full term babies at birth or occurring in the following days/months after suffering at birth.

The experimental model is performed in 7 day-old rat pups (Wistar strain). Unilateral transient focal ischemia is induced in P7 Wistar rats of both sexes, as previously described (Renolleau S et al Stroke. 1998 Jul;29(7): 1454- 60). Seven-day-old Wistar rats (Janvier, Le Genest-St-Isle, France) are anesthetized with chloral hydrate (i.p., 350 mg/kg) or gas anaesthesia. Briefly, the left Middle Cerebral Artery is coagulated at the inferior level of the cerebral vein, and then a clip is placed to occlude the left common carotid artery. After 50 min, the clip is removed and carotid blood flow restoration is verified by microscopy. During the surgical procedure, body temperature is maintained at 37-38°C. The test item is administered according to the route defined with the Sponsor at the time of reperfusion (corresponding to lh post-ischemia onset). Importantly, vehicle and test item doses must be randomly assorted within a litter to take into account and to minimize differences of responses between litters. The pups are then transferred to an incubator (37°C) until recovery, and then returned to their dams.

Rat pups are killed from reperfusion to 48h post-reperfusion and their brains are removed. An infarct lesion (pale zone) appears progressively after reperfusion, reflecting the course of brain damages in the ipsilateral hemisphere, and should be reduced in case of efficient drug treatment.

To establish caspase-2 activity kinetics and determine whether level of caspase-2 activity after stroke is modified by TRP701 versus TRP701 treatment, enzymatic assay is performed from brains. Importantly, caspase-2 in vivo inhibition in the brain allows correlating with brain tissue protection. To proceed, caspase-2 activity must be measured in ischaemic penumbra only corresponding to the injured tissue. To conveniently measure any difference of activity, brains of rat pups (24h post-ischemia) are removed just after decapitation. Contralateral (CL) and ipsilateral (IL, containing the infarct) hemispheres are rapidly frozen and kept at -80°C. After thawing, the ipsilateral hemisphere is rapidly micro-dissected in order to take exclusively the penumbra area which exhibited more or less pronounced white colour according to the time of post-reperfusion. The corresponding area is also taken in the contralateral counterpart to measure the threshold of intrinsic caspase activity (caspase-2, C2; caspase-3, C3 : Caspase-9, C9). Each tissue is then quickly processed in ice. Sample (1-2 mm3) is put into a glass tube containing 800 μΐ of buffer B (HEPES lOmM pH 7.4, KC1 42mM, MgC12 5mM, DTT ImM, CHAPS 0.5%, EDTA 0.1 mM) extemporaneously supplemented with protease inhibitors: PMSF ImM, leupeptin 1 μg/ml, pepstatin A 1 μg/ml, cytochalasin B 1 μΜ, chymopapain 10 μg/ml, antipain 1 μg/ml. The tube is placed in an ice-bath and a manual crushing is performed with a glass Potter. The crushed tissues are then kept 24 h at - 80°C at least, prior to thawing and elimination of debris (10 min, 4°C, 2000g). 100 μ of supernatant is diluted in the buffer A (specific caspase activity buffer) and incubated for 2-3 hours at 37°C in presence of specific commercially available caspase substrates (50 μΜ): for Caspase-2 (C2), caspase-3 (C3) and caspase-9 (C9). Caspase activities are monitored by spectrofluorimetry 465 nm).

To establish protective effect of TRP 701, rat pups are killed at 48 h post-reperfusion and their brains are removed. The brains are then fixed for 2 days in 4% PFA. 50 μιη coronal brain sections are cut on a cryostat and collected on gelatin-coated slides. Eighteen sections from anterior striatum to posterior hippocampus (Bregma +3 mm to -6.5 mm) may be selected, taken at equally spaced 0.5 mm intervals. Lesion areas are measured on cresyl violet-stained sections using an image analyzer, and distances between respective coronal sections are used to calculate the infarct volume and the % infarct volume in the ipsilateral hemisphere (based on the Cavalieri principle).