AND AT LEAST ONE OF TRYPTOPHAN, TYROSINE AND

PHENYLALANINE AND USES THEREOF

This invention relates to ingestible formulations for use in the following tests: acute tryptophan depletion (ATD), acute tryptophan loading (ATL), acute tyrosine and phenylalanine depletion (ATPD), acute tyrosine plus phenylalanine loading (ATPL), combined ATL and ATPL, and combined ATD and ATPD and their control formulation test. The invention also relates to associated methods of investigating behavioural processes in humans and other mammals associated with brain monoamines. The invention relates further to ingestible formulations for use in the treatment of psychotic and manic disorders.

The brain chemicals known as the monoamines control many physiological functions, not only in the central nervous system, but also in the periphery. Many behavioural processes in the brain are associated with these monoamines, particularly 5-hydroxytryptamine (5-HT or serotonin) and the catecholamines dopamine (DA) and noradrenaline (NA). These processes include affect, aggression, anxiety, appetite, arousal, cognition, drive, emotions, impulsivity, mood, movement, reward and self-control. The acute tryptophan (Trp) depletion (ATD) and loading (ATL) tests (Young et a/., 1985) are powerful research and diagnostic tools for studying the role of serotonin in some of the above behaviours in healthy volunteers and in those with psychiatric and other behavioural disorders. Thus, hundreds of such studies have administered these tests to examine behaviour in healthy volunteers and many disease conditions, including aggressive behaviour, alcoholism, Alzheimer's disease, anorexia nervosa, anxiety disorders, autistic disorder, bipolar affective disorder, bulimia nervosa, depression, insomnia, irritable bowel syndrome, obsessive-compulsive disorder, panic disorder, premenstrual syndrome, schizophrenia, and seasonal affective disorders.

The acute tyrosine (Tyr) and phenylalanine (Phe) depletion (ATPD) test (Sheehan et al., 1996) is similarly used to assess the role of the catecholamines dopamine (DA) and noradrenaline (NA) in normal subjects and those with psychiatric and other behavioural disorders. Because of the preferential role of serotonin in many of the conditions described above and as the ATPD test was developed by the above authors 1 1 years after the ATD test (Young et al., 1985), fewer studies of the ATPD test have been performed, e.g. in healthy volunteers, cigarette smokers, depression and manic illness.

More recently, some investigators have attempted to investigate behavioural measures under conditions leading to simultaneous depletion of serotonin, dopamine and noradrenaline by performing combined ATD and ATPD tests by giving an amino acid mixture lacking in all three relevant amino acids, namely Trp, Phe and Tyr (Leyton et al., 2003; Harrison et al., 2004; Nathan et al., 2004; Scholes et al., 2007).

Although most investigations of the ATD, ATPD and related tests have been performed in human subjects, the tests have also been conducted in other mammals, e.g. ATD in rats (Moja et al., 1989; Ardis et al., 2009) and ATPD in vervet monkeys (Palmour et al., 1998).

Both the ATD and ATPD tests involve administration to the subjects under study of a mixture of up to 15 amino acids (AA) (both essential and non- essential) which lack tryptophan (Trp) (for the ATD test) or lack phenylalanine (Phe) and tyrosine (Tyr) (for the ATPD test). By the same token, when loading, as opposed to depletion, is required, the amino acid mixture will then contain an excess of Trp (for acute Trp loading or ATL) or an excess of Phe and Tyr (for acute Tyr plus Phe loading or ATPL) and moderate amounts of the corresponding amino acids (Phe and Tyr for the ATL and Trp for the ATPL tests). Additionally, in either the depletion or loading studies, a "balanced" amino acid mixture is also administered as a control treatment. This control formulation will contain the same AA used in the depletion or loading tests, but with moderate amounts of Trp, Phe and Tyr. Table 1 shows the amino acid (AA) composition of the ATD, ATL and control balanced formulation originally published by Young et al. (1985) and gives amounts for both the traditional 100g dose and a 50g dose.

Table 1 Amino acid composition of the original acute tryptophan depletion, loading and control formulations of Young et al. (1985)

Amino Formulation 50g 100g Amino Formulation 50g 100g acid Acid

Trp Depletion 0 0 Arg All 2.45 4.90

Loading 5.15 10.30 Cys ^* 1 .35 2.70

Balanced 1 .15 2.30 Gly 1 .60 3.20

Leu All 6.75 13.50 His 1 .60 3.20

Val 4.45 8.90 Lys 4.45 8.90

He 4.00 8.00 Met ^* 1 .50 3.00

Tyr 3.45 6.90 Pro 6.10 12.20

Phe 2.85 5.70 Ser 3.45 6.90

Ala 2.75 5.50 Threo 3.25 6.50 Amounts of amino acids are in g per a 50g or a 10Og formulation. The ^* denotes that these two amino acids are given in capsules due to their offensive odours and tastes. For the ATPD test, two AA formulations have been used. In one

(Sheehan et al., 1996) only 7 AA were used, which were supplemented with Phe and Tyr in the control formulation. The AA composition of the ATPD test and its control as reported by Sheehan et al. (1996) is shown in Table 2. Because the original amounts of AA added up to only 45g in the depletion and to 57.5g in the control, formulation, the AA amounts shown in Table 2 here have been adjusted to a total dose of 50g or 100g.

Table 2 Amino acid composition of the original acute tyrosine plus phenylalanine depletion and control formulations of Sheehan et al. (1996)

Composition Control Formulation ATPD Depletion

AA 50g 100g 50g 100g

Trp 1 .09 2.17 1 .39 2.78

Leu 9.78 19.56 12.50 25.00

Val 7.61 15.22 9.72 19.44

He 6.53 13.05 8.33 16.67

Phe 5.43 10.87 0.00 0.00

Tyr 5.43 10.87 0.00 0.00

Lys 7.61 15.21 9.72 19.44

Met 2.17 4.35 2.78 5.56

Threo 4.35 8.70 5.56 1 1 .1 1

Total 50.00 100.00 50.00 100.00 Amounts of amino acids are in g per a 50g or a 10Og formulation.

In the other formulation for the ATPD test, reported by the group of S N Young (Ellenbogen et al. (1996), the composition resembled more closely that used in the ATD and ATL tests, namely containing the same 15 AA originally used. Thus, the composition of the ATPD and its control formulation by Ellenbogen et al. (1996) as detailed by Leyton et al. (2003) is shown in Table 3 below. Because the total amount of AA in the control formulation was only 85.5g, the contents in Table 3 have been adjusted to a 100g or a 50g total for the control formulation, but to only 87.6g or 43.80g total for the ATPD formulation.

Table 3 Amino acid composition of the acute tyrosine plus phenylalanine depletion and control formulations of Ellenbogen et al. (1996)

Composition Control Formulation ATPD Depletion

AA 50g 100g 43.9g 87.9g

Trp 1 .09 2.17 1 .09 2.17

Leu 6.47 12.93 6.47 12.93

Val 4.23 8.47 4.23 8.47

He 3.84 7.66 3.84 7.66

Phe 2.75 5.49 0.00 0.00

Tyr 3.32 6.64 0.00 0.00

Lys 5.26 10.53 5.26 10.53

Met 1 .43 2.86 1 .43 2.86

Threo 3.09 6.18 3.09 6.18

Ala 2.63 5.26 2.63 5.26

Arg 2.34 4.69 2.34 4.69

Cys 1 .31 2.63 1 .31 2.63 Gly 1 .54 3.09 1 .54 3.09

His 1 .55 3.09 1 .55 3.09

Pro 5.83 1 1 .67 5.83 1 1 .67

Ser 3.32 6.64 3.32 6.64

Amounts of amino acids are in g per the total doses shown for the two formulations in the Table.

From the data in Tables 1 -3, it is clear that there are 6 amino acids which must all be present in the control formulation, but that some of them (namely Trp, Phe and Tyr) must be absent in the depletion, or present in excess in the loading, formulations. The remaining 3 amino acids always present in all formulations are the branched-chain amino acids (BCAA) valine (Val), leucine (Leu) and isoleucine (lie). Their concentrations, sums and proportions in the various formulations are shown in Table 4 and their significance will be discussed below. The concentrations of all 3 BCAA shown in Table 4 are in 100g formulations, except the ATPD formulation of Ellenbogen et al. (1996), which was 87.9g. As shown, the % of BCAA in the formulations by the group of Young is around 30%, whereas that by Sheehan et al., is much higher, at 48- 61 %.

Table 4 Contents of the branched-chain amino acids valine, leucine and isoleucine in the various original depletion, loading and control formulations

Young et al. Sheehan et al. Ellenbogen et al.

Parameter (1985) (1996) (1996)

Test ATD/ATL Control ATPD Control ATPD Control [Leu] 13.50 13.50 25.00 19.56 12.93 12.93

[Val] 8.90 8.90 19.44 15.22 8.47 8.47

[lie] 8.00 8.00 16.67 13.05 7.66 7.66

[BCAA] 30.40 30.40 61 .1 1 47.83 29.06 29.06

% BCAA 30.40 30.40 61 .1 1 47.83 33.00 29.06

The amounts of the 3 branched-chain amino acids are in g per 100g or per 87.9g of formulation

There are 4 main disadvantages of current amino acid mixtures used in the ATD and ATPD tests, their loading counterparts and their control formulations: (1 ) cost; (2) un-palatability; (3) other side effects; (4) lack of specificity.

More significant side effects than unpalatability vary between a slight nausea and drowsiness to severe nausea and vomiting, which cause some individuals to drop-out of a study. Thus these side effects could confound interpretation of behavioural findings in subjects who remain in the study, while drop-out leads to smaller numbers of participants than is required for sample sizes. The problems of side-effects and resultant drop-out (attrition) have been addressed in a study (Dougherty et ai, 2008) comparing the traditional 100g dose with a 50g dose of the formulations for ATD and ATL and a 50g control formulation. It was found that the 50g dose was well tolerated by all study subjects and therefore did not result in attrition. Drop-out was confined to the 100g dose irrespective of depletion or loading and the above authors (Douigherty et ai., 2008) proposed the use of the 50g dose of the amino acid formulations for ATD, ATL and their control test. Specificity of the above tests implies that only the rate of synthesis of the monoamine under study is altered in the desired direction. The rate of serotonin synthesis in the brain can be predicted from the ratio of concentration in plasma or serum of either free Trp or total Trp to the sum of those of the 5 competing amino acids (CAA), which compete with Trp for entry into the brain, namely Val, Leu, lie, Phe and Tyr, i.e. the [Free Trp]/[CAA] and [Total Trp]/[CAA] ratios. The rate of dopamine and noradrenaline synthesis in the brain can similarly be predicted from the ratio of the sum of Tyr plus Phe to that of total Trp plus BCAA, i.e. the [Tyr + Phe]/[Total Trp + BCAA] ratio. Thus, specificity of the ATD or ATL test formulation implies that only the rate of serotonin synthesis will be decreased or increased respectively, with no change to the rate of dopamine or noradrenaline synthesis. Therefore one would expect that, whereas the [Free Trp]/[CAA] and [Total Trp]/[CAA] ratios will be either decreased (after ATD) or increased (after ATL), that of [Phe + Tyr]/[BCAA + Trp] should remain unaltered from the baseline value before intake of the AA formulations. By the same token, specificity of the ATPD or ATPL tests implies that, whereas the [Phe + Tyr]/[BCAA + Trp] ratio is either decreased (after ATPD) or increased (after ATPL), those of [Free Trp]/[CAA] and [Total Trp]/[CAA] should remain unaltered from baseline. As regards the control formulation(s) for the ATD, ATL, ATPD or ATPL tests, all three ratios should remain unaltered from baseline. In practice, existing AA formulations do not show this desired specificity. This lack of specificity across all these test formulations has been demonstrated and discussed in detail (Badawy et al., 2007, 2010a).

The main, if not only, reason for the above undesirable decreases in the [Phe + Tyr]/[BCAA + Trp] and [Trp]/[CAA] ratios in the control or the corresponding relevant depletion (or loading) formulations is the relatively larger contents of the three BCAA (i.e., Leu, Val and lie), compared with those of Phe, Tyr and/or Trp, in the original Trp (Young et al., 1985) or Tyr + Phe (Sheehan et al., 1996) formulation.

The above decreases in the [Trp]/[CAA] and [Phe + Tyr]/[BCAA + Trp] ratios therefore suggest that 5-HT, DA and/or possibly NA synthesis could be inhibited by the control formulation and the corresponding ones for ATD, ATPD and their loading counterparts, an effect that could confound interpretation of behavioural changes (or lack of them).

In general, behaviour does not seem to be influenced in normal volunteers of either gender by the ATPD or its control formulation (Lythe et al., 2005). Behaviour is also not influenced by the control formulation for ATD or ATL in normal males or females, nor in males undergoing ATD: although ATD in normal females may influence behaviour. The situation in patient populations is, however, different, not only regarding ATD, which can precipitate a depressive episode in recovered depressed subjects, but also the control formulation itself. Thus, e.g. in depressed patients, the free and/or total [Trp]/[CAA] ratio is already known to be decreased by 16-36% relative to controls. Thus, in depressed individuals, a further decrease of 50-60% induced by the control formulation might lead to an even greater depletion of brain serotonin. Even if monoamine- dependent behaviour does not appear to be influenced when measured by existing instruments, investigators using a control formulation in the knowledge that it will decrease the Trp and Tyr ratios by ~ 50% in control subjects could not rule out a greater decrease in patient populations, which may precipitate significant behavioural changes. Three previous attempts have been made to overcome the above ratio changes (Weltzin et al., 1994; Krahn et al., 1996; Booij et a/., 2005), but with only partial success. Thus, Weltzin et al. (1994) succeeded in maintaining the baseline [Trp]/[CAA] ratio by increasing the Trp content to 4.6g/100g of the traditional amino acid mixture. However, they did not measure the [Phe + Tyr]/[BCAA + Trp] ratio and it is almost certain that, with this level of Trp loading (which is 45% of that of the ATL dose), or even without it, this latter ratio will have been decreased. Booij et al. (2005) used a low dose ATD (25% of the normal one) as a control [based on a previous design by Krahn et al. (1996)]. However, although this low-dose mixture did not alter the [Tyr]/[CAA] ratio, it still decreased the [Trp]/[CAA] ratio by 42%, against a 92% decrease by the full dose. However, interpretation of some, or all, of these biochemical changes is difficult because the subjects consumed a lunch during the test procedure. Still, while the use of a low-dose mixture may be useful in studies examining the effects of sub-optimal depletion of Trp and 5-HT, it cannot be considered an appropriate control for the ATD test dose. As regards the ATPD and its control formulation, no previous attempts have been made to address the issue of their specificity.

The question of specificity of control and test amino acid formulations has been discussed and addressed experimentally (Badawy et al., 2007, 2010a, b). As the large content of BCAA (29-61 %) of the above formulations (see Table 4) appears to be the main, if not the only, reason for the undesirable changes in the above ratios, it was suggested (Badawy et al., 2007, 2010a) that the best and most logical strategy for normalisation of these ratios is by decreasing the content of BCAA in the formulations by ~ 30%. In a recent study (Badawy et al., 20106), this latter strategy was applied to the control formulation in the first instance, to be applied subsequently to the depletion and loading formulations. This is because the control formulation: (1 ) is common to both the ATD and ATPD tests and their loading counterparts, as it shares the same amino acid components, except when depletion and loading are required; (2) suffers undesirable decreases in both the [Trp]/[CAA] and [Phe + Tyr]/[BCAA + Trp] ratios. The above three ratios were determined in a detailed study (Badawy et al., 20106) in four groups of normal human subjects (n=12 each) before and hourly for 7 hours after ingestion of a control amino acid mixture with the traditional composition and three other mixtures with 20%, 30% and 40% less of the BCAA. Full normalisation of the [Free Trp]/[CAA], [Total Trp]/[CAA] and [Phe + Tyr]/[Total Trp + BCAA] ratios was observed with the formulation containing 40% less of the BCAA, thus achieving the desired specificity. As a result, we proposed that the amino acid composition of the control formulation for the above depletion and loading tests should be altered from the traditional formula to the recommended formula, as shown in Table 5.

Table 5 Composition of the traditional and recommended control amino acid formulations

Amino acids Traditional Recommended

Tryptophan 1 .15 1 .15

Phenylalanine 2.85 2.85

Tyrosine 3.45 3.45 Leucine ^* 6.75 4.05

Valine ^* 4.55 2.73

Isoleucine ^* 4.00 2.40

Alanine† 2.75 3.34

Arginine† 2.45 2.98

Cysteine† 1 .35 1 .64

Glycine† 1 .60 1 .94

Histidine† 1 .60 1 .94

Lysine† 4.45 5.41

Methionine† 1 .50 1 .82

Proline† 6.10 7.41

Serine† 3.45 4.19

Threonine† 3.25 3.95

51 .25

TOTALS 51 .25

^* Branched-chain amino acids, the decreases in which were compensated for proportionately across the remaining amino acids† in the recommended formulations.

From the amino acid composition of the two control formulations in Table 5, it can be calculated that the BCAA content of the traditional formulation (29.8%) is decreased in the recommended formulation to 17.9%.

Despite the above improvements in tolerability and specificity, it will be appreciated that there is still a need for alternative formulations. Such need has been recognized in a few previous studies. Two successful attempts have already been made to use proteins to provide new formulations. These investigations relate only to ATD and ATL. The first was using the Trp-rich protein a-lactalbumin as a means for acute Trp loading (Markus et al., 2002, 2008; Odontiadis ef al., 2003; Scrutton et al., 2007; Nieuwenhuizen et al., 2009) and the second using the Trp-deficient collagen-derived protein (or peptide) gelatine as a means for acute Trp depletion (Evers et al., 2005; Sambeth et al., 2009). In these studies, it was shown that the Trp-rich protein a-lactalbumin increased plasma Trp concentration as effectively as a Trp load, whereas the Trp-poor protein gelatine decreased plasma Trp as effectively as the amino acid mixture used in the ATD test. This suggests that administration of a Trp-rich or a Trp-poor protein can induce changes in Trp disposition similar to those observed after ATL or ATD respectively.

No attempt has been made to identify a protein(s) which may be useful for the acute tyrosine plus phenylalanine depletion or loading tests, nor for the combined depletion of Trp, Tyr and Phe.

The invention further relates to the use of one or more of these formulations as a treatment for psychotic and manic disorders.

In relation to manic illness, the ATPD test was administered, not as a diagnostic test, but as a potential therapy, in two studies. In the first (McTavish et al. 2001 ), an amino acid mixture of the composition described by Sheehan et al. (1996) (see below) was administered, whereas in the second study (Scarna et al. 2003) only a branched-chain amino acid (BCAA) mixture was administered. In both studies, manic symptoms were decreased following intake of these mixtures. This improvement occurred rapidly (beginning within 2h and lasting until 6h, the longest observation period) after acute intake of the mixtures in both studies. This acute improvement varied between 30 and 35% over and above that induced by the standard neuroleptic medication and is likely to have remained for several more hours. In the second study, the BCAA mixture was given daily for 7 days and improvement was observed both acutely (as stated above) and also on test days 4 and 7. Moreover, this improvement continued when tested 1 week later (on day 15) in patients given the BCAA mixture, but not in the placebo control group. Manic illness, whether in patients with bipolar affective disorder (manic-depressive disorder) or in those with psychosis (schizophrenia) is characterised by a central dopamine hyperactivity (Jacobs & Silverstone, 1986; Jimerson, 1987; Losonczy et al. 1987; Laruelle, 1998) hence the use in these conditions of antipsychotic (neuroleptic) drugs, which act by blocking dopamine receptors thereby diminishing dopamine actions. In the above two studies (McTavish et al. 2001 ; Scarna et al. 2003), the amino acid mixtures were administered to manic patients receiving antipsychotic medication and it is at first sight remarkable that, despite such standard medication, the amino acid mixtures were still capable of eliciting a clinical improvement over and above that exerted by the standard medication, thus suggesting the effectiveness of amino acid mixtures lacking Tyr and Phe as adjuncts to pharmacotherapy. In fact, their effectiveness can be rationalised, as they tackle the primary biochemical cause of the mania, namely the high dopamine levels, by reducing dopamine synthesis via Tyr and Phe depletion. However, a disadvantage of these two formulations is that they are also associated with Trp depletion, an effect that could precipitate an acute depressive episode in manic- depressive patients.

The present invention, in at least some of its embodiments, addresses the above named problems and needs, including the problem of lack of specificity. According to a broad aspect of the invention, there is provided the use of glyconnacropeptide in the preparation of an ingestible formulation for investigating behavioural processes in mammals, principally but not exclusively humans associated with monoamines, and ingestible formulations including glycomacropeptide which can be employed for these purposes.

According to a first aspect of the invention, there is provided an ingestible formulation including glycomacropeptide and at least one of tryptophan, tyrosine and phenylalanine.

One class of ingestible formulation of the invention includes glycomacropeptide, tryptophan, tyrosine and phenylalanine. Formulations belonging to this class can be used as a control formulation in any of the tests described herein, and as active formulations in ATL, ATPL and combined ATL and ATLPL tests.

The ingestible formulation may include by weight 79.9 to 87.0% of glycomacropeptide, 2.0 to 3.6% of tryptophan, 5.0 to 7.5% of phenylalanine and 6.0 to 9.0% of tyrosine, preferably 84.0 to 86.9% of glycomacropeptide, 2.1 to 3.0% of tryptophan, 5.0 to 6.0% of phenylalanine and 6.0 to 7.0% of tyrosine, most preferably about 84.7 to 86.7% of glycomacropeptide, 2.3% of tryptophan, 5.0 to 6.0% of phenylalanine and 6.0 to 7.0% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%. These formulations can be used as a control formulation, and as a placebo in clinic trials in manic and psychotic illnesses against ingestible formulations of the type described below which include glycomacropeptide and tryptophan, but do not include tyrosine and phenylalanine. The ingestible formulation may include by weight 69.2 to 80.0% of glycomacropeptide, 9.0 to 12.3% of tryptophan, 5.0 to 8.5% of phenylalanine and 6.0 to 10.0% of tyrosine, preferably 75.3 to 88.2% of glycomacropeptide, 10.1 to 1 1 .0% of tryptophan, 5.3 to 5.6% of phenylalanine, and 6.4 to 7.4% of tyrosine, most preferably about 77% of glycomacropeptide, 10.3% of tryptophan, 5.8% of phenylalanine and 6.9% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%. These formulations can be used in an ATL test.

The ingestible formulation may include by weight 68.0 to 80.5% of glycomacropeptide, 3.0 to 6.0% of tryptophan, 7.5 to 1 1 .0% of phenylalanine and 9.0 to 15.0% of tyrosine, preferably 76.1 to 79.1 % of glycomacropeptide, 3.1 to 4.1 % of tryptophan, 8.1 to 9.1 % of phenylalanine, and 9.7 to 10.7% of tyrosine, most preferably about 77.6% of glycomacropeptide, 3.6% of tryptophan, 8.6% of phenylalanine and 10.2% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%. These formulations can be used in an ATPL test.

The ingestible formulation may include by weight 60.0 to 74.5% of glycomacropeptide, 9.0 to 14.0% of tryptophan, 7.5 to 1 1 .0% of phenylalanine, and 9.0 to 15.0% of tyrosine, preferably 69.4 to 72.4% of glycomacropeptide, 9.8 to 10.8% of tryptophan, 8.1 to 9.1 % of phenylalanine, and 9.7 to 10.7% of tyrosine, most preferably about 70.9% of glycomacropeptide, 10.3% of tryptophan, 8.6% of phenylalanine, and 10.2% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%. These formulations may be used for combined ATL and ATPL tests. Another class of ingestible formulations includes glycomacropeptide, tyrosine and phenylalanine, but does not include tryptophan. The ingestible formulation may include by weight 83.5 to 89.0% of glycomacropeptide, 5.0 to 7.5% of phenylalanine, and 6.0 to 9.0% of tyrosine, most preferably about 89.0% of glycomacropeptide, 5.0% of phenylalanine, and 6.0% of tyrosine, with the combined amount of glycomacropeptide, phenylalanine and tyrosine totalling 100%. These formulations may be used in ATD tests.

Yet another class of ingestible formulations includes glycomacropeptide and tryptophan, but does not include tyrosine and phenylalanine. The ingestible formulation may include by weight 96.4 to 98.0% of glycomacropeptide and 2.0 to 3.6% of tryptophan, preferably 97.0 to 97.9% of glycomacropeptide and 2.1 to 3.0% of tryptophan, most preferably about 97.7% of glycomacropeptide and 2.3% of tryptophan, with the combined amount of glycomacropeptide and tryptophan totalling 100%. These formulations may be used in ATPD tests, and may be used also in the treatment of manic and psychotic illnesses such as bipolar affective disorder (manic-depressive disorder) and schizophrenia. The present inventor has realised that formulations which include glycomacropeptide and tryptophan but do not include tyrosine and phenylalanine can be used to treat manic and psychotic illnesses by reducing dopamine synthesis through Tyr and Phe depletion. Because Trp is present, Trp depletion is prevented, thereby at least reducing the likelihood of an acute depressive episode.

According to a second aspect of the invention there is provided an ingestible formulation including glycomacropeptide for use in a method of investigating behavioural processes in humans and other mammals associated with monoamines. The ingestible formulation may consist essentially of glycomacropeptide for use in a combined acute tryptophan and tyrosine plus phenylalanine depletion test.

The ingestible formulation may include glycomacropeptide, tryptophan, tyrosine and phenylalanine for use as a control treatment in a method of investigating behavioural processes in humans and other mammals associated with monoamines. The ingestible formulation may include by weight 79.9 to 87.0% of glycomacropeptide, 2.0 to 3.6% of tryptophan 5.0 to 7.5% of phenylalanine and 6.0 to 9.0% of tyrosine, preferably 84.0 to 86.9% of glycomacropeptide, 2.1 to 3.0% of tryptophan, 5.0 to 6.0% of phenylalanine and 6.0 to 7.0% of tyrosine, most preferably about 84.7 to 86.7% of glycomacropeptide, 2.3% of tryptophan, 5.0 to 6.0% of phenylalanine and 6.0 to 7.0% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%.

The ingestible formulation may include glycomacropeptide, tryptophan, tyrosine and phenylalanine for use in an acute tryptophan loading test. The ingestible formulation may include by weight 69.2 to 80.0% of glycomacropeptide, 9.0 to 12.3% of tryptophan, 5.0 to 8.5% of phenylalanine and 6.0 to 10.0% of tyrosine, preferably 75.3 to 88.2% of glycomacropeptide, 10.1 to 1 1 .0% of tryptophan, 5.3 to 5.6% of phenylalanine, and 6.4 to 7.4% of tyrosine, most preferably about 77% of glycomacropeptide, 10.3% of tryptophan, 5.8% of phenylalanine and 6.9% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%.

The ingestible formulation may include glycomacropeptide, tryptophan, tyrosine and phenylalanine for use in an acute tyrosine plus phenylalanine loading test. The ingestible formulation may include by weight 68.0 to 80.5% of glycomacropeptide, 3.0 to 6.0% of tryptophan, 7.5 to 1 1 .0% of phenylalanine and 9.0 to 15.0% of tyrosine, preferably 76.1 to 79.1 % of glycomacropeptide, 3.1 to 4.1 % of tryptophan, 8.1 to 9.1 % of phenylalanine, and 9.7 to 10.7% of tyrosine, most preferably about 77.6% of glycomacropeptide, 3.6% of tryptophan, 8.6% of phenylalanine and 10.2% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%.

The ingestible formulation may include glycomacropeptide, tryptophan, tyrosine and phenylalanine for use in a combined acute tryptophan and tyrosine plus phenylalanine loading test. The ingestible formulation may include by weight 60.0 to 74.5% of glycomacropeptide, 9.0 to 14.0% of tryptophan, 7.5 to 1 1 .0% of phenylalanine, and 9.0 to 15.0% of tyrosine, preferably 69.4 to 72.4% of glycomacropeptide, 9.8 to 10.8% of tryptophan, 8.1 to 9.1 % of phenylalanine, and 9.7 to 10.7% of tyrosine, most preferably about 70.9% of glycomacropeptide, 10.3% of tryptophan, 8.6% of phenylalanine, and 10.2% of tyrosine, with the combined amount of glycomacropeptide, tryptophan, phenylalanine and tyrosine totalling 100%.

The ingestible formulation may include glycomacropeptide, tyrosine and phenylalanine for use in an acute tryptophan depletion test. The ingestible formulation may include by weight 83.5 to 89.0% of glycomacropeptide, 5.0 to 7.5% of phenylalanine, and 6.0 to 9.0% of tyrosine, most preferably about 89.0% of glycomacropeptide, 5.0% of phenylalanine, and 6.0% of tyrosine, with the combined amount of glycomacropeptide, phenylalanine and tyrosine totalling 100%. The ingestible formulation may include glycomacropeptide and tryptophan for use in an acute tyrosine plus phenylalanine depletion test or for use in the treatment of manic and psychotic illnesses associated with monoamines. The ingestible formulation may include by weight 96.4 to 98.0% of glycomacropeptide and 2.0 to 3.6% of tryptophan, preferably 97.0 to 97.9% of glycomacropeptide and 2.1 to 3.0% of tryptophan, most preferably about 97.7% of glycomacropeptide and 2.3% of tryptophan, with the combined amount of glycomacropeptide and tryptophan totalling 100%.

According to a third aspect of the invention there is provided a method of investigating behavioural processes in humans and other mammals associated with monoamines, the method including the step of orally administering an ingestible mixture of amino acids to a human or another mammalian subject , and subsequently observing one or more behaviours of the subject , in which the mixture of amino acids includes glycomacropeptide.

The behavioural process investigated may be a behavioural process in the brain associated with monoamines. Other behaviours, such as irritable bowel syndrome, might be investigated.

According to a fourth aspect of the invention there is provided a method of treating manic and psychotic illnesses in humans and other mammals associated with monoamines including the step of orally administering an ingestible mixture of amino acids to a human or other mammalian subject, in which the mixture of amino acids includes glycomacropeptide and tryptophan. The illness may be bipolar affective disorder (manic-depressive disorder) or schizophrenia. According to a fifth aspect of the invention there is provided the use of glycomacropeptide in the preparation of an ingestible formulation for investigating behavioural processes or treating manic or psychotic illnesses in humans or other mammals associated with monoamines.

Whilst the invention has been described above, it extends to any inventive combination of the features set out above, or in the following description, drawings or claims.

Embodiments of ingestible formulations, methods and uses in accordance with the invention will now be described.

Glycomacropeptide (GMP) is a highly bioactive protein isolated from whey during cheese making. It is a light-coloured, mild tasting free flowing powder that can be used as a dietary supplement or in functional foods and beverages. Its usefulness for the above tests derives from its structure, which is rich in branched-chain amino acids (BCAA), but devoid of the three aromatic amino acids tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). Any traces of these amino acids found in a GMP preparation are attributable to small contaminations from casein, from which GMP is derived, or from the cheese curd.

GMP is claimed to have many uses. Because of the absence of Phe, it is recommended as a protein source for patients with phenylketonurea (PKU), which is a genetic defect in the metabolic handling of Phe. Because of the additional absence of the other two aromatic amino acids Tyr and Trp, GMP is recommended also as a protein source for patients with liver diseases to avoid the deleterious effects of these amino acids on brain and other bodily functions. Other uses of GMP include improved oral hygiene, tooth remineralisation and dental plaque reduction, weight management, and relief of inflammatory bowel disease.

GMP is a peptide consisting of the 64 amino acid (AA) residues shown in Table 6.

Table 6 Amino acid residues in glycomacropeptide

Of these 64 amino acid residues, there are 13 BCAA, or 20.3%. The absolute amounts (in g) per 10Og of GMP of the above amino acids are shown in Table 7.

Table 7 Amino acid (AA) composition of glycomacropeptide AA Manufacturer's Calculated from

Data AA Composition

Trp 0.00 0.00

Phe 0.50 0.00

Tyr 0.10 0.00

Leu 2.60 1 .70

Val 8.00 9.00

He 10.10 10.00

Ala 5.50 5.68

Arg 0.50 0.00

Cys 0.10 0.00

Gly 1 .10 0.96

His 0.30 0.00

Lys 5.90 5.59

Met ^* 1 .80 1 .89

Pro 12.50 1 1 .75

Ser 6.10 8.04

Threo 15.80 18.23

Asp ^* 8.60 3.39

Asn ^* 0.00 5.05

Glu ^* 20.50 15.01

Gin ^* 0.00 3.73

Total (100.00) (100.02)

BCAA 20.70 20.70

As shown in Table 7, the amounts based on the AA analysis by one manufacturer are compared with values calculated on the basis of the AA residues present as outlined in Table 6, using exact molecular weights and transforming data to a 100g amount of GMP. Thus, the calculated content is the more accurate one and discrepancies between the two sets of data can be explained as follows. (1 ) The presence of traces of Phe, Tyr, arginine (Arg), cysteine (Cys) and Histidine (His) is due to contamination with other whey, or the cheese curd, proteins. (2) The absence of Asn and Gin from the manufacturer's data is due to their hydrolysis to Asp and Glu during the analytical procedure, as suggested by the higher levels of Asp and Glu provided by the manufacturer, as compared with their theoretical amounts and the observation in Table 7 that concentrations of Asp and Glu provided by the manufacturer equal roughly the sums of the two pairs (i.e. Asp + Asn and Glu + Gin). (3) The small differences in all other remaining amino acids reflect the analytical methodology used by the manufacturer. Finally, the content of BCAA in GMP is 20.70g and 20.70%, a value close to that (20.3%) mentioned above under Table 6 based on BCAA as a proportion of the total amino acid residues.

The amino acid data in Table 8 below compare the AA contents in GMP with the formulation recommended earlier to achieve specificity of the control AA formulation for acute Trp and Tyr + Phe depletion and loading purposes. A comparison of the two sets of data in Table 8 reveals a number of interesting aspects. (1 ) As stated earlier, because GMP is devoid of Trp, Phe and Tyr (and also Arg, Cys and His), the presence of traces of these amino acids is due to contamination from other proteins. (2) Although GMP has only a fifth of the Leu content of the recommended AA formulation, this is compensated for by the presence in GMP of almost twice as much Val and lie, such that the sum of all 3 BCAA in GMP is 7% higher than in the AA formulation. (3) The lower contents in GMP, compared to the recommended AA formulation, of Arg, Gly, Lys, Met and Pro is compensated for by the higher contents of Threo, Asp, Asn, Glu and Gin, such that the total amounts of amino acids are comparable in a 50g amount. (4) The BCAA content of GMP is naturally very favourably comparable with that of our recommended AA formulation (shown in Table 5), which is designed to achieve specificity, namely 19.6% of the total AA content in GMP, versus 17.9% in our proposed AA formulation. This translates into a decrease in the [BCAA], in comparison with the original formulation by Young et al. (1985), of 35% in GMP, close enough to the 40% decrease proposed in our recommended AA formulation.

Table 8 Comparison of the amino acid composition of glycomacropeptide (GMP) with the recommended amino acid mixture proposed for enhancing the specificity of the control formulation

AA Recommended Control

AA formulation GMP

Trp 1 .15 0.00

Phe 2.85 0.25

Tyr 3.45 0.05

Leu 4.05 0.85

Val 2.73 4.50

He 2.40 4.50

(BCAA) (9.18) (9.85)

Ala 3.34 2.84

Arg 2.98 0.25

Cys 1 .64 0.05 Gly 1 .94 0.48

His 1 .94 0.15

Lys 5.41 2.79

Met 1 .82 0.94

Pro 7.41 5.87

Ser 4.19 4.02

Threo 3.95 9.1 1

Asp + Asn 0.00 4.30

Glu + Gin 0.00 9.36

Total 51 .25 50.31

BCAA (% of Total) (17.91 ) (19.57)

GMP is a unique protein in that it lacks Trp, Tyr and Phe, but is rich in branched-chain amino acids (BCAA), whose content is 35% lower than that of the traditional amino acid formulation of Young et al. (1985). This gives rise to advantageous properties in the context of the present invention, including: (1 ) the ability to be useful as the test material for the combined acute tryptophan and tyrosine plus phenylalanine depletion test; (2) the ability to achieve specificity of the individual tests (ATD, ATL, ATPD, ATPL and control) when formulated for the purposes of these individual tests. Further advantages associated with the invention are: 1 . The first protein to achieve combined ATD and ATPD; 2. With supplements of Trp, Phe and Tyr, its use can be extended 6-fold; 3. Palatability and hence acceptability (no drop-outs). 4. Use of only one or three pure amino acids (mixed with the protein); 5. No cysteine or methionine capsules are needed (it contains 50% of the Met in the AA formulation). In accordance with the invention, glycomacropeptide is provided as a template for a range of formulations for depletion, loading and control tests. In particular, GMP forms the basis of 7 different formulations for the ATD, ATPD, Combined depletion, ATL, ATPL, combined loading and control tests. An outline of each application is provided below.

Combined acute tryptophan and tyrosine plus phenylalanine depletion

Based on its amino acid composition, GMP as it stands represents a single formulation (Formulation 1 ) for use to achieve combined depletion of Trp, Tyr and Phe. It is a simple and palatable powder that can be suitably flavoured as a drink and is thus free from undesirable taste or odour properties and from both the moderate and more serious side effects of amino acid formulations. It can be used for this combined depletion application on its own in the amounts described in Table 9.

Various additions of Tyr, Phe and/or Trp to GMP could widen its use by 6- fold as follows.

The control treatment

For the control treatment (Formulation 2) in the above and other tests, GMP can be supplemented with the three amino acids, namely Trp, Phe and Tyr in the amounts shown in Table 9 for every 100g total amount of the active ingredients of this GMP-containing formulation. For amounts different from 100g, the amounts of these 3 amino acids should be adjusted to maintain their proportions (percentages) as in the 100g dose.

Acute tryptophan depletion only

For ATD only (Formulation 3), GMP should be supplemented only with Phe and Tyr, in the amounts shown in Table 9 for every 10Og total amount of the active ingredients of this formulation. No Trp should be added. For amounts different from 100g, the amounts of Phe and Tyr should be adjusted to maintain their proportions (percentages) as in the 100g dose.

Acute tyrosine plus phenylalanine depletion only and treatment of manic and psychotic illnesses

For ATPD only and for the treatment of manic and psychotic illnesses (Formulation 4), no Phe or Tyr should be added to GMP. Only Trp should be added in the amounts shown in Table 9 per 100g total amount of the active ingredients of this formulation. For amounts different from 100g, the amount of Trp should be adjusted to maintain its proportion (percentage) as in the 100g dose.

Acute tryptophan loading only

For ATL only (Formulation 5), the amounts of Trp, Phe and Tyr shown in

Table 9 should be added per 100g total amount of the active ingredients of this formulation. For amounts different from 100g, the amounts of all three amino acids (Trp, Phe and Tyr) should be adjusted to maintain their proportions

(percentages) as in the 100g dose.

Acute tyrosine plus phenylalanine loading only

For ATPL only (Formulation 6), the amounts of Trp, Phe and Tyr shown in Table 9 should be added for every 100g total amount of the active ingredients of this formulation. For amounts different from 100g, the amounts of all three amino acids (Trp, Phe and Tyr) should be adjusted to maintain their proportions

(percentages) as in the 100g dose.

Combined acute tryptophan and tyrosine plus phenylalanine loading For combined loading with Trp, Tyr and Phe (Formulation 7), these 3 amino acids should be added in the amounts shown in Table 9 for every 100g total amount of the active ingredients of this formulation. For amounts different from 100g, the amounts of all three amino acids (Trp, Phe and Tyr) should be adjusted to maintain their proportions (percentages) as in the 100g dose.

Table 9. Compositions of the Different GMP-Containing Formulations No Formulation Total amount GMP range Tryptophan Phenylalanine Tyrosine

(Amino acid amounts per 100g total)

Combined ATD 20-200g 20-200 g

and ATPD

2 Control 10Og 79.9-86.7g 2.3-3.6g 5.0-7.5g 6.0-9.0g

3 ATD only 100g 83.5-89.0g 5.0-7.5g 6.0-9.0g

4 ATPD only ^* 100g 96.4-97.7g 2.3-3.6g

5 ATL only 100g 69.2-78.7g 10.3-12.3 5.0-8.5g 6.0-10.Og

6 ATPL only 100g 68.0-77.6g 3.6-6.0g 8.6-1 1 .0g 10.2-15.0g

7 Combined ATL 100g 60.0-70.9g 10.3-14.0g 8.6-1 1 .0g 10.2-15.0g and ATPL

^* and treatment of manic and psychotic illnesses

The combined ATD and ATPD formulation (Formulation 1 ) contains only GMP without added amino acids. In all the other six formulations, the amounts of the three amino acids tryptophan, phenylalanine and tyrosine shown above are given per 100g of the total amounts of the active ingredients of the GMP- containing formulations. If any of these six formulations is given in a total amount different from 100g of active ingredients, the amounts of each of the above three amino acids should be adjusted accordingly to maintain their proportions (percentages) as those in the total 100g doses. It will be appreciated that the 100g dose is a typical dose which is generally suitable for an average human adult of around 70kg weight. A greater or a smaller dose might be provided for humans of different weight. Additionally, a greater or smaller dose might be provided for other mammals, taking into account the size and inherent physiological characteristics of the mammal. For example, it is known from ATD tests and other studies in rats that rats require about 3 to 10 times the doses that humans require, when referenced to body mass.

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