AU661399B2 - Use of alpha-ketoglutarate - Google Patents

Description

OPI DATE 13/12/93 APPLN. ID 40989/93 AOJP DATE 24/02/94 PCT NUMBER PCT/SE93/00426 AU9340989 IN'I RNAI'IONAL APPILICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Pagent Classification 5 00i International Publication Namber: WO 93/23027 A61IK 31/19, 31/195 Al1 (43) Internailonal Publication Date: 25 November 1993 (25.11.93) (21) Internatlonal Aplication Number; PCT/SE93/00l26 (74) Agents: TANNERFELDT, Agneta et al.; Kabl Pharmacia (22)N~nternatlonal Filing Date: I14 May 1993 (14.05.93) AB -128Stchl

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(81) Design~ated States: AU, CA, NO, US, Europecan patent Priorlty data: BE, ClH, D)E, DK, FS. FR, GB, OR, IC, IT, LU, 920,584.1 20 May 1992 (20,05.92) SE MC, NL, PT, SE).

(71) Applicant ('for all designated States except LISP All ERI K Published VINNARS [SE/S1El: Oregrundsgatan 24, S-115 59 Wifth international search report.

Stockholm (SE), (72) I nventors;- and Inventors/Applicants (for US o1nlY- VINNARS, Erik [SE/ 6 9 SE); Viringavllgen 17 C, S-193 00 Sigtuna WERN- ERMAN, Jan [SE/SEJ; Tomntebogntan 23, S-113 38 Stockholm (SE).

(54) Title:, USE OF A LPi-IA. KETOG LUTARATE (57) Abstract The Invention relates to the use of aI lha.ketogluta rate or analogues thereo(' in the preparation or at medicament ('or treat.

mnent or critically Ill patients ror Improving protein synthesis capacity, maintaining energy level, preserving the lean body mass and ('or Improving the glutamnine content In skeletal muscle, Tie medicament contains preferably ti lha.'getogluta rate in such an amount, so that It provides morc than 0,25g/kg body weight/day or alpha-ketoglutarate, when administered to thle patient, Thle medicament can also comprise conventional amnino acid solution and/or glutamine or analogues thereof, L-asparaglne and/or acetoacetate. The invention also relates to at composition comprising convent lonal~z nmino acid mixture and alpho-ketogltarale or analogues thereof, in such an amount, so that It provides more than 0.25g/kg body weight/day of alplia-ketoglutarate, whenl ad.

ministered to the patient, optionally with the addition of glutaine or analogues thereof, L-aspiraglne and,'or acetoacetate, giu.

cose and/or rat, WO 93/23027 PCT/SE93/00426 1 USE OF ALPHA-KETOGLUTARATE The present application relates to the use of alpha-ketoglutarate In the preparation of a medicament for treatment of critically ill patients for Improving protein synthesis capacity and preserving the lean body mass and for improving the glutamine content and maintaining energy status in skeletal muscle, especially of a medicament containing alpha-ketoglutarate in such an amount, so that It provides more than 0.25 g/kg body weight /day of alphaketoglutarate, when administered to the patient.

It also reiates to a composition containing conventional amino acid mixture and alpha-ketoglutarate in such an amount, so that it provides more than 17,5 g/day, 17,5 g/L) of alpha-ketoglutarate, optionally with the addition of glutamine or analogues thereof, glucose and/or fat.

The glutamine content In skeletal muscle of critically III patients ,who are treated with TPN (Total Parenteral Nutrition) according to common method of today is not Influenced by this conventional treatment. Also when glutamine Is given additionally In an amount of P^g/day per person, only a moderate influence on the skeletal muscle glutamine of critically III patients could be established, It must be regarded as surprising that the use of alpha-ketoglutarate has an influence on the glutamine content In skeletal muscle of critically ill patients.

Backaround of the Invention In states of Illness, surgical operations and Injuries, profound changes are Induced in the energy and protein metabolism of the human body. This means, for example, loss of active cellular mass, leading to muscular fatigue, pronounced apathy and loss of appetite, and a period of convalescence involving general weakness which, for Instance after a billary tract operation, may last 5-6 weeks before the patient has regained his normal function. The cellular mass which is broken down very rapidly in different states of Illness

I

i i WO 93/23027 PCT/SE93/00426 2 will need a time for re-establishment which is about four times as long as the time of breakdown for the same mass.

In critical states of Illness and injuries, parenteral nutritional support is generally applied. In the past, preparations for intravenous nutritional support generally contained an aqueous solution of a high caloric content carbohydrate, such as glucose and the like, fat and electrolytes. In prolonged states of illness or in injuries the nitrogen balance of the body must however be considered, i.e. the ratio of nitrogen loss to nitrogen intake. In the case of negative nitrogen balance, the parenteral nutritional support can be supplemented with amino acid supply to Improve the nitrogen balance.

Different amino acid compositions for parenteral supply are previously known, see e.g. SE Patent Application 8203965-2 and DE-A 25 30 246 concerning amino acid nutrient compositions in renal failure, WO 82/00411 concerning a nutrient composition containing branched-chain amino acids, and WO 83/03969 concerning an aqueous nutrient solution consisting of L-amino acids.

From a survey made of the free amino acid pattern in the muscles, it has been found that skeletal muscle, which is the major body tissue in respect of weight, has a free amino acid pool, 62% of which consists of glutamlne, see Bergstr8m et al: Intracellular free amino acid concentration in human muscle tissue, J. of Appl. Physiol., Vol 36, No 6, 1874. In states of illness, Injuries or surgical operations, this content decreases by 40 50%, see Vinnars et al: Influence of the postoperative state on the intracellular free amino acids in human muscle tissue. Annals of Surg., Vol 182, 6:665-671, 1975 and in states of blood poisoning, even more,.

It has been found that this glutamine reduction cannot be affected by enteral or parenteral nutritional support according to the methods hitherto available, see Vinnars et al: Metabolic effects of four Intravenous nutritional regiments in patients undergoing elective surgery. II. Muscle amino acids and energy rich phosphates. Clin, Nutr, 2:3-11, 1983. There probably Is a correlation between the Inability immediately postoperatively to make a negative nitrogen balance positive and to normalise the exhausted intracellular glutamine pool and the reduced muscular mass and strength. This reduction probably depends on a reduced protein synthesis capacity post traumatically in skeletal muscle, see Werneman et al: Protein synthesis after trauma as studied by muscle I mod WO 93/23027 PCT/SE93/00426 3 ribosome profiles. Proceedings in the 7th ESPEN Conpress. Ed, Dletze et al, Karger, Basel.

The addition to the nutritional support of a dipeptlde of the type ornitHinealpha-ketoglutarate to a commercial amino acid solution has been found to improve to some extent, whereas not to normalise the intracellular glutamine pool, see Leander et al: Nitrogen sparing effect of Ornicetll In the immediate postoperative state. Olin. Nutr. 4:43-51, 1985. This preparation is however very expensive, and it has not been possible so far to evaluate whether Its use in parenteral nutrition confers a clinical advantage.

When a patient is critically III, It becomes necessary to resort to intravenous feeding. The nutrition substrates available for energy metabolism are various sugar solutions and fatty emulsions, which today seem appropriate. However, the amino acid solutions commercial available are inadequate, because they lack or have too low concentration of important amino acids such as tyrosine, cystelne, asparagIne or glutamine. This Is due to difficulties in heat-sterilising solutions of the amides, and also to the fact that the amides are unstable when stored. Another problem is that some of these compounds are relatively sparingly soluble and therefore require large amounts of water when being prepared, After elective surgery, for instance biliary tract operations, It has been found that the negative nitrogen balance primarily depends on reduced protein synthesis which is assessed by determining the ribosome activity in skeletal muscle, see Wernerman et al: Protein synthesis in skeletal muscle after abdominal surgery: The effect of total parenteral nutrition. JPEN, 1985. An Increased protein breakdown occurs only in very critical traumas and primarily in septic states. This reduced protein synthesis capacity cannot be affected by conventional Intravenous or enteral nutritional support.

WO 89/03688 discloses that alpha- ketoglutarate has the same effect as glutamine when given to postoperative patients, Preliminary tests on patients subjected to a biliary tract operation showed that an addition of alphaketoglutarate to a conventional parenteral nutritional support program improves the nitrogen balance of the patients. Besides, the pathological WO 93/23027 PCT/SE93/00426 4 amino acid changes which normally occur after Injury or surgical operation are normalised and, also, the reduction of the ribosome activity Is prevented.

Critically ill patients is a group of patients who are very 1ll. They have one or multiple organ failure, such as respiratory problem, renal, liver and/or Intestinal Insufficiency, a general protein catabolism and must be under Intensive car,. This group is different from the group of postoperative patients, who often has normal glutamine and protein values before the operation and for who the drop in glutamlne level Is due to the operation. Critically III patients have a pronounced protein catabolism and a lower skeletal glutamine content than postoperative patients.

Critically Ill patients have a decrease of at least 50 of the normal glutamine concentration in skeletal muscle. In extreme cases there Is a drop to 70-80 Roth et al (Olin Nutr 1:25-42, 1982) have shown that there Is correlation of mortality of the patients with a decrease of more than 70 Jeppson et al (Am J Physlol 1988, 255, E166-172) has shown a correlation between the protein synthesis and glutamine level In skeletal muscle, By improving the glutamine level In skeletal muscle the protein synthesis capacity is Improved and the lean body mass is preserved. This correlation is of Importance for the Interpretation of the results given In the example below.

Earlier studies (J Karner and E Roth, Clin Nutr. Vol 9, 1990, 43-44) have shown that when alanyl-glutamine Is given In an amount of 20 g/day per patient to two patients, no Influence on the skeletal muscle of critically ill patients could be established and when 40 g/day per patient was given to |I two patients, a marginal improvement of the muscle glutamine concentration could be seen. When 60 g/da was given to two patients an Improvement of 50-100 was found.

It is also of importance to maintain the energy status in the skee!tal muscle tissue for critical II patients. The energy status is coupled to the protein synthesis, but the mechanism is not totally known, Th-iaventlon WO 93/23027 PET/S E93/00426 It has now been demonstrated for the first time that the addition of alphaketoglutarate, alone or in combination with conventional amino acid solutions to a parenteral nutrition program can improve the reduction of the protein synthesis capacity for critically Ill patients, maintain energy status in the skeletal muscle tissue and also Improve the muscle glutamine concentration with almost 100 A larger effect has been obtained compared to glutamine given in equal amount, (J Karner and E Roth). This must be regarded as surprising, The abnormal amino acid pattern Intracellularly in skeletal muscle for critically Ill patients, and especially the 50% reduction of the glutamine pool Involved, can then, by the addition of aipha-ketogiutarate, be partially normaiiseI, The present Invention relates to the use of alpha..ketoglutarate or analogues thereof In the preparation of a medicament for treatment of critically Ill patients for Improving protein synthesis capacity, maintaining energy level and preserving the lean body mass and for Improving the glutamino content, especially in such an amount so that it provides more than 0,25 g/kg body So: weight (bw) /day of aipha-ketoglutarate whon administered to the patient. The medicament can also comprise conventional amino acid solution and or glutamine or analogues thereof, L-asparagine and/or acetoacetate.

TV=, in accor~nce with a f i=s aspect of tha prozont invcnticn, thmr is provided a nnthod for t trmamwnt of such criticalJly ill patients that have miffere-I n're thtn a 50% reucticn of tho intsollutx glut~an in skcmlotal r~nucles which rmthd copisa nitrng 1*o such patients we than O-2S gkRg bty vwight/day of alpiaotcglutarae or on analogm ti10of, in 0%ft to in~rve the protein synth~ is o.pacity, mntaining fth eerV~ levol, anl to prv, fth Ion bcdy n=s.

Alpha-ketoglutarate may be givon alone or in combination with a conventional amino acid solution, optionally with the addition of L-glutamine oy analogues thermefnt eagoasalts, anter addietidceaes.~ s aalrft oy analogues th eantof, g alsagne and OOitpactdtG luoe n ft ~AL4 k? WO 93/23027 PCT/S E93/00426 6 And4 in a~rdanca with a seccrid aspect of thex present inventicon, th~ze is3 provided a carpoitin for tb3 treatrrent of such critically ill patients that have suffered nore than a 50% reduction of, the glutaTn~m level in Oketa).

inu~les, in orcbr to ixrprove protein sythezis capacity, nma~ntaining the energy level, and to preserve thz lean bcdy mass, said cmpxiticn oaiprising a axwenticnal andn acid mixture and nore than 25 g/l of alplia-}'ntjlutarte or analogms ther~f, cjptiamaIy with thx3 additioni of glutwiine or analox-ue= thro~f, L.-aqp~gine and/or acxewo-tate, glucace ard/or fat.

V An upper limit of the dose of alpha-ketoglutarato Is related to the tolerated level for the patient, No Investigations have boon done, but an upper limit could be estimated to be about 83 -100 g/ day per patient.

The use of alpha..ketoglutarate Is not limited to parontoral administration but can also be administered orally. The suggested doses for nipha-ketoglutarate Is applicable both for parentoral and oral administration, if The conventional amino acid solution is given paronterally.

H By critically Ill patients is meant a group of patients who have one or multiple organ failure, and a general protein catabolism. They are treated in intensive care units, often under mechanical ventilation and/or dialysis.

The amount of alpha-kotoglutarate is here calculated on the need of the patient per day 24 hours). It is iho only proper way of defining the amount, as the amount always must depend on body weight (bw) Mf the patient and the time pertod for the amount, Normally the alpha-katoglutarate or its analogue is given together with the solution comprising conventional amino acids. The amount for such a solution is often about one litre per day, but this very much depending on concentration of the solution and the amount of liquid that can be given to the patient.

All scientific report regaraing amino aid admi nist ration and stupplemont thereto are calculating the amount in g per body Weight and time or in C, N perI body weight and time. This tS the Only proper way for a doctor to de.cOO the amount for the patient.

WO 93/23027 PCT/SE93/00426 7 When giving the alpha-ketoglutarate parenterally together with amino acid solution and other additives, the products can either be heat sterilised or brought into a form suitable for administration by sterile filtration of an aqueous solution, followed by rapid cooling and cold storage limited to a few months. One alternative is freeze-drying of the sterile-filtered solution, yielding a sterile powder. Immediately before administration, this powder can be added to a conventional amino acid mixture. Also other forms of powder sterilisation, not relying on heat, are conceivable. The possibility of using the sodium salt or esters of the compounds in order to Increase the solubility has also been considered.

The concentration of at least 0.25 g/kg body weight/ day corresponds to at least 17,5 g component/L aqueous solution if 1 L amino acid solution/day Is given to a patient weighing 70 kg, One example of a conventional amino acid solution expressed In g dry component/L aqueous solution is: glycine 1-12 aspartame 1.10 glutamate 2-12 alanlno 2-20 arginine 2-14 cystelno/cystlne 0.4-2.0 histidine 2-8 isoleuclne 2-8 leucine 2-8 lyslne 2-12 methlonine 1-6 phenylalanlne 4-10 prollne 4-10 serine 2-10 threonino 2-8 tryptophan 1.3 tyrosine 0.2-1 valine 2.8 I k WO 93/23027 PCr/SE93/00420 8 and optionally 5-30 g/l L-giutamine or analogues thereof and/or 0.5-10 g/l Lasparagine and/or 0.5-10 gI acetoacetate, or salts or esters thereof.

The added amount of L-glutamine to be given together with this amino solution Is at least 17,5 gIL and an especially preferred amount Is morn) then gIL.

When sparingly soluble amino acids are given as dipeptides, the amounts given above for e.g. glutamIne, cysteine and tyrosine, can be higher.

Pibferred compositions could Include the following suitable components (expressed In g dry component/i aqueous solution):.

Amino acid solution 1 2 3 4 glycine 5.9 5.9 5.9 5.9 5.9 aspartate 4.8 4.8 4.8 4.8 4.8 glutamate 6.8 6.8 6.8 6.8 6.8 alanIne 12.0 12.0 12.0 12.0 1210 arginine 8.4 8.4 8.4 8.4 8.4 cystelne/cystine 0.42 0.42 0.42 0.42 0.42 histidine 5.1 5.1 5.1 5.1 5.1 isoleusine 4.2 4.2 4.2 4.2 4.2 Meaine 519 5.9 5.9 5.9 5.9 lysine 6.8 6.8 6.8 6.8 6.8 methionino 4.2 4.2 4.2 4.2 4.2 N phenylalanine 5.9 5.9 5.9 5.9 519 proilne 8.0 8.0 8.0 8.0 serine 6.0 6.0 6.0 6.0 threonino 4.2 4.2 4.2 4.2 4.2 tryptophan 1.4 1.4 1.4 1.4 tyrosine 0.4 0.4 0,4 0.4 0.4 vallne BIB 5.5 5.5 5,5 asparagine 2.0 acetoacotate 2.0 I 1 WO 93/23027 I'CT/SE93/00426 9 alpha-ketoglutarate should then be added to these solutions in an amount of more than 17,5 g/L, preferably 25 g/L or more, When alpha-ketoglutarate Is Included in the composition, it must be added in the form of Its sodium salt or its esters, since It is otherwise extremely sparingly soluble and unstable. Glutamine can also be added in the form of the sodium salt thereof, thus improving Its solubility, implementation of the Invention When preparing a composition Including alpha-ketoglutarate, heat sterilization can be used if the components are not heat sensitive. When Including heat sensitive components, such as L-glutamlne, they can be dissolved In sterile pyrogen-free water at 30-50 0 0. The solution is sterilefiltered and rapidly cooled and may thereafter be stored for a few months in a solution In a cooled state or for an even longer time In the frozen state, or stored after freeze-drying for several years In sterile powder form, until It should be used together with an amino acid solution of conventional commercial type, for Instance of the Vamin® type (amino acid nutrient composition from Kabl Pharmacla AB). Carbohydrates and fatty substances can also be added to the infusion solution. When using alpha-ketoglutarate, this must be added in the form of its sodium salt or Its esters, which Is also possible, but not necessary, in the case of L-glutamlno.

A new prepared composition as above, either in large bags or in separate vials for each substrate, is then administrated to patients exhibiting disordered nitrogen balance with a critical illness The administration being conducted during a period of from 2.4 days to several weeks e.g. with an addition of nutrition to reach a dosage of 120-170 kJ/kg body weight/day, including 0.1.

0.2 g amino acid nitrogen/kg body weight/day.

By Improving glutamlne concentration in skeletal muscle and maintaining the energy level, a critically iII patient can be faster mobilisod with less complications and thereby the patient need a shorter time for convalescence.

The treatment with alpha-ketoglutarate reduces a lowering of glutamine level In skeletal muscle.

;I I WO 93/23027 PCT/SE93/00426 By preserving the lean body mass of the patients they are less susceptible for complications during Illness and can recover faster from convalescence.

1 Example I 19 patients in acutely critical catabolic conditions with low muscle glutamine level I were studied.

SDuring five to six days a TPN program was given. The energy (70-130 kJ/bw/day) was given as equal amounts of glucose and fat together with amino acids (0.1-0.2 gN/kg bw/day). 13 patients served as controls (Group A) and 6 patients (Group B) were given an addition of 0.28 g/kg bw/day alpha.ketoglutarate (AKG) Percutaneous muscle samples were takerni Uom the quadriceps femoris muscle before and after TPN treatment. The concentrations of amino acids were determined by Ion exchange chromatography and expressed as mmol/kg wet weight (ww) muscle, Results, In both groups low glutomlne levels were noted prior to TPN treatment, 3.00±0.46 and 3.04±0.34 mmol/kg ww (wet weight) muscle In the control- and AKGgroups respectively. These values should be compared with 12.61±0.64 observed In otherwise healthy patients undergoing elective abdominal surgery. In the control the values remained unchanged throughout the study period (2.98±0,36 mmol/kg ww), whereas the glutamino concentration increased to 5.18±1.10 mrol/kg ww (P<0.05) In the AKG-group.

Table 1 Group A Group B control AKG AKG, g/kg bw/day 0.28 glutamine level mmol/kg Prior treatment 3.00±0.46 3.04±0.34 After treatment 2.98±0.36 5.18±1.10 Conclusion. The very low glutamine values in critically ill Intensive care patients are possible to Increase by AKG supplementation. This indicates that the skeletal muscle is the target organ for the AKG treatment and moreover and evidence for the metabolisatlon of AKG to glutamino in skeletal muscle tissue.

i WO 93/23027 PCT/SE93/00426 11 Example 2 12 critically III patients on mechanical ventilation under intensive care were studied during five days whilst given TPN including 95-125 kcal/24 hours and 0.1 0.15 g N /24 hours adjusted to the basal energy expenditure. The patients were randomised to receive supplementation with glutamlne (gin), 0.28g/kg bw/24 h or 0.28 AKG g/kg bw/24 h, in which case the conventional nutrition was reduced In order to obtain an Isocaloric and isonltrogenous support between the groups.

Percutaneous muscle blosles were taken before and after the study period from the lateral portion of the quadriceps femoris muscle. The muscle specimens were analysed for their content of DNA, RNA (Munro Fleck), alkali soluble protein (ASP) (Lowry) and ATP, creatlne (Cr) and phosphocreatine (PCr) (Hultman).

Or and PCr Indicates the energy status In the cell.

Result. The content of DNA, RNA and ASP in muscle was unaltered during the study period although the levels were low as compared to healthy controls. The levels of ATP were 20.5±1.3 mmol/kg fat-free solid (FFS) for the control group, 22.011.6 for the glutamlne group (Gin) and 19.1±0.8 for the AKG group respectively. This level did not change significantly during the treatment period. The oreatine (Cr) and phosphocroatlne (PCr) contents are given In Table 2.

Table 2 Controls Gin AKG PCr mmol/kg FFS Day 0 77.0±4.4 82.1±11.1 00.2±6.7 Day 5 67,1 t5,1 75.6±6.7 66.31:13.2 Cr mmol/kg FFS Day 0 71.1±12.9 66.6±6.9 75.0±1.7 Day 5 71.1±12.3 71.2±7.7 83.3±6,2* significant Conclusion. TPN supplemented with glutamlne or AGK does not alter the content of protein or RNA in muscle of critically III patients during 5 days of treatment, However, supplementation with AKG Improves significantly the energy status of the tissue In terms of maintained levels of ATP and POr in parallel with an elevation of the free creatine level, This finding is a very important and surprising finding, which can have a great impact for the treatment of critically ill patients,

Claims (5)

1. A method for the treatment of such critically ill patients that have suffered more than a 50% reduction of the intracellular glutamine in skeletal muscle-, which method comprises administering to such patients more than 0.25 g/kg body weight/day of alpha-ketoglutarate or an analogue thereof, in order to improve the protein synthesis capacity, maintaining the energy level, and to preserve the lean body mass,

2. A method of claim 1, wherein a composition providing more than 17.5 g/day of alpha-kotoglutarate is administered.

3. A method of "laim 2, wherein the administered composition besides alpha-ketoglutarate comprises glutamine or analogues thereof, L-asparagine and/or acetoacotate.

4. A method of claim 3 wherein the composition further comprises a conventional amino acid mixture. A method of claim 1, substantially as hereinbefore described in any of ito aspects. Composition for the treatment of such critically ill patients that have suffered more than a 50% reduction of the glutamine level in skeletal muscles, in order to improve !zs protein oyntheois capacity, maintaining the energy level, and to preserve the lean body mass, said composition comprising a conventional amino acid mixture and more than 25 g/l of alpha-ketoglutarate or analogues thereof, optionally wit i the addition of glutamine or analogues thereof, L-asparagine and/or acetoacetate, glucose and/or fat. A$^TLIA, WO 93/23027 PCT/SE93/00426

7. Composition of claim 6, substantially as herein- before described in any of its aspects. DATED this 18th day of MayI 1995 AB ERIK VINNARS, By its Patent Attorneys, E.F WE 1, 7 15N 'O S. Wellington) S. I I S S 4 0 A/JD/3503 94TE RNATIONAL SEARCH REPORT Iinternatonal application No. PCT/SE 93/00426 A. CLASSIFICATION OF SUBJECT MATTER IPC5: A61K 31/19, A61K 31/195 According to international P~atent classification (IPC) or to both national classification and IPC B, FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K Documentation searched other than minimum documentation to the extent that such documents are included in the fields searchod SE,DK,FI,NO classes as above Electronic data bAse consulted during the International search (name or data base and, where practicable, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category$ Citation of document, with Indication, where appropriate, or the relevant pm'sages Relevant to claim No. X WO, Al, 8903688 (AB ERIK VINNARS), 5 May 1989 1-6,9-12 (05.05.89), page 4, line 14 page 22 X WO, Al, 8703806 (VEECHI, RICHARD, 2 July 1987 1-6,9-12 (02.07.87), page 20, line 22 page 26 rurdhet documents are listed In ithe continuation or Box C. j See patent ramliy annex. Special categortej or cited documients. liter documient published after the Internatioalt filhn date or piority A document OefNngteeeattteote wtb, data sd not In conflict With the ApplaCAtlca but titelol hdrtn to be or patiu At r te nteavna fh tho principle or theory underlying tinvention It' flie dcumnt ut ublihedon r Aterthelateeaton~ fiingdai OW document ot particullar rlevane! the clhiaimed invention cannot be document which hifly throw doubts an~ ibi conidered nvil ot eannot be considered to lnvolve an inventive Cited to establish the publicaion data orf mother citation or other*~ he h oumcti ae ln special MAJORl (41 Ipecifled) 'Y document at Particular retvAncte, the elaimed invenoo cannot be O0' document rtecrring to An oral dtidosure, we. exhibition or other tOdsidftd to InVolve A tftvfntiVt It,~ %id the (Idaent It meaM combined with one ot more 6ter I=e docuiments, such tomboitton *P document published prior to the iterationat flhinl date but tater than hotel b4oAm to a person skilled to the art tile priority date claimed document member ot the same pAtifit family Dnto or the acttual comnpletion or ate intration a I sear chI Date or inaiiing or tho Intarriationai seircl repot Nam~e and imailing addreis or ili ISA/ Authotited Wflet gwadIah Patent Offiea Box 606 S-iO2 42 STOCKHOLM Wafrn Kari sOn ,jPacshnille No. +46 9 666 01 R6 Wet one No. +46 A 792I SI Potra PdrilSA1O (tecorid Ahme) (July 1002) p.- wnI'ERNATiONAL SEARCH REPORT international application No. PCT/SE 93/00426 C (Continuation), DOCUMENTS CONSIDERED TO BE RELEVANT Category* I Citation of document, with Indication, whore appropriate, of tie relevant passages 7 Relevnt to claim No, Chemical Abstracts, Volume ill, No 19, 6 November 1989 (06.11.89), (Columbus, Ohio, USA), Wernerman Jan et al, "Glutamine and ornithina-alfa-ketoglutarate but not branched-chain amino acids reduce the loss off muscle glutamine after surgical trauma", THE ABSTRACT No 172866p, Motab., Clin. Exp. 1989, 38~ 63-66 1-6,9-12 Ptm I'czrlISAJ21 (MUN~nu'Otr WORI~ d atet) (MuY 1992) I II I. INTERNATIONAL SEARCH REPORT Inkeatonal Lpplicailon No. PCT/SE 93/00426 I Box I Observations where ccrf-dln claims were ind unsearchable (Continuation of item I of first sheet) This intcrnational search report has not been established In respect or certain clims under Artilie 17(2)(a) for the following reasons: 1. W claims Nom. 7-8 because they relate to subject matter not required to be searched by this Authority, narneir A meth~od for treatment of the human or animal body by therapy, siee rule 39.1. 2. CaJmt Nom, because they relate to parts of the International application that do niot comfplyI with the prescribed requirements to such sun extent that no meaningful International search cat be carried out, specifl ly. 3. Claims No. because they are dependent clnis and are not drafted In accordance with the secoW, and third sentences of Rule 6.4(a). Box 11 Observations where unity of Invention Is la~cking (Continuation of Item 2 of first sheet) This International Searching Authority found multiple inventions In ths initernational application, ats followst 1. M As all required additional search feet were timely paid by the applicant, this internatinl search report covers ill searchablo claims. At all searchable claims could be searches without Wfort Jutufyg an additional fee, this Authority did not invite patyment of any additionatl fev. 3. El Aonlt somne of the requlred additional search feet were timely paid b the applicat, thIs international search report coes1nythose claimt fot which rest were paid 1 rpetflcally clims N'om* 4. No requlred additional stitch fees were Utielaid by the appUCLnt Cornequentiy, thIs Intetrntional search report bt reattlete to the InvenUon ftlrs metionaed In te elmil It It covered by clais Now. t I I I I Rettit to Wowes 11 The idtonil seareh ft were atripmWe by the appUcant's protest 13 No protest uw~mpmttei the payment of' ddonal search teet, Peorm Pe~lgAt~lo (tormilnuatlon of fitsi thto; 11)) (Julsy 1901) INTE RNATIONAL sEARCH REPORT Information on patent family members inteornational application No. 02/07/93 PCT/SE 93/00426 Patent document Publication Patent famliy IPublication cited In search reoport d ate member(s) date- WO-Al- 890368 18 05/05/89 AU-A- 2623888 23/05/89 DE-A- 3875322 19/11/92 tEP-A,B- 0318446 31/05/89 SE-T3- 0318446 WO-Al- 8703806 02/07/87 AU-A- 6777487 15/07/87 EPAw 0250559 07/01/88 FOMt PGrJISARzlO wpatet nifit R"IUte) VWYiu7991)