`
` LUPIN
`EX. 1021
`(Part 2 of 6)
`
`
`
`wo 20o6m5a79-4
`
`PCTlGB2005.='00-£539
`
`30
`
`Figure 16 shows an alternative by which OIP may act is through a reduction in
`
`the ammoniagenic amino acid, Glycine. Substantial reduction in Glycine is observed
`
`only in the OIP group.
`
`Figure 17 shows the isoleucine levels are very low to start with in each of the
`
`5
`
`groups but increases to twice normal values in the Isoleucine treated groups. The
`
`concentration in the Placebo group remains low and unchanged.
`
`Figure 18 shows the changes in the Omithine levels hi the patients over the
`
`course of treatment showing marked sustained increase in the concentrations of
`
`Ornithine which are significantly reduced to basal values on stopping the drug
`
`10
`
`indicating uptake in the different tissues.
`
`Example 13: The effect of ornithine and Qhenylbutyrate in the bile duct ligated
`
`l'_=i.t.
`
`15 Methods
`
`Induction of cirrhosis by bile duct ligation (BDL)
`
`Male Sprague—Dawley rats (200-250g) were used for this procedure. Following
`
`anaesthetisation, a rnid-line laparotarny was performed, the bile duct was exposed, triply
`
`ligated with 4.0 silk suture, and severed between the second and third ligature. The
`wound was closed in layers with absorb able suture, and the animal allowed to recover in
`
`20
`
`a quiet room before being returned to the animal storage facility. Animals were kept at a
`
`constant temperature (20°C) in a 12 hour lightfdark cycle with access to water and
`
`standard rodent chow ad libitum.
`
`After five weeks post BDL (or sham procedure) the animals were switched from
`
`25
`
`rodent chow to a complete liquid diet (Liquidiet, Bio—Serv, Frenchtown NJ, USA) to
`
`which was added an amino acid mixture mimicking the composition of haemoglobin
`
`(2.8g/Kg/day, Nutricia Cuijk, The Netherlands,Produ'ct No. 24143). At six weeks, under
`
`anaesthesia a right carotid arterial catheter was inserted and used to collect repeated
`
`blood samples. Following this procedure a baseline sample was collected prior to
`
`30
`
`administration of the study formulations by IP injection. The study groups were: BDL
`
`control + Saline (n=5), BDL + ornithine (0.22g/Kg, n=6) in saline IP, BDL +
`
`phenylbutyrate (0.3 g/Kg, n=7) in saline ]P, BDL + OP (0.22g/Kg 2’ 0.3 g/Kg, n=7) in
`
`saline IP.
`
`127
`
`127
`
`
`
`W0 200 GIDSG794
`
`PC TIC BZOOSIO 04539
`
`31
`
`Blood samples were collected into pre-cooled heparinsed tubes and stored on ice
`
`prior to processing. Plasma was collected following centrifugation (3,000rpm, 10 mins)
`
`and stored at -80°C prior to analyses.
`
`Ammonia, glucose, lactate and urea were measured using a COBAS Mira S
`
`according to manufacturers instructions. Amino acids were quantified by HPLC with
`
`fluorescence detection.
`
`Results
`
`In the cirrhotic bile duct ligated rat model there is a substantial increase in the
`
`10
`
`arterial plasma ammonia level (205 i 11 umolesr’L, mean :f: SEM) compared with
`
`healthy controls (25.6 :1: 2 p.mo1esfL, p<0.00l data, not shown). In this model we found
`
`that there was no change in the arterial ammonia levels over three hours in the saline
`
`treated placebo group.
`Figure 19 shows the change iI1 arterial plasma ammonia levels in BDL cirrhotic
`
`15
`
`rats following IP injections of saline (BDL control, n=5), omithine (Om, 0.22g/Kg,
`n=6), phenylbutyrate (PB, 0.3g/Kg, n=7) and ornithine phenylbutyrate (OP, 0.22g/Kg +
`0.3g/Kg, n='}'). * signifies p<0.05 for OP vs Om at 3 hours (2 way ANOVA).
`This figure shows that in the ornithine treated animals a slight decrease in
`
`ammonia concentration was detected, though this was not found to be different from
`
`20
`
`placebo. In the phenylbutyrate treated group a significant increase in plasma ammonia
`was found after 1 hour (p<0.01 vs all other groups), though this difference was found to
`
`be smaller at the three hour time point. This finding fits with the hypothesis that
`phenylbutyrate (phenylacetate) is only effective in subjects with raised glutamine
`concentrations. In the animals without ornithine supplementation which can be
`
`25
`
`metabolised to form glutamine the effects of P alone are undesirable and are potentially
`
`harmful. A significantly lower ammonia level was observed in the ornithine plus
`
`phenylbutyrate (OP) treated group. In these animals a sustained lowering of amrnonia
`was measured over the three hour duration of the study the levels of which were found
`
`to be significantly less than those in the ornithine only group at the end of the study
`
`30
`
`(P-<0.05).
`This clearly demonstrates that the combination of OP has greater efficacy in
`
`reducing plasma ammonia than either 0 or P alone. Furthermore, the increased plasma
`
`levels of ammonia may be detrimental in the P alone treated animals.
`
`128
`
`128
`
`
`
`W0 20o6m5s79-4
`
`PCTlGB2005.='00-£539
`
`32
`
`In a sub set of samples we examined the uptake of ornithine into the blood stream
`
`following I? injection of O or OP. Figure 20 shows the arterial ornithine concentration
`
`in the supplemented groups. It can be clearly seen that in both groups the plasma
`
`omithine concentration is markedly increased at 1 hour following the [P inj ection,
`
`which is subsequently reduced at 3 hours as this ornithine is rnetabolised in the body.
`
`No significant difference was found in plasma omithine concentration between these
`
`groups at any time point.
`
`This finding is important as it demonstrates that the chosen method of
`
`administration is effective in delivering ornithine in these animals. Furthermore, the
`
`10
`
`rapid uptake and observed decrease in plasma levels indicate that active metabolism of
`
`this amino acid is occurring.
`
`Example 14: The effect of ornithine, Qhenylbutyrate and isoleucine in the bile
`
`duct ligated rat
`
`15
`
`20
`
`Methods
`
`I
`
`Male Sprague-Dawley rats (200—250g) were used for this procedure. For the 48
`hrs prior to sacrifice the animals were switched from standard rodent chow to a
`complete liquid diet (Liquidiet, Bio-Serv, Frenchtown NJ, USA) to which was added an
`amino acid mixture mimicking the composition of haemoglobin (2.8g/Kg, Nutricia
`
`Cuijk, The Nether1ands,Product No. 24143). Acute liver failure (ALF) was induced 24
`hours prior to sacrifice by [P injection of galactosamine (1 g/Kg, Sigma, Poole UK) in
`
`saline (n=5 ir1 each group). Three hours prior to sacrifice animals were treated with
`
`either a formulation of OIP (ornithine 0.22g/Kg, isoleucine 0.25 g!Kg, phenylbutyrate
`
`25
`
`0.3g,:'Kg, in saline IP) or saline control. At the termination of the experiment arterial
`
`blood was collected into pre-cooled heparinised tubes and stored on ice until processing.
`
`Plasma was collected and stored as above. Ammonia was determined as above.
`
`Results
`
`30
`
`Arterial ammonia levels were found to be significantly reduced in acute liver
`
`failure rats treated with OIP compared with placebo controls (Pig. 21). This study was
`
`designed to test whether isoleucine in combination with omithine and phenylbutyrate
`
`(phenylacetate) would be able to effectively lower plasma ammonia. It has been
`
`129
`
`129
`
`
`
`W0 200 GIDSG794
`
`PC TIC BZOOSIO 04539
`
`33
`
`previously demonstrated that isoleucine alone does not effect amrnonia levels in human
`studies, though its efficacy in combination with O and P has not been previously tested.
`
`Figure 21 shows arterial plasma ammonia levels in a hyperammonaemic acute
`
`liver failure model for saline placebo (ALF) and 011’ treated (ALF + OIP). A
`
`significance level ofp<0.01 was found between these two groups (T—Test).
`This finding supports the hypothesis that isoleucine in combination with
`
`ornithine and phenylbutyrate is effective in reducing ammonia levels. These are in
`
`addition to the beneficial efiects of isoleucine previously described for protein
`
`synthesis.
`
`10
`
`Example 15: The effect of ornithine and pl: en1lbug_r1_-ate in the devascularized pig
`model
`
`Methods
`
`15
`
`Five pigs were randomised into four groups: acute liver failure
`
`(ALF)+p1acebo+placebo (n=2); ALF+Omithine-I-placebo; ALF+ Phenylbutyrate
`+placebo; ALF+OInithine and Phenylbutyrate. Pigs had catheters inserted into the
`femoral artery and vein, portal vein, renal vein and pulmonary artery. The experiment
`
`started at time= -lhr, when placebo or treatment infusions were started.
`
`20
`
`1.
`
`2.
`
`3.
`
`Placebo: 5% Dextrose over 3 hours, oral water placebo
`
`Ornithine alone: 0.3 g/Kg, 5% dextrose over 3hours intravascular drip
`
`Phenylbutyrate: 0.3g/Kg, 5% dextrose over 3hours intragastric feed
`
`Omithine + Phenylbutyrate: 0.3 g/Kg, 5% dextrose over 3houIs intravascular drip,
`
`0.3g/Kg, 5% dextrose over 3hoursi11tragastric feed.
`ALF was induced by portal vein anastamosis to the inferior vena cava and
`
`25
`
`subsequent hepatic artery ligation (devascularisation) at time= Ohr; infusions were
`stopped at t= +2hr and the experiment was terminated at time=8hr. Blood and urine
`samples were collected at time= O, 1, 3, 5, 7 and 9hr for the measurement of regional
`annnonia and amino acid changes. At the end of the experiment a section of frontal
`
`30
`
`cortex was removed for brain water measurements.
`
`130
`
`130
`
`
`
`W0 2006i'056'?94
`
`PC TIC B2005l0 04539
`
`34
`
`Results
`
`Following ornithine infusion generating intracellular glutamate and the
`
`intragasuic supply of conjugating phenylacetate results suggest profound alteration in
`
`overall ammonia levels and glutamine utilization in this catastrophic model of liver
`
`failure.
`
`There is a consistent rise in the arterial ammonia concentration with time from
`
`devascularisation in the placebo treated animal (Figure 22), with some muscle
`
`production (Figure 23) and a large amount of ammonia coming from the gut (Figure
`
`24). This animal shows a modest muscle glutamine release (Figure 25) and appreciable
`
`10
`
`gut glutamine uptake (Figure 26).
`
`In the case ofthe omithine alone treated animal, the early ammonia rise is
`
`initially blunted, but rises thereafter to be the highest at termination of the experiment
`
`(Figure 22). There is a net uptake of ammonia by the muscle in this animal (Figure 24),
`
`with a comparable amount of glutamine being released from muscle — compared to the
`
`15
`
`placebo treated animal (Figure 25) with an increased gut uptake of glutamine (Figure
`
`26).
`
`Phenylbutyrate alone also shows an initial blunting of arterial ammonia levels,
`
`which quickly rises to levels comparable with ornithine alone at experiment termination
`
`(Figure 22) with little change in muscle ammonia uptake (Figure 23), but appreciable
`
`2.0
`
`gut production of ammonia (Figure 24). Interestingly, there is a net removal of
`
`glutamine by muscle with Phenylbutyrate alone treatment (Figure 25) with little overt
`
`effect on gut glutamine uptake, compared to placebo treated animal (Figure 26).
`
`The combination of ornithine and Phenylbutyrate has the greatest impact on
`
`arterial ammonia levels with an impressive reduction in circulating levels at the end of
`
`25
`
`the experiment compared to all the other animals (Figure 22). Ammonia is actively
`
`removed from the blood by muscle in this animal (Figure 23) with a greatly reduced gut
`
`ammonia production (Figure 24). It is interesting to note that the muscle glutamine
`release is increased compared to both the placebo and omithine alone treated animals
`
`(Figure 25). Despite this increased glutamine production i.n the muscle the gut
`
`30
`
`glutamine uptake is substantially reduced (Figure 26).
`
`A demonstration of increased circulating levels of ornithine in the ornithine
`
`treated animals is shown in Figure 27.
`
`131
`
`131
`
`
`
`W0 2006i'056'?94
`
`PC TIC B2005l0 04539
`
`The impact of the devascularisation and treatment interventions on arterial
`
`glutamine are shown in Figure 28. There is an increase in the circulating level of
`
`glutamine in the ornithine treated animal, which is ameliorated by the co-administration
`of phenylacetate. An interesting finding was the substantial amelioration of the arterial
`
`glycine levels that was found in the animal treated with both omithine and
`
`phenylbutyrate (Figure 29).
`
`At the end of the experiment the frontal cortex of the brain was removed and
`
`brain water content measured (Figure 30).
`
`An independent pathologist reported on the cellular anatomy of the brain in
`
`10
`
`these experimental animals. His report is summarized below.
`
`ALF: Microvessels with perivascular oedema with surrounding vesicles. Neuron
`
`with necrotic changes surrounded by vesicles.
`
`ALF + O+P: Microvessels with perivascular oedema with surrounding vesicles
`
`(less than from ALF without any treatment). Intracellular edema.
`
`15
`
`Sham: Brain tissue with minimal ultrastructural changes=normal brain tissue.
`
`Conclusions
`
`The inventors have found that simulation of some of the symptoms of an acute
`
`attack associated with chronic liver disease, such as increasing the concentration of
`
`20
`
`ammonia or simulating a gastrointestinal bleed, results in reduction of neutrophil
`
`function and this reduction can be partially reversed by ornithine or isoleucine. Rescue
`
`of neutrophil function by both omithine and isoleucine plays an important role in the
`
`prevention of sepsis which is a common precipitating factor ii: the progression of liver
`
`decompensation.
`
`25
`
`Furthennore, the inventors have found that isoleucine does not affect the rise in
`
`concentration of ammonia following a simulated gastrointestinal bleed. Therefore,
`
`contrary to the hypothesis that ammonia levels will decrease upon administration of
`
`isoleucine because of stimulation of protein synthesis, ammonia levels are unaffected.
`
`Thus, use of isoleucine in combination with ornithine, which is known to lower
`
`30
`
`ammonia levels, is particularly advantageous.
`
`Therefore, administration of ornithine and isoleucine prevent the metabolic
`
`consequences of a gastrointestinal bleed. Rising ammonia levels are blunted, the
`
`deficiency in isoleucine is corrected and neutrophil fiinction is rescued. The combined
`
`132
`
`132
`
`
`
`W0 200 GIDSG794
`
`PC TIC BZOOSIO 04539
`
`36
`
`use of ornithine and isoleucine therefore provides a new treatment for patients following
`
`a precipitating event to prevent liver decompensation fi-om occurring.
`The inventors have also found that L-ornithine L-aspartate (LOLA), which is
`
`used to reduce ammonia in patients with hepatic encephalopathy, does not reverse the
`
`effect of ammonia on neutrophil function. Thus, use of ornithine alone is more
`
`advantageous than use of LOLA, since omithine can both reduce ammonia and rescue
`neutrophil function. Also, the aspartate component of LOLA accumulates in the body.
`This accumulation of aspartate may actually by harmful to patients since aspartate
`
`worsens the effect of ammonia on neutrophil function, further reducing neutrophil
`
`10
`
`function. Accordingly, preventing or delaying the onset of liver decompensation can be
`
`achieved using ornithine in combination with isoleucine, preferably in the absence of
`
`aspartate.
`Furthennore, the inventors have found that treatment of patients with hepatic
`encephalopathy (I-IE) with L—o1nithine L-aspartate (LOLA) reduces ammonia levels and
`as a consequence, increases glutamine levels. However, glutamine is only a temporary
`ammonia buffer as it can recycle and regenerate ammonia in the kidney and the small
`
`15
`
`intestine. Therefore, treatment with LOLA alone can lead to a secondary rise in
`
`ammonia levels, further contributing to the pathology of hepatic encephalopathy.
`
`Use of phenylacetate or phenylbutyrate in children with urea cycle disorders
`reduces_ the abnormally high levels of glutamine. In contrast, patients suffering from HE
`
`20
`
`have normal levels of glutamine unless, as shown in Example 1, they are being treated
`
`with LOLA which reduces levels of ammonia but increases levels of glutamine.
`Therefore, use ofphenylacetate andfor phenylbutyrate allows for the removal of
`glutamine to prevent the secondary rise in ammonia levels in patients with HE.
`Accordingly, an improved treatment for hepatic encephalopathy can be achieved
`by administration of ornithine in combination with at least one of phenylacetate and
`
`25
`
`phenylbutyrate, preferably in the absence of aspartate.
`Our extensive investigations in animal models and also in humans with cirrhosis
`
`support the view that the major organ removing ammonia in patients with cirrhosis is
`the muscle, converting ammonia to glutamine, a reaction in which glutamate is utilised.
`
`30
`
`In liver failure, the enzyme responsible for this reaction, glutamine synthetase is
`
`induced and the provision of glutamate would increase ammonia detoxification.
`
`133
`
`133
`
`
`
`W0 20o6m5a79-4
`
`PCTlGB2005.='00-L539
`
`37
`
`Ornithine, a precursor of glutamate, detoxifies ammonia by transformation to
`
`glutamine. However, our preliminary studies have shown that this glutamine,
`
`recirculates and regenerates ammonia. Our invention provides a novel method of not
`
`only detoxifying ammonia into glutamine but also eliminating the excess glutamine that
`
`is generated. Thus, OP reduces ammonia concentration in patients with cirrhosis and
`
`hyperamrnonemia significantly more markedly than either alone. The effect is clearly
`
`synergistic rather than additive. In addition, postprandial increase in ammonia is
`
`abolished by administration of OP. This may allow for feeding of patients with
`
`decompensated cirrhosis with protein-rich diets without the risk of hyperammonemia.
`The reduction in ammonia was associated with improvement in the mental state.
`
`10
`
`It achieves reduction in ammonia concentration by preventing an increase in glutamine.
`
`This is consistent with the hypothesis that Ornithine is driving glutamine production in
`
`the muscle (thereby trapping 1 molecule of ammonia) but this glutamine is excreted
`
`(possibly as an adduct of phenylacetate) preventing a rise in systemic glutamine,
`
`15
`
`thereby preventing rebound hyperannnonemia.
`
`The established wisdom that phenylacetate reduces ammonia in the
`
`hyperammcnaemic infant presenting with urea cycle disorders is that the ammonia is
`trapped into glutamine and that the glutamine is shuttled to the kidneys for excretion as
`the phenylacetateglutamine adduct. These infants present with high ammonia and,
`importantly, high glutamine. Conversely the cirrhotic patient presents with high
`ammonia and normal to low glutamine. The pig model described above does not have a
`
`20
`
`raised glutamine and the ammonia levels increase dramatically after the liver is isolated.
`Treatment with omithine alone increases blood glutamine whereas ammonia
`
`levels are unaffected. Phenylbutyrate alone marginally increases glutamine and again
`
`25
`
`has insignificant effects on ammonia levels. In dramatic contrast, in this catastrophic
`
`model of escalating hyperammonaemia the combination of both ornithine and
`
`phenylbutyrate (OP) brings about an appreciable reduction in the circulating annnonia
`and ameliorates the increase in glutamine seen with ornithine alone. Glycine, an
`
`ammonia generating amino acid increased in all the animals, however, the rise in this
`
`30
`
`amino acid was substantially blunted only in the OP treated animal, suggesting
`
`additional benefit for this form of intervention. An established consequence of elevated
`
`amrnonia is brain swelling as water content of the brain increases. The brain from
`
`ornithine alone treated pig shows considerable increase in water content while the
`
`134
`
`134
`
`
`
`wo zoosm-56794
`
`PCTfGB2005.='00-L539
`
`3s
`
`onfithjne and phenylbutyrate combined reduces brain water content. I-Iistologically,
`
`there is less apparent injury in the microstructure of the brain of the ornithine and
`
`phenylbutyrate combined treatment animal compared to the placebo treated animal.
`
`135
`
`135
`
`
`
`W0 2006i'056'?94
`
`PCTlGB2005.='00-L539
`
`39
`
`CLAIMS
`
`1.
`
`Use of ornithine in the manufacture of a medicament for use in combination
`
`with at least one of phenylacetate and phenylbutyrate for preventing or treating liver
`
`decompensation or hepatic encephalopathy.
`
`2.
`
`Use of at least one of phenylacetate and phenylbutyrate in the manufacture of a
`
`medicament for use in combination with ornithine for preventing or treating liver
`
`decompensation or hepatic encephalopathy.
`
`10
`
`3.
`
`Use of ornithine and at least one of phenylacetate and phenylbutyrate in the
`
`manufacture of a medicament for preventing or treating liver decompensation or hepatic
`
`encephalopathy.
`
`15
`
`4.
`
`Use according to any one of the preceding claims wherein said liver
`
`decompensation is in a patient with chronic liver disease.
`
`5.
`
`Use according to any one of the preceding claims wherein said prevention or
`
`treatment involves delaying the onset of liver decompensation.
`
`20
`
`6.
`
`Use according to any one of the preceding claims wherein the patient has had or
`
`is suspected of having had a precipitating event.
`
`7.
`
`Use according to claim 6, wherein said precipitating event is gastrointestinal
`
`25
`
`bleeding, infection, portal vein thrombosis or dehydration.
`
`8.
`
`Use according to claim 6 or 7, wherein the medicament is administered within 6
`
`hours of the sy1nptom(s) of a said precipitating event or suspected precipitating event
`
`having been detected.
`
`30
`
`9.
`
`Use according to any one of the preceding claims wherein hepatic
`
`encephalopathy is treated in a patient with chronic liver disease or acute liver failure.
`
`136
`
`136
`
`
`
`W0 200GIDSG794
`
`40
`
`PCTICBZOOSIO04539
`
`10.
`
`Use according to any one of the preceding claims wherein said ornithine is
`
`present as a flee monomeric amino acid or physiologically acceptable salt.
`
`11.
`
`Use according to any one of the preceding claims wherein the at least one of
`
`5
`
`phenylacetate and phenylbutyrate is present as sodium phenylacetate or sodium
`
`phenylbutyrate.
`
`12.
`
`Use according to any one of the preceding claims wherein said medicament
`
`further comprises isoleucine.
`
`1 0
`
`13.
`
`Use according to claim 12 wherein said isoleucine is present as a free
`
`monomeric amino acid or physiologically acceptable salt.
`
`14.
`
`Use according to any one of the preceding claims wherein said medicament
`
`15
`
`contains substantially no other amino acid.
`
`15.
`
`Use according to any one of the preceding claims wherein the medicament is
`
`formulated for intravenous, intraperitoneal, intragastric, intravascular or oral
`
`administration.
`
`20
`
`16.
`
`Products containing ornithine and at least one of phenylacetate and
`
`phenylbutyrate as a combined preparation for simultaneous, separate or sequential use
`for preventing or treating liver decompensation or hepatic encephalopathy.
`
`25
`
`17.
`
`Products according to claim 16 which further comprise isoleucine.
`
`18.
`
`Products according to claim 16 or 17 which comprises substantially no other
`
`amino acid.
`
`30
`
`19.
`
`A pharmaceutical composition comprising ornitliine and at least one of
`
`phenylacetate and phenylbutyrate.
`
`137
`
`137
`
`
`
`W0 zoosm-56794
`
`PCTfGB2005.='00-£539
`
`41
`
`20.
`
`A pharmaceutical composition according to claiIn 19 which further comprises
`
`isoleucine.
`
`21.
`
`A pharmaceutical composition according to claim 19 or 20 which comprises
`
`substantially no other amino acid.
`
`22.
`
`A pharmaceutical composition as defined in any one of claims 19 to 21 for use
`
`in a method of preventing or treating liver decompensation or hepatic encephalopathy.
`
`10
`
`23.
`
`An agent for preventing or treating liver decompensation or hepatic
`
`encephalopathy, comprising ornithine and at least one of phenylacetate and
`
`phenylbutyrate.
`
`24.
`
`An agent according to claim 23 which further comprises isoleucine.
`
`15
`
`25.
`
`A method of treating a patient having or at risk of having liver decompensation
`
`or hepatic encephalopathy, which method comprises administering an effective amount
`
`of ornithine and at least one ofphenylacetate and phenylbutyrate to said patient.
`
`20
`
`26.
`
`A method according to claim 25 which further comprises administering an
`
`effective amount of isoleucine to said patient.
`
`27.
`
`A method according to claim 26 wherein said patient has an isoleucine
`
`deficiency attributable to gastrointestinal bleeding.
`
`138
`
`138
`
`
`
`W0 2‘""”'“5“794
`
`PCT/GB2005m04539
`
`1115
`
`Figure 1
`
`.1. D 0
`
` N U1 %IncreaseInOxidativeBurst
`
`NI 0'!
`
`U1 0
`
`
`
`AH+
`
`Decomp
`
`Comp
`
`Normal
`
`phagocytosis
`%Neutrophil
`
`Norm
`
`40-so
`
`59-190
`
`>1 on
`
`Ammonia (pM)
`
`139
`
`139
`
`
`
`W0 2"‘W"5“7'94
`
`PCT/GB2ousmo4539
`
`2:15
`
`Figure 3
`
`25
`
`
`
`
`
`NeutrophilChemotaxis(hpf)
`
`Normal
`
`40-60
`
`60-100
`
`>1 OD
`
`Ammonia (mm)
`
`Figure 4
`
`*p=0.1 vs NH3
`
`p<0.001 vs NH3
`
`P<0.05 vs NH3
`
`
`
`Contra!
`
`NH3, 500
`NH3.5OCI NH3.5U0 NH3.50D_ NH3. 500
`+ LOLA
`+ Glulamme + Aspartate + Ornithine
`
`
`
`%neutrophilsphagocytosing
`
`140
`
`140
`
`
`
`W0 2(l(l6;"056?94
`
`PCT/GB2l)05mI)4539
`
`ms
`
`Figure5
`
`
`
`Pre
`
`Placebo
`
`Sim Bleed
`
`Sim B|eecl+lsoIeucine
`
`Figurefi
`
`Q 40
`.C
`--'
`35
`
`30
`25
`
`20
`15
`
`10
`
`5
`
`0
`
`lg
`><
`G
`‘I-5
`O
`
`E_
`
`:
`2
`
`‘E
`2..
`:3
`g
`
`500 I-
`
`400 -
`
`
`
`(nmollkgbwlmin)
`
`300
`
`200 r
`
`100 L
`
`Wb Ra
`
`Oxidation
`
`141
`
`141
`
`
`
`W0 2006l056'?94
`
`PCT;GB2005m04539
`
`4:15
`
`Figure 7
`
`—— lsoleucine
`"""“' Placebo
`
`
`
`150
`
`,...
`
`E E
`
`:
`
`,9 100
`
`:c E E
`
`‘E
`
`50
`
`0
`
`0
`
`1
`
`2
`
`3
`
`4
`
`Time
`
`Figure 8
`
`0A: 40g!day
`over 12 hr
`
`515°” 0A
`
`1600
`
`140
`
`E 1200
`_§
`300
`‘g
`
`400
`
`6
`
`I
`
`Arnmony xx"
`I
`
`80
`
`130 --
`
`120 %
`110 -E
`100 E
`
`90
`
`__
`
`142
`
`142
`
`
`
`wo 2006l056794
`
`PCT;GB2o05mu4539
`
`sns
`
`Figure 9
`
`0A: 40g!day
`over 12 hr
`
`1200
`
`Stop OA
`
`Y
`
`120
`
`110 _
`5
`
`CE
`
`100 3
`S
`E
`q
`
`an
`
`30
`
`70
`
`360
`
`320
`
`230
`
`240
`
`200
`
`160
`
`__
`E
`_g
`5
`E
`E
`-at
`
`
`
`Figure 10
`
`l.V. Phenylacetate
`
`OA: 40g!day over 12 hr
`
`—--~
`
`"""
`
`E 900
`.3
`E
`3
`(3
`
`000
`
`300
`
`2100
`
`1800
`
`1500
`
`E
`--
`g
`E
`55
`.2
`(D
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Days
`
`143
`
`
`
`W0 2006:'056794
`
`PC TIC B2005l0 0-I-539
`
`—u— Placebo
`
`
`
`
`
`-0- Ornithine
`
`-#~ Phenylbutyrate
`
`
`
`‘F Ornithine-
`
`Phenylbutyrate
`
`
`
`
`
`
`
`6i'15
`
`Figure 11
`
`L Post prandial samples
`
`Treatments
`
`E 18
`
`
`
`3a
`
`o E E
`
`<
`
`s
`-E 15
`
`12
`
`9
`
`6
`
`O
`
`20
`
`40
`
`60
`
`100
`
`120
`
`Time (hr)
`
`Figure 12
`
`—n— Placebo
`
`-0- Ornithine
`
`—A+ Phenylbutyrate
`-‘F Omithine-
`
`Phenylbutyrate
`
`l Post prandial samples
`
`Treatments
`
`
`
`7:)
`
`60
`
`50
`
`40
`
`E0
`
`E E £
`
`53
`
`(D
`
`100
`
`120
`
`144
`
`144
`
`
`
`W0 2006l056'?94
`
`PCT;‘GB2005.='00-L539
`
`‘H15
`
`Figure 13
`
`Placebo
`
`Phenylbutyrate
`
`4
`
`:
`
`9
`
`"
`
`2
`
`3
`
`4
`
`Days
`
`.
`.
`Ormthme
`
`i
`
`0
`
`T1 T?
`1
`2
`Days
`
`-IT IT
`3
`4
`
`3
`
`UJ
`:1:
`fl:
`1%
`5 1
`
`2
`
`0
`
`4
`
`0
`
`1
`
`2
`
`3
`
`4
`
`Days
`
`Ornithine
`Phonylbutyrate
`
`Lu
`:1:
`‘*5
`-g
`E
`0
`
`3
`
`2
`
`
`
`Figure 14
`
`
`
`
`
`
`—~o— OIP
`-0- OIP
`
`
`
`—“‘“ Placebo
`"*7" ‘ Placebo
`—--69-- -~
`lsoleucine
`<-‘--
`Isoleucine
`
`Lu
`:1:
`3
`E
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`
`4
`
`3
`
`Lu
`I
`‘*5
`% 2
`E
`(3
`1
`
`0
`
`fl"|I-
`
`E ‘
`
`*3
`.9:
`
`o E
`
`IX)45
`
`NI 0
`
`16
`
`12
`
`
`
`E
`-cl:
`
`3
`
`4
`
`0
`
`10
`
`20
`
`30
`Hours
`
`40
`
`so
`
`60
`
`145
`
`145
`
`
`
`W0 2006l056794
`
`PC TIGBZOOSIU 0-I-539
`
`8i'15
`
`Figure 15
`
`43" Placebo
`"“"" Placebo
`--6
`lsoleucine
`.......o_
`
`—o—- OIP
`-9- OIF’
`
`
`
`120
`
`E105
`3:
`
`75
`
`E 33
`
`(D
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`“"“*—"' Placebo
`-'-‘B’-F Isoleucine
`-43- -
`Isoleucine
`
`-4- OIP
`
`50
`
`
`
`Glycine(pllll)83
`
`20
`
`Hours
`
`146
`
`146
`
`
`
`W0 200 61056794
`
`PC TIGBZOOSIU 0-I-539
`
`'3' Piacebo
`"““"“ Placebo
`lsoleuoine
`lsoleucine
`
`-ow ou=
`
`+ OIP
`
`_|. N
`
`{D
`
`
`
`lsoleucine(pM}
`
`9:1 5
`
`Figure 17
`
`Figure 18
`
`Placebo
`"F1
`'““*'*"“"' Placebo
`~?=
`[soleucine
`
`lsoleucine
`—'<%“'
`—o— OIP
`
`.
`
`To
`
`—9-" OIP
`
`
`
`15
`
`_\. N5
`
`_|. D
`
`
`
`Ornithine(uM)
`
`Hours
`
`147
`
`147
`
`
`
`W0 200 GIOSG794
`
`PC TIC B2005l0 04539
`
`10:" 1 5
`
`Figure 19
`
`Arterial ammonia in BDL rats
`
`--I— OP
`
`-' 1%“ °' Om
`- =W- - PB
`"<>'- BDL control
`
`
`
`360
`
`____
`E
`lg
`5 260
`
`E E
`
`<32
`
`160
`
`Time (hours)
`
`Figure 20
`
`Plasma Ornithine (|.IM)
`
`
`
`Ornithine(pm)
`
`1000
`
`03O388
`
`400
`200
`
`0
`
`_
`
`T
`
`D
`
`0.5
`
`1
`
`‘l .5
`
`2
`
`2.5
`
`3
`
`3.5
`
`Time (hr)
`
`148
`
`148
`
`
`
`W0 2006!056794
`
`PC TIGBZOOSIOU-I-539
`
`1 H15
`
`Figure 21
`
`(pM)
`
`Ammonia
`
`ALF
`
`(W-'5)
`
`ALF + DIP
`
`(|'I=5)
`
`Figure 22
`
`
`
`Treatment
`
`t_,_____-—
`
`_
`_
`-0- Ormthlne
`
`
`
`—A— Phenyfbutyrate
`
`
`
`Phenylbutyrate
`
`"'* 0rnithine—
`
`149
`
`149
`
`700
`
`600
`500
`
`400
`
`300
`
`200
`
`100
`
`
`
`Ammonia(pM)
`
`
`
`W0 200 61056794
`
`PC TIC B2005l0 04539
`
`1 2!] 5
`
`Figure 23
`
` —-— Placebo
`
`Ornithine
`
`Femoral Vein - Artery
`Phenylbutyrate
`
`Ornith ine-
`
`
`Phenylbutyrate
`
`
`
`
`SD
`
`40
`
`20
`
`uhO
`
`-40
`
`-so
`
`-80
`
`
`
`Ammonia(pm)
`
`I
`0
`
`r
`
`r
`2
`
`**-"
`
`'1
`4
`
`I
`6
`
`=
`
`a ——-
`3
`
`Time (hr)
`
`Figure 24
`
`—u— Placebo
`
`
`
`-0- Ornithine
`
`
`
`
`
`—A— Phenylbutyrate
`
`
`—"'— Ornithine-
`
`
`Phenylbutyrate
`
`
`
`
`
`0
`
`-
`
`T
`
`,
`
`8
`
`
`
`250 Portal drained viscera - Artery
`
`-I
`
`‘
`
`I
`
` #“”f
`
`O
`
`2
`
`4
`
`6
`
`Time (hr)
`
`150
`
`150
`
`200
`
`150
`
`100
`
`50
`
`O
`
`E‘
`E
`-E
`
`E E
`
`;
`
`
`
`W0 2006l056794
`
`PC TIGBZOOSIOU-I-539
`
`131'] 5
`
`Figure 25
`
`Phenylbutyrate
`
`uction!ReIease
`MuscieGlutamineProd
`
`
`
`Placebo Ornithine
`
`I
`
`5-
`
`Ornithine -
`Phenylbutyrate
`
`Figure 26
`
`Placebo Ornithine
`
`Phanyl-
`butyrate
`
`Ornithine -
`Phenylbutyrate
`
`
`GutUptakeofGlutamine(_uM) E
`
`
`
`151
`
`151
`
`
`
`W0 2006l056'?94
`
`PC TIC B2005!!! 04539
`
`14:" 1 5
`
`Figure 27
`
`1 800
`
`1500
`
`‘I20
`
`
`Ornithine
`
`
`Phenylbutyrate
`Ornithine-
`
`Placebo
`
`
`
`Phenylbutyrate
`
`900
`
`sun
`
`300
`
`D
`
`0
`
`2
`
`4
`
`6
`
`8
`
`Time (hr)
`
`Figure 28
`
`
`
`Ornithine(pM)
`
`
`
`
`
`ArterialGlutamine(M1)
`
`600
`
`400
`
`200
`
`Placebo
`Omithine
`
`Phenylbutyrate
`Ornithine—
`
`Phenylbutyrate
`
`152
`
`152
`
`
`
`W0 200 6J'l)56 794
`
`PC TIC B2005r'00-I-539
`
`153' 15
`
`Figure 29
`
`2500-‘
`
`2000“
`
`
`
`
`
`1 500-
`
`1000"
`
`50%
`
`(pm) ArterialGlycine
`
`—1-- Placebo
`
`—I— Ornithine
`
`-A~ Phenylbutyrate
`
`—o— Ornilhine-
`Phenylbutyrate
`
`flg&Ml
`
`%differenceinbrainwater
`
`_-
`Ornithlne -
`.
`Pm‘-‘flY'bUtY|'flt9
`-
`'
`Ornithine
`
`\
`
`relativetocontrol
`
`Phenylbutyrate
`
`153
`
`153
`
`
`
`INTERNATIONAL SEARCH REPORT
`A. CLASSIFICATION OF UBJECT MATIEFI
`A61P1 16
`A61K31/192
`
`A61K31/198
`
`Inter
`tai application No
`PC” ‘$2005/004539
`
`According to international Patent Classification (I530) orto both national classification and IF-‘C
`
`a HELDSSEAHCHED
`Minimum documentation searched (oiassiiication system followed by clasailicalicn symbols)
`A61K A61?
`
`'
`
`Documentation searched other than minimum documentation to the extent that such documents are included In the Fields searched
`
`Electronic data base consulted during the tntemattonal search (name of data base and. where practical, search terms used)
`
`EPO-Internai, CHEM ABS Data, NPI Data
`
`'3. DOCUMENTS CONSIDEFIED TO BE RELEVANT
`
`Category‘
`
`Citation of document, with indication. where approprlale, ofthe relevant passages
`
`Fieievani to claim No.
`
`(BURZYNSKI STANISLAW R)
`Us 2005/182064 Al
`18 August 2005 (2005-08-18)
`paragraph ‘U056!
`ciaims
`
`US 4 228 099 A CWALSER ET AL)
`14 October 1930 (1980-10-14)
`abstract
`
`DATABASE NPI
`Section Ch, Meek 200331
`Derwent Publications Ltd., London, GB;
`Ciass B05, AN 2003-314385
`XP002364873
`
`& CN 1 383 815 A (LIU M)
`11 December 2002 (2002-12-11)
`abstract
`
`'
`
`Further documents are listed in the continuation of Box C.
`‘l
`'
`f
`1
`cl
`1
`:
`Spec” cmegoneso med acumen S
`'A' document defining the general state orttie art wttiott is not
`considered to be 01 particular relevance
`‘E’ earlier document but published on or after the international
`"mg dam
`'L' document which may throw doubts on priority ciatrnts) or
`which is cited to establish the publication date or another
`or (as specmau:
`‘0‘ document reterrtng to an oral disclosure. use. exhibition or
`other means
`'P" document published prior to the International
`later than the priority date claimed
`Date of the actual completion oftlte inlemational search
`
`filing dole out
`
`See potent family annex.
`
`.
`
`'T‘ later document published attertire intemationai filing date
`gi::éi?;flgn%:tr:‘:'::é 11?; I3rfi%?g|1gIa‘:jm;2:, ififlgabflfig $1‘:
`“wanna”
`.x. document of pamcmar relevance; me claimed invention
`cannot be considered novel or cannot be considered to
`irwotve an inventhre step when the document is taken alone
`.
`.
`Y “£2223?=tt°;3::c::::,::*?,r:.i3:-gitflztrrs LT'r§S‘1lIE2r.
`document is combined with one or more other such docu-
`meow. such combination being obvious to a person skilled
`In "'9 3"-
`'8.’ document member of the same patent family
`Date or mailing of the lntematlonal search report
`
`27 January 2006
`Name and mailing address of the ISA!‘
`European Patent Office. FEEL 581 B Patettiiean 2
`NL - 2280 HV Fiijawiik
`E::f:3;:3§’3,°:fafs°:PeT“' 3‘ 65‘ °"° ""
`
`03/02/2006
`Authorized oiiicer
`
`skafii demand, c
`
`154
`
`154
`
`
`
`H§TEFUWATK)NA1.SEAJH3HIREPCMQT
`
`Inter
`
`nal application No
`
`PCn;uB2UU5/004539
`
`Clcontinuationi.
`
`DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Category‘
`
`Cilailon of document. with Indication. where appropriate. of the relevant passages
`
`Flelevant to claim No.
`
`(SUMMAR MARSHALL L ET
`us 2004/229948 Al
`AL) 18 November 2004 (2004-11-18)
`the M1019 document
`
`155
`
`155
`
`
`
`
`
`ationai application No.
`PCT/932005/004539
`
`iNTEFtNATt0NAL SEARCH REPORT
`
`Box No. It Observations where certain claims were tound unsearchable (continuation or item 2 of first sheet)
`
`This international search report has not been established in respect of certain claims under Article 1?'{2)la) for the iollowing reasons:
`
`
`
`
`Claims Nos.:
`because they relate to subject matter not required to be searched by this Authority, namely:
`
`Although claims 25-27 are directed to a method of treatment of the
`human/animal body,
`the Search has been carried out and based on the alleged
`effects of the compound/composition.
`
`
`
`
`2. D Claims Nos.:
`because they relate to parts of the international appli