WO2012019295A1

WO2012019295A1 - Bioactive compounds in camel urine and milk

Info

Publication number: WO2012019295A1
Application number: PCT/CA2011/000953
Authority: WO
Inventors: Tony Antakly
Original assignee: Tony Antakly
Priority date: 2010-08-13
Filing date: 2011-08-12
Publication date: 2012-02-16

Abstract

The present document describes extracts from camel urine and/or milk comprising benzoate, phenylacetate and other molecular species. Also described are methods of use of the extracts and the identified compounds for the treatment of diseases, and methods of isolating the extracts.

Description

Title: BIOACTIVE COMPOUNDS IN CAMEL URINE AND MILK

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from US provisional patent applications 61/373,300, filed August 13; 2010, and 61/384,516, filed

September 20, 2010 the specifications of which is hereby incorporated by reference.

BACKGROUND

(a) Field

[0002] The subject matter disclosed generally relates to extracts from camel urine and milk. More specifically, the subject matter relates to bioactive extracts from camel urine comprising benzoate; phenylacetate and several other molecular species.

(b) Related Prior Art

[0003] The use of animal urine and milk as a source of therapeutic ingredients has been known for centuries. The use of camel urine in traditional Arab medicine has been well acknowledged since pre-medieval era, as best documented by several authors including Avicenna who, in the book "laws of medicine" described a number of medicinal ingredients from various plant and animal sources including those from human and camel urines [Avicena or Ibn Sina "Qanun fi-al-tibb"

(http://almashriq.hiof.no/ddc/projects/saab/avicenna/896/html/S1_005.html)], and Prioreschi, 2001 , Horatius press, Omaha, Nebraska).

[0004] Despite the acknowledgement of its medicinal properties, the various bioactive molecules in human urine have not yet been thoroughly characterized. This is surprising since it is now well established that urine contains many biomedically relevant compounds namely hormones, growth factors and neuropeptides. The most outstanding of those are premarin consisting of conjugated estrogens extracted from pregnant mare's urine (Stefanick, M.L., 2005, Am. J. Med. 118 SuppI 12B, 64-73), one of the most prescribed drugs in the USA used in hormone replacement therapy. Another one is human chorionic gonadotropin (hCG) approved since more than 40 years for clinical treatment of infertility where it is used to induce ovulation or stimulate testicular development and spermatogenesis. Of particular importance is the characterization of anti-cancer molecules in human urine which contains low molecular weight compounds which we named HIP (HCG- like Inhibitory Products) purified from urine extracts of pregnant women which potently block the growth of tumour cells in HIV-related Kaposi sarcoma (Kachra et al., 1997, Endocrinology 138, 4038-4041 ).

[0005] The urine of the camel is of particular interest. Desert Bedouins in Asia are known to use camel urine and milk for healing several diseases such as fever, infections, abdominal swelling, liver diseases and a variety of other ailments (Guthier-Pilters and Dagg, 1981 , University of Chicago Press, Chicago; Prioreschi, 2001 , Horatius press, Omaha, Nebraska). Such healing properties of camel urine and milk are particularly reported in Islamic literature called "Hadith" (Guthier-Pilters and Dagg, 1981 , University of Chicago Press, Chicago; Prioreschi, 2001 , Horatius press, Omaha, Nebraska) , Although these claims remain to be substantiated by scientific proof. Some of these medicinal ingredients are used even today. For example, it has been witnessed that in Saudi Arabia, people come to camel sheppards on the side of local highways to purchase urine and milk for consumption as medicinal products.

[0006] The physiology of camels is interesting. Arabian camels are called the desert ships because they can walk in the desert for days without drinking and can withstand extreme high temperature of 40°C. Camels not only survive the harsh hot weather but also resist many viral and other infections presumably due to the unique molecular composition of their immunoglobulins (Deschacht et al., 2010, J. Immunol. 184, 5696-5704). It is believed that the camel's peculiar natural antibodies display a wide antigen- recognizing repertoire may be responsible for infection resistance. It is suggested that camels display very low rates of water excretion as compared to other animals who share the same habitat (donkeys, horses, sheep) Guthier-Pilters and Dagg, 1981 , University of Chicago Press, Chicago). This results in the concentration of solutes and metabolites in the urine. Since camels do not urinate for a long time, their urine represents a valuable source of metabolites.

[0007] There is a need for bioactive compounds isolated from camel urine.

[0008] There is a need for bioactive compounds isolated from camel urine for the treatment of diseases, including cancers.

SUMMARY

[0009] According to an embodiment, there is provided an extract from camel urine comprising an NMR spectrum as set forth in figure 1.

[0010] According to an embodiment, there is provided an extract from camel urine comprising a HPLC fractionation spectrum as set forth in figure 8A.

[0011] The extract may comprise a fraction C of said HPLC fractionation spectrum as set forth in figure 8A.

[0012] According to another embodiment, there is provided extract from camel milk comprising at least one compound chosen from benzoate, lactate and citrate.

[0013] The extract may be further comprising at least one compound chosen from acetoacetic acid, fumaric acid, glyceric acid, homovanillic acid, oxalic acid, oxoprolic acid, phenylpyruvic acid, propionylglycinic acid, pyruvic acid, 2-hydroxyglutaric acid, 2-oxoadipic acid, 2-oxoglutaric, 3-hydroxybutiric acid, 3 hydroxypropionic acid, 4-hydroxyphenyllactic acid, and 4- hydroxyphenylpyruvic acid.

[0014] According to another embodiment, there is provided a pharmaceutical composition comprising an extract from camel urine, camel milk, or combination thereof and a pharmaceutically acceptable carrier. [0015] According to another embodiment, there is provided a pharmaceutical composition comprising at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof; and a pharmaceutically acceptable carrier.

[0016] According to another embodiment, there is provided a use of an extract from camel urine, camel milk, or combination thereof for the preparation of a medicament for the treatment of a disease.

[0017] According to another embodiment, there is provided a use of an extract from camel urine, camel milk, or combination thereof for the treatment of a disease.

[0018] According to another embodiment, there is provided a use of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof for the preparation of a medicament for the treatment of a disease.

[0019] According to another embodiment, there is provided a use of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof for the treatment of a disease.

[0020] The at least one compound may be benzoate, phenylacetate or a combination thereof. The at least one compound may be chosen from Butyrylglycinic acid, Citric acid, Ethylmalonic acid, Glyceric acid, Glycolic acid, Glutaric acid, Hexanoylglycenic acid, Hippuric acid, Homovanillic acid Homogentisic acid, Isobutyrylglycinic acid, Isovalerylglycinic acid, Lactic acid, Malonic acid, Methylcitric acid, Methylmalonic acid, Methylsuccinic acid, N- acetylaspartic acid, Oxalic acid, Oxoprolinic (pyroglutamic) acid, Phenylpropioniglycinic acid, Phenyllactic, Propionylglycinic acid, Pyruvic acid, Sebacic acid, Suberic acid, 2-hydroxyadipic acid, 2-hydroxyglutaric acid, 2- hydroxyisovaleric acid, 2-hydroxyphenylacetic acid, 2-methyl-3-hydroxybutiric acid, 2-methylacetoacetique acid, 2-methylbutyrylglycinic acid, 3- hydroxyisovaleric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxy butyric acid, 3-hydroxypropionic acid, 3-methylcrotonylglycinic acid, 3-methylglutaconic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid.

[0021] The disease may be a cancer, a neoplasm including multidrug resistant tumors, various haematological malignancies, and the cancer may be chosen from a breast cancer, colon cancer, a prostate cancer, malignant gliomas, and a thyroid cancer .

[0022] The disease may be a metabolic defect in a urea-cycle enzyme.

[0023] The disease may be any metabolic disease such as diabetes or pathologic obesity that is regulated by the Peroxisome Proliferator-activiated Receptor (PPAR) which belongs to the steroid receptor superfamily

[0024] The disease may be any metabolic disease such as diabetes or pathologic obesity that is regulated or by the DNA binding partner of the Peroxisome Proliferator-activiated Receptor (PPAR) which is the retinoid X receptors (RXR),

[0025] The disease may be chosen from multiple sclerosis, sickle cell anaemia, amyotrophic lateral sclerosis, and Huntington's disease.

[0026] According to another embodiment, there is provided a method of treating a disease in a subject in need thereof by administering to the subject a therapeutically effective amount of an extract camel urine, camel milk, or combination thereof.

[0027] According to another embodiment, there is provided a method of treating a disease in a subject in need thereof by administering to the subject a therapeutically effective amount of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof.

[0028] The compound may be benzoate, phenylacetate or a combination thereof. The at least one compound may be chosen from Butyrylglycinic acid, Citric acid, Ethylmalonic acid, Glyceric acid, Glycolic acid, Glutaric acid, Hexanoylglycenic acid, Hippuric acid, Homovanillic acid Homogentisic acid, Isobutyrylglycinic acid, Isovalerylglycinic acid, Lactic acid, Malonic acid, Methylcitric acid, Methylmalonic acid, Methylsuccinic acid, N- acetylaspartic acid, Oxalic acid, Oxoprolinic (pyroglutamic) acid, Phenylpropioniglycinic acid, Phenyllactic, Propionylglycinic acid, Pyruvic acid, Sebacic acid, Suberic acid, 2-hydroxyadipic acid, 2-hydroxyglutaric acid, 2- hydroxyisovaleric acid, 2-hydroxyphenylacetic acid, 2-methyl-3-hydroxybutiric acid, 2-methylacetoacetique acid, 2-methylbutyrylglycinic acid, 3- hydroxyisovaleric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybutyric acid, 3-hydroxypropionic acid, 3-methylcrotonylglycinic acid, 3-methylglutaconic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyl lactic acid, and retinoid derivative such as 9-cis retinoic acid.

[0029] The disease may be a cancer, a neoplasm, a multidrug resistant tumors, a haematological malignancy, and the cancer may be a breast cancer, a colon cancer, a prostate cancer, a malignant glyoma, and a thyroid cancer.

[0030] The disease may be a metabolic defect in a urea-cycle enzyme.

[0031] The disease may be chosen from multiple sclerosis, sickle cell anemia, amyotrophic lateral sclerosis, and Huntington's disease.

[0032] According to another embodiment, there is provided a method of producing an extract from camel urine comprising an NMR spectrum as defined in figure 1 by removing a fraction of at least 10 kDa from a camel urine sample to produce an extract having at least one molecule of molecular weight lower than 10 kDa.

[0033] The removal of the molecules may be done by filtration of said camel urine sample. The filtration may be with a filter membrane. The filter membrane may have pores preventing passage of molecules having at least 10 kDa or more. The filter membrane may be treated with water prior to filtration of said camel urine sample. [0034] The following terms are defined below.

[0035] The term "molecular weight cut-off' or cut-off value is defined as the molecular weight at which a where the membrane or filter will reject, or prevent the passage of 90% of the solutes. Hence, a filter with a molecular weight cut-off of about 10 kDa will prevent the passage of molecules having molecular weight of at least 10 kDa.

[0036] The term "pharmaceutically acceptable carrier" is intended to mean a preservative solution, a saline solution, an isotonic (about 0.9%) saline solution, or about a 5% albumin solution, suspension, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic substances suitable for delivery to a patient may be included in the compositions of the present invention. The compositions may be solutions, suspensions or any appropriate formulation suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques.

[0037] The term "nutraceutical" is intended to mean a food or food product that provides health and medical benefits, including the prevention and treatment of disease.

[0038] The term "a non-food ingredient" is intended to mean an ingredient that may be added to food but that may not be a food ingredient contributing to the caloric content of the food per se.

[0039] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

[0041] Fig. 1 illustrates the ¹H NMR spectra of camel urine compared to human urine. The full spectra are shown in the lower part of the figure. The expanded spectra of the region 7.1-7.9 (boxed area) are shown on the top. The phenylacetate and benzoate NMR peaks are more intense in camel than human. The numbers 1a, 1 b, 1c and 2b, 2c, 2d refer to the characteristic peaks emanating from the carbons identified by the same numbers on the chemical atomic structures shown. Other compounds labeled by the indicated numbers correspond to the chemicals shown in the boxed area. Creatinine, which constitutes a standard reference in urine analysis, is also shown. The star (*) identified peak is present only in camel but not human.

[0042] Figs. 2A and 2B illustrate the GS-MS spectra of benzoic acid and phenylacetic acid identified in camel urine using GS-MS. Fig. 2A shows the entire spectrum of camel urine. Fig. 2B shows spectra identified in camel urine. Right panel shows the mass spectrum of benzoic acid on El ionization, showing molecular ions C₇H₆0₂ ^'+ at m/z= 122, C₆H₅CO⁺ benzoyl ions at m/z=105, C₆H₅ ⁺ ions at m/z=77 and C₄H₃ ⁺ ions at m/z=51. Left panel corresponds to phenylacetic acid on El ionization, showing molecular ions C₈H₈02⁺ at m/z= 36 and strong peak at m/z=91 which is characterized by C₆H₅CH₂ ⁺ ions.

[0043] Fig. 3 illustrates the mechanisms of ammonia diversion from the urea cycle with the administration of phenylacetate and benzoate.

[0044] Figs. 4A and 4B illustrates (A) the fractionation of camel urine using reversed phase-HPLC and (B) the bioassay of the collected fractions following HPLC separation. Panel A shows the elution profile of camel urine from the HPLC column. Y axis indicates absorbency at 220 nm. x-axis indicated time of elution in minutes. The contents of the indicated elution tubes are pooled into five fractions named A, B, C, D, E, then lyophilized. Panel B shows the bioactivity of the five fractions as tested in vitro for cell proliferation using the MCF7 cells. Y-axis shows % cell proliferation of the test material as compared to the Vehicle (Veh), i.e a control consisting of culture media alone.

[0045] It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In embodiments there is disclosed an extract from camel urine. The extract of the present invention of the present invention comprise the NMR spectrum as defined in Fig. 1.

[0047] Referring now to Fig. 1 , a ¹H NMR spectra of camel urine showing significant differences as compared to human urine. The most striking finding in the camel urine is marked, high levels of both benzoate and phenylacetate (Fig. 1 ). In contrast, human, rat, elephant and dromedary urines display very little expression of these two compounds as shown in Table 1.

Table 1. Concentrations of four relevant metabolites in the urine of three adult male camels as compared to the corresponding average values in human *. Values are expressed in mol/mmol of creatinine

Benzoate Phenylacetate Hippurate Citrate

Camel 1 794 85 91 2

Camel 2 1374 28 6436 11

Camel 3 2284 47 2166 BDL

Human 2 BDL 252 311

Elephant 9 2 9836 UD

Dromedary 1 3 UD 7285 UD

Dromedary 2 6 UD 5790 3

Rat 2 1 507 1833

Notes:* The numbers shown for "human" are average values in urines from 13 adult men (average age 34). UD: means undetectable. Actual values are shown in Table 2

[0048] The number of NMR signals in camel urines is much less than the corresponding signal numbers in human urines. The spectra show that the camel urine has lower level of sugar compared to human samples. More notable is the presence of peaks that are only present in camel urine. For example, an intense peak around 5.3 ppm is always present in the camel urine spectra and it is absent from any human urine spectra, indicating that some metabolites are unique to camel. Other intense peaks appear at 1.85 ppm, 2.255 ppm, 2.93 ppm, 3.205 and 3.91 ppm repeatedly demonstrated in camel spectra.

[0049] While the urea peak is quite comparable in camel and human urine, creatinine and citrate signals demonstrate higher concentration in human urine than camel. NMR results show that the level of benzoate (benzoic acid) in camel urine is higher than in human urine. Similarly, for phenylacetate there is a significant difference between camel and human. In contrast, the creatinine level in human urine is slightly higher than camel. Quantitative assays are done using established methods according to Sweetman, 1990. After normalizing the creatinine level we found that camel contains remarkably more benzoate than human (Table 1 ). Similarly, phenylacetate is significantly higher in camel (Table 1). The results confirm that the detected metabolites in camel urine have a unique profile with respect to their type, number and concentrations. The high level of benzoate and phenylacetate represent an exceptional difference of camel urine.

[0050] The clinical use of phenylacetate (PA) and benzoate in lowering plasma ammonium levels in patients with hyperammonemia represent the hallmark of their therapeutic action (Enns et a/. , 2007, N. Engl. J. Med. 356, 2282-2292). In certain metabolic diseases resulting from defects in urea-cycle enzymes, ammonium which cannot be converted to urea accumulates to a toxic level that can be lethal. This drug combination of PA and benzoate, marketed as Ammonul®, is useful to treat patients with inborn errors of metabolism of the urea-cycle enzyme and prevents several complications such as encephalopathy and death (Enns et a/., 2007, N. Engl. J. Med. 356, 2282-2292). In fact, phenylacetate, through mitochondrial conjugation with glutamine, results in phenylacetyglutamine. Similarly, benzoate combines with glycine thus forming benzoylglycine (hippurate) (see Fig. 3). These two nontoxic compounds are easily eliminated in the urine. Using this well established drug combination consisting of phenylacetate and benzoate, Enns (Enns et a/. , 2007, N. Engl. J. Med. 356, 2282-2292) reported results of a 25 year clinical study demonstrating an overall survival of 84 % following the treatment.

[0051] In addition to their usefulness in treating urea cycle enzyme defects, both phenylacetate and benzoate are found to be potentially useful for the treatment of multiple sclerosis, as disease characterized by demyelination presumably due to a T-cell auto-immune response. In a mouse model of multiple sclerosis (Brahmachari et a/. , 2009, J. Immunol. 183, 5917- 5927), both compounds exert immunomodulatory and anti-inflammatory effects which are mediated by immune T-cells and involve among others, suppression of NF-κΒ and nitric oxide synthetase; these actions ultimately result in the alleviation of the disease. [0052] Phenylacetate (PA) and phenyl butyrate (PB), the parent compound from which it is metabolized in humans and animals, both display other important bioactivities other than those cited above. Chemically, these metabolites (PA and PB) belong to a group of aromatic fatty acids having a stable phenyl ring. They are proven useful in many diseases, ranging from cancer, sickle cell anemia, ALS and Huntington's disease.

[0053] Phenylacetate is originally discovered as a plant hormone that regulates cell growth it has been studied in the past two decades as anticancer arid cellular differentiating compounds. PA inhibits the growth of several cancer cell types of different lineages and, in some instances, it promotes their differentiation to a non-cancerous phenotype. Of interest are the effects of PA and PB on gliomas and neuroblastomas, originally thought to be mediated by inhibition of protein prenylation cholesterol and fatty acid biosynthesis. Subsequent studies have demonstrated that PA and PB inhibits the growth of several neoplastic cell types including breast cancer (Liu et a/., 2007, Cancer Chemother. Pharmacol. 59, 217-225), prostate cancer, colon cancer and thyroid carcinoma. Furthermore, PA and PB can potentiate the action of other hormones such as estrogens and retinoids in regulating cancer cell growth. These anti-cancer actions of PA and PB have prompted clinical trials especially that these compounds display little toxicity (Lin et a/., 2009, Clin. Cancer Res. 15, 6241-6249; Cudkowicz et al., 2009, Amyotroph. Lateral. Scler. 10, 99-106). The overall picture that emerges is that PA and PB act as mild agents that synergize or cooperate with other bioactive agents, some of which may be endogenous. In fact, physiological or pharmacologic actions of PA and PB can act alone or in synergy with differentiating agents such as retinoid to down-regulate key cell-cycle genes as well as angiogenic and growth factors and that promote tumor cell growth. Studies regarding the molecular mechanisms of action of PA has shed light on two important pathways for its action: induction of histone acetylation which regulates chromatin structure and regulation of steroid / nuclear receptor gene expression. In fact, PA and PB can bind and activate the Peroxisome Proliferator-activiated Receptor γ (PPAR) which belongs to the steroid receptor superfamily of ligand-activated transcription factors (Samid et al., 2000, Clin. Cancer Res. 6, 933-941). The latter finding would suggest that these compounds act similar to steroidal hormonal drugs and would explain the pleotropic action of PA and PB compounds in different diseases and different physiological settings to regulate metabolism, inflammation, cancer growth and cell differentiation.

[0054] Thus, although camel urine has been known for its healing properties in the Arabic traditional medicine which is practiced even today in Saudi Arabia, the identification of its active molecules has not yet been reported.

[0055] There is also disclosed a method of preparing camel urine extracts. The extracts may be prepared through the fractionation of camel urine samples using techniques well known in the art. For example, the sample may be fractionated using exclusion chromatography techniques or any other method known in the art for the separation of molecules based on their size, shape, hydrophilicity, hydrophobicity, charge, polarity, or any other inherent physical characteristics which may be employed for the isolation (or exclusion) of molecules from the urine sample of interest. The preferred method for the preparation of extracts according to the present invention is by using size exclusion filtering membranes excluding a fraction of molecules having a molecular weight larger than a set weight (i.e. having a predetermined molecular weight cut-off). The urine samples are filtered with the filtering membrane to fractionate the sample in a filtrate comprising smaller molecules and a retentate of larger molecules. Suitable molecular weight for exclusion are from at least 100 kDa, or from at least 50, or from at least 30 kDa, or from at least 10 kDa, or from at least 3 kDa. The preferred molecular weight for exclusion (i.e. the preferred molecular weight cut-off) is from at least 0 kDa.

[0056] In embodiments, there is described an extract from camel urine which comprises an NMR spectrum as set forth in Fig. 1. The extract may be used for the preparation of medicaments and/or for the treatment of various diseases such as cancer, including but not limited to breast cancer, prostate cancer, and thyroid cancer; metabolic diseases such as defects in the urea- cycle enzymes; multiple sclerosis, sickle cell anaemia, amyotrophic lateral sclerosis and Huntington's disease.

[0057] In embodiments, there is described an extract from camel urine which comprises a HPLC fractionation spectrum as set forth in Fig. 8A, and preferably comprising fraction C of said HPLC fractionation spectrum as set forth in Fig. 8A. The extract may be used for the preparation of medicaments and/or for the treatment of various diseases such as cancer, including but not limited to breast cancer, prostate cancer, and thyroid cancer; metabolic diseases such as defects in the urea-cycle enzymes; multiple sclerosis, sickle cell anaemia, amyotrophic lateral sclerosis and Huntington's disease.

[0058] In embodiments, there is described an extract from camel milk prepared according to the method described above. Desert Bedouins and even ordinary people living in Saudi Arabia today are also known to mix camel urine and milk to get therapeutic effects. We therefore examined whether camel milk contains any remarquable molecules. Indeed, the milk from adult camels contains significant amounts of benzoate (table 5) albeit in lower amounts than in camel urine. However, camel milk is devoid of phenyl acetate.

[0059] In use, the extracts of the present invention may be used alone, or combined to each other for any therapeutic use for which they may be useful. According to another embodiment, the extracts of the present invention may also be used in food composition, such as for example nutraceutical compositions. Nutraceuticals are food or food product that provides health and medical benefits, including the prevention and treatment of disease. Products according to the present invention may range from isolated nutrients, dietary supplements, prebiotic, specific diets and herbal product supplement, and processed foods such as cereals, soups, and beverages. The compositions of the present invention may also be used as a functional food, a food ingredient, a food additive, a natural food additive, a non-food ingredient, a cosmeto-food, a pharmaceutical, and a food supplement."

[0060] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE 1

Animals

[0061] Urine are collected from 15 male and 5 female adult camels (Camelus dromedarius) raised in separate geographic areas in Saudi Arabia, Jordan, Egypt, and Bahrain. In addition, urines are obtained from 2 adult dromedaries, 2 alpaca, and one llama and two elephants from a zoo near Montreal, Canada. Furthermore, urines are obtained from two adult male Sprague-Dawley laboratory rats. The urine samples are transported to the laboratory either on ice or after freezing.

EXAMPLE 2

NMR Spectroscopy

[0062] A urine sample (0.5 ml) is centrifuge-filtered at 2,000 g and 4°C for 15 minutes by using 10 kDa centrifugal filter tubes (Millipore, Billerica, MA, USA) to remove molecules and particles in the sample that have molecular weights of at least 10 kDa. The centrifugal filters are washed several times with 0.5 ml water, then centrifuged at 12000 x g at 4°C to remove glycerol from the filter membrane until no NMR signal is observed in the filtrate. Four hundred microliters of the filtrate are transferred to a 5 mm NMR tube and 100 μΙ of D₂0 (Cambridge Isotope Laboratories, Inc.) are added to the NMR tube.

[0063] The NMR spectra are obtained using a Varian Inova 600 MHz instrument (Palo Alto, California). All NMR experiments are obtained using a one-dimensional presaturation sequence with gradients pulse sequence (zgpr in the standard Bruker pulse sequence library). The 1 H NMR spectra are recorded by collecting 64 and 128 free induction decays (FIDs) and digitized into 64 K complex data points over a spectral width of 12 ppm. The recycle delay time is set to 5 sec and the receiver gain is kept at a constant value of 32. Before Fourier transform, the FID values are multiplied by an exponential function equivalent to 1.0 Hz line broadening factor. All spectra are then visually phased and adjusted manually where necessary.

EXAMPLE 3

Identification of urine compounds using NMR spectroscopy

[0064] Select spectra are examined using the Chenomx NMR Suite (Chenomx Inc., Edmonton, Alberta, Canada). The metabolites' signals are examined and the assignments are made by fitting and comparing of the experimental spectra with reference spectra from the database using the Chenomx NMR Profiler software.

EXAMPLE 4

Identification of phenylacetate and benzoate in camel urine using Mass

Spectrometry

[0065] Target chemicals are determined using an Agilent 7890 A GC (or equivalent) with 5975C MSD equipped with an Agilent 7683B automatic liquid sampler and an HP-5MS GC (or equivalent) column (30 m, 0.25mm i.d., 0.25 Mm film thickness). Helium is used as the carrier gas, with a column flow rate of 1.0 ml/min in constant flow mode. Injector temperature is 280°C. The GC-MSD interface quadruple and the ion source temperatures are set at 280, 170 and 230°C, respectively. The GC oven temperature is kept at 50°C for 0.5 min, followed by the first ramp at 5°C / min to 225°C, second ramp at 3°C / min to 280°C, and holding for 1 min. Prior to quantification process, mass spectra and GC retention times of each compound from mlz 50 to 550 are obtained in full scan mode. The mass spectrometer used is operating in electron impact with an ionization voltage of 70 EV. The sample is injected in pulsed splitless mode.

[0066] The demonstration of BNZ and PAA in camel urine are independently confirmed by three laboratories using Gas Chromatography- Mass Spectrometry {GC-MS) (Fig. 2). The MS at the top of each peak is searched using NIST (National Institute of Standards and Technology, an agency of the U.S. Commerce Department) library with the best fit. Usually the best-matched 25 compounds are displayed. The mass spectrum of benzoic acid on El ionization shows molecular ions CyHe * at m/z= 122. Their intensity is 95% of the base peak, which is characterized by C₆H₅CO⁺ benzoyl ions at m/z=105. Other prominent fragments are C₆H₅ ⁺ ions are at m/z=77 (90%) and C₄H₃ ⁺ ions at m/z=51 (70%). The mass spectrum of phenylacetic acid on El ionization shows molecular ions C₈H₈02⁺ at m/z=136. Their intensity is 30% of the base peak, which is characterized by C₆H₅CH2⁺ ions at m/z=91.

EXAMPLE 5

Identification of other components of camel urine using Mass

Spectrometry

[0067] Urine samples from 2 camels are subjected to mass spectrometry as described above in Example 4. EXAMPLE 6

Identification of other components of camel urine using Mass

Spectrometry compared to human urine

Table 3. Organic Acids and Metabolites in Urine

Metabolite name (acid) Camel Human

Average SEM Average SEM

ACETOACETIC 0.00 0.00 1.55 0.19

ADIPIC 1.33 0.22 1.00 0.08

BENZOIC 1484 00 124.82 2 45 ^■

BUTYRYLGLYCINE 1.67 0.22 0.00 0.00

CITRIC 4.33 0.88 310.82 19.16

ETHYL ALONIC 2.00 0.17 2.82 0.19

FU ARIC 0.33 0.08 0.55 0.06

GLUTARIC 0.67 0.17 0.45 0.05

GLYCERIC 4.33 0.36 4.00 0.46

GLYCOLIC 6.00 0.54 43.73 1.64

HEXANOYLGLYCINE 0.67 0.08 0.00 0.00

HIPPURIC 2897.67 458.52 251.73 24.35

HOMOGENTISIC 0.67 0.08 0.00 0.00

HO OVANILLIC (HVA) 1.33 0.11 2.00 0.07

ISOBUTYRYLGLYCINE 5.00 0.38 0.27 0.03

ISOVALERYLGLYCINE 3.00 0.23 0.73 0.08

LACTIC 60.67 5.65 22.45 2.12

MALONIC 0.33 0.08 0.09 0.02 ETHYLCITRIC 4.00 0.39 1.36 0.05

METHYLMALONIC 3.00 0.38 0.91 0.02

METHYLSUCCINIC 9.33 0.73 0.91 0.05

MEVALONOLACTONE 0.00 0.00 0.18 0.04

N-ACETYLASPARTIC 0.33 0.08 4.36 0.17

OXALIC 102.00 8.66 22.27 0.63

OXOPROLINE(PYROGLUTAMIC) 3.67 0.33 27.36 1.08

PHENYLPROPIONYLGLYCINE 0.67 0.17 0.00 0.00

PHENYLACETIC 53.33 4 64 U UU 0 00

PHENYLLACTIC . 0.33 0.08 0.00 0.00

PHENYLPYRUVIC 0.00 0.00 0.00 0.00

PROPIONYLGLYCINE 0.67 0.17 0.09 0.02

PYRUVIC 1.00 0.09 11.55 1.28

SEBACIC 2.00 0.17 0.00 0.00

SUBERIC 2.67 0.24 1.09 0.12

SUCCINYLACETONE 0.00 0.00 0.00 0.00

VANILLYMANDELIC(VMA) 0.00 0.00 1.36 0.07

2-HYDROXYADIPIC 1.00 0.09 0.00 0.00

2-HYDROXYGLUTARIC 1.33 0.11 3.18 0.18

2-HYDROXYISOCAPROIC 0.00 0.00 0.00 0.00

2-HYDROXYISOVALERIC 1.00 0.14 0.09 0.02

2-HYDROXYPHENYLACETIC 1.67 0.13 0.45 0.07

2-METHYL-3-HYDROXYBUTYRIC 8.67 0.66 2.55 0.09

2-METHYLACETOACETIC 2.33 0.19 1.91 0.06 Table 3. Organic Acids and Metabo ites in Ur ne (continued)

2- ETHYLBUTYRYLGLYCINE 1.00 0.14 0.00 0.00

2-OXOADIPIC 0.00 0.00 0.55 0.05

2-OXOGLUTARIC 0.00 0.00 19.09 1.58

2-OXOISOVALERIC 0.00 0.00 0.73 0.17

3-HYDROXYBUTYRIC 1.67 0.22 1.00 0.05

3-HYDROXYGLUTARIC 0.00 0.00 0.64 0.05

3-HYDROXYISOVALERIC 30.33 2.30 22.00 0.86

3-HYDROXY-3- ETHYLGLUTARIC 61.67 6.35 23.91 0.96

3-HYDROXYPROPIONIC 20.00 1.89 20.27 1.02

3-METHYLCROTONYLGLYCINE 0.33 0.08 0.18 0.04

3-METHYLGLUTACONIC 5.00 0.43 2.55 0.12

3-METHYLGLUTARIC 0.00 0.00 0.09 0.02

4-HYDROXYBUTYRIC 0.00 0.00 0.00 0.00

4-HYDROXYPHENYLACETIC 52.67 4.48 11.55 0.49

4-HYDROXYPHENYLLACTIC 7.33 0.56 0.36 0.04

4-HYDROXYPHENYLPYRUVIC 0.00 0.00 2.00 0.10

Values are in μηιοΙ/mmol of creatinine, Number of subjects: Camel, N=3, Human, N=13

EXAMPLE 7

Comparison of four metabolites in the urine of animals related to camels using Mass Spectrometry

Table 4. Concentratration of Benzoate, Phenylacetate, Hippurate and

Citrate in urine of animals related to camel

ote: : means undetectable.

[0068] Concentrations of four relevant metabolites in the urine of animals raised in the zoo. The alpaca and llama are species related to the camel. Rats were raised in a laboratory. A total of 57 metabolites are assayed but this table shows only 4. Values are expressed in pmol/mmol of creatinine.

EXAMPLE 8

Fractionation of camel urine using reversed phase-HPLC

[0069] Adult male camel urine previously lyophilized is reconstituted with an equivalent amount of 0.1 % trifluoroacetic acid (TFA) in water, then placed on ice and processed for HPLC within two hours. 10 to 100 microliters of this reconstituted urine solution is injected into either a Waters™ HPLC apparatus or an equivalent Beckman "Gold"™ HPLC apparatus. Both apparatuses are fitted with a 300 mm C-18™ column. Elution from the column is done using an increasing linear isocratic gradient of acetonitrile in water containing 0.1 % TFA. The gradient is increased from 5% to 75% acetonitrile. The absorbancy is monitored at 220 nm wavelength during the elution and fractions are collected manually into plastic tubes. After collection, the fractions are promptly placed in a Savant™ Speed-vac apparatus in order to dry the samples. Now referring to Fig. 4A, the gradient is shown with the x- axis indicating time, in minutes. The absorbency at 220 nm is recorded on the y-axis.

EXAMPLE 9

Bioassay of the collected fractions following HPLC separation

[0070] The fractions (peaks) indicated by arrows on Fig. 4B. are lyophilized and each is reconstituted in one (1) ml of RPMI culture medium (without serum) and tested for biological activity using MCF7 cells. The biological activity is tested in absence (Vehicle control, i.e. RPMI medium alone) or presence of 10 microliters of the reconstituted fractions. It can be seen from Fig. 4B that fraction C display an inhibitory activity. EXAMPLE 10

Assessment of cell proliferation

[0071] The breast cancer cell line called MCF7 is obtained from the American Type Tissue Culture Collection. The KS cells are passaged and the culture medium is changed every other day in presence or in absence of any of the samples mentioned above for the indicated periods ranging from 24-96 hrs. ³H-thymidine incorporation is measured as described (Guo WX et al., 1996, Am J Pathol 148: 1999-2008). In most experiments, data are reported as means ± SEM of sextuplet determinations. Statistical analysis is determined by student t-test.

EXAMPLE 11

Comparison of four metabolites in the milk of camels using Mass

Spectrometry

Table 5. Concentrations of Benzoate, Phenylacetate, Hippurate and

Citrate in the milk of 5 adult camels.

Benzoate Phenylacetate Lactate Citrate

Camel #EG2 20 UD 30 3326

Camel #EG3 23 UD 42 3091

Camel #EG4 1 1 UD 55 3087

Camel #EG5 38 UD 46 3461

Camel #EG6 17 UD 27 3455

Note:^*UD: means undetectable.

[0072] Concentrations of four relevant metabolites in the milk of 5 adult camels. A total of 57 metabolites are assayed but this table shows only 4. Values are expressed in mol/mmol of creatinine* EXAMPLE 12

Identification of other components of camel milk using Mass

Spectrometry

Table 6. Organic Acids and Metabolites in Milk

# ACIDES mol/L

1 ACETOACETIQUE 1

2 ADIPIQUE 0

3 BENZOIQUE 38

4 BUTYRYLGLYCINE 0

5 CITRIQUE 3461

6 ETHYLMALONIQUE 0

7 FUMARIQUE 1

8 GLUTARIQUE 0

9 GLYCERIQUE 2

10 GLYCOLIQUE 0

11 HEXANOYLGLYCINE 0

12 HIPPURIQUE 0

13 HOMOGENTISIQUE 0

14 HOMOVANILLIQUE (HVA) 1

15 ISOBUTYRYLGLYCINE 0

16 ISOVALERYLGLYCINE 0

17 LACTIQUE 46

18 MALONIQUE 0

19 METHYLCITRIQUE 0

20 ETHYLMALONIQUE 0

21 METHYLSUCCINIQUE 0

22 EVALONOLACTONE 0

23 N-ACETYLASPARTIQUE 0

24 OXAUQUE 9

25 OXOPROLINE(PYROGLUTA IQUE) 4

26 PHENYLPROPIONYLGLYCINE 0

27 PHENYLACETIQUE 0

28 PHENYLLACTIQUE 0

29 PHENYLPYRUVIQUE 18

30 PROPIONYLGLYCINE 1

31 PYRUVIQUE 10

32 SEBACIQUE 0

33 SUBERIQUE 0

34 SUCCINYLACETONE 0

35 VANILLYMANDEUQUE (VMA) 0

36 2-HYDROXYADIPIQUE 0

37 2-HYDROXYGLUTARIQUE 1

38 2-HYDROXYISOCAPROIQUE 0

39 2-HYDROXYISOVALERIQUE 0

40 2-HYDROXYPHENYLACETIQUE 0

41 2-METHYL-3-HYDROXYBUTYRIQUE 0

42 2-METHYLACETOACETIQUE * 0

43 2-METHYLBUTYRYLGLYCINE 0

44 2-OXOADIPIQUE 7

45 2-OXOGLUTARIQUE 15

46 2-OXOISOVALERIQUE 0

47 3-HYDROXYBUTYRIQUE 1

48 3-HYDROXYGLUTARIQUE 0

49 3-HYDROXYISOVALERIQUE 0

50 3-HYDROXY-3-METHYLGLUTARIQUE 0

51 3-HYDROXYPROPIONIQUE 33

52 3- ETHYLCROTONYLGLYCINE 0

53 3-METHYLGLUTACONIQUE 0

54 3-METHYLGLUTARIQUE 0

55 4-HYDROXYBUTYRIQUE 0

56 4-HYDROXYPHENYLACETIQUE 0

57 4-HYDROXYPHENYLLACTIQUE 1

58 4-HYDROXYPHENYLPYRUVIQUE 26 EXAMPLE 13

Identification of components of rat urine using Mass Spectrometry

[0073] Urine from two adult male Sprague-Dawley laboratory rats is obtained and subjected to mass spectrometry as described above. The organic acid content is determined:

Table 7. Organic Acids in rat urine

Table 7. Organic Acids in rat urine (continued)

[0074] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.

Claims

CLAIMS:

1. An extract from camel urine comprising an NMR spectrum as set forth in figure 1.

2. An extract from camel urine comprising a HPLC fractionation spectrum as set forth in figure 8A.

3. The extract according to claim 2, wherein said extract comprises a fraction C of said HPLC fractionation spectrum as set forth in figure 8A.

4. An extract from camel milk comprising at least one compound chosen from benzoate, lactate and citrate.

5. The extract according to claim 4, further comprising at least one compound chosen from acetoacetic acid, fumaric acid, glyceric acid, homovanillic acid, oxalic acid, oxoprolic acid, phenylpyruvic acid, propionylglycinic acid, pyruvic acid, 2-hydroxyglutaric acid, 2-oxoadipic acid, 2-oxoglutaric, 3-hydroxybutiric acid, 3 hydroxypropionic acid, 4- hydroxyphenyllactic acid, and 4-hydroxyphenylpyruvic acid.

6. A pharmaceutical composition comprising:

• an extract from camel according to any one of claims 1 to 5, or combinations thereof; and

• a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising:

• at least one compound as identified by a peak of an NMR spectrum as defined in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof; and

• a pharmaceutically acceptable carrier.

8. The use of an extract from camel according to any one of claims 1 to 5, or combination thereof, for the preparation of a medicament for the treatment of a disease.

9. The use of an extract from camel according to any one of claims 1 to 5, or combination thereof, for the treatment of a disease.

10. The use of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof for the preparation of a medicament for the treatment of a disease.

11. The use of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , a HPLC fractionation spectrum as set forth in figure 8A, or a fraction C thereof for the treatment of a disease.

12. The use according to any one of claims 10 - 11 , wherein said at least one compound is benzoate, phenylacetate or a combination thereof.

13. The use according to any one of claims 10 - 11 , wherein said at least one compound is chosen from Butyrylglycinic acid, Citric acid, Ethylmalonic acid, Glyceric acid, Glycolic acid, Glutaric acid, Hexanoylglycenic acid, Hippuric acid, Homovanillic acid Homogentisic acid, Isobutyrylglycinic acid, Isovalerylglycinic acid, Lactic acid, Malonic acid, Methylcitric acid, Methylmalonic acid, Methylsuccinic acid, N-acetylaspartic acid, Oxalic acid, Oxoprolinic (pyroglutamic) acid, Phenylpropioniglycinic acid, Phenyllactic, Propionylglycinic acid, Pyruvic acid, Sebacic acid, Suberic acid, 2- hydroxyadipic acid, 2-hydroxyglutaric acid, 2-hydroxyisovaleric acid, 2- hydroxyphenylacetic acid, 2-methyl-3-hydroxybutiric acid, 2- methylacetoacetique acid, 2-methylbutyrylglycinic acid, 3-hydroxyisovaleric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybutyric acid, 3- hydroxypropionic acid, 3-methylcrotonylglycinic acid, 3-methylglutaconic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyl lactic acid, retinoic acid derivatives.

14. The use according to any one of claims 8 - 13, wherein said disease is chosen from a cancer, a neoplasm, a multidrug resistant tumor, and a haematological malignancy.

15. The use according to claim 14, wherein said cancer is chosen from a breast cancer.a colon cancer, a prostate cancer, a malignant glyoma, and a thyroid cancer.

16. The use according to any one of claims 8 - 1 1 and 13, wherein said disease is a metabolic defect in a urea-cycle enzyme.

17. The use according to any one of claims 8 - 13, wherein said disease is chosen from multiple sclerosis, sickle cell anaemia, amyotrophic lateral sclerosis, and Huntington's disease.

18. A method of treating a disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of of an extract from camel according to any one of claims 1 to 5 or combination thereof.

19. A method of treating a disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of at least one compound as identified by a peak of an NMR spectrum as set forth in figure 1 , an HPLC fractionation spectrum as set forth in figure 8A or a fraction C thereof.

20. The method according to claim 19, wherein said at least one compound is benzoate, phenylacetate or a combination thereof.

21. The method according to claim 19, wherein said at least one compound is chosen from Butyrylglycinic acid, Citric acid, Ethylmalonic acid, Glyceric acid, Glycolic acid, Glutaric acid, Hexanoylglycenic acid, Hippuric acid, Homovanillic acid Homogentisic acid, Isobutyrylglycinic acid, Isovalerylglycinic acid, Lactic acid, Malonic acid, Methylcitric acid, Methylmalonic acid, Methylsuccinic acid, N-acetylaspartic acid, Oxalic acid, Oxoprolinic (pyroglutamic) acid, Phenylpropioniglycinic acid, Phenyllactic, Propionylglycinic acid, Pyruvic acid, Sebacic acid, Suberic acid, 2- hydroxyadipic acid, 2-hydroxyglutaric acid, 2-hydroxyisovaleric acid, 2- hydroxyphenylacetic acid, 2-methyl-3-hydroxybutiric acid, 2- methylacetoacetique acid, 2-methylbutyrylglycin^'ic acid, 3-hydroxyisovaleric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxybutyric acid, 3- hydroxypropionic acid, 3-methylcrotonylglycinic acid, 3-methylglutaconic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid.

22. The method according to any one of claims 18 - 21 , wherein said disease is chosen from a cancer, a neoplasm, a multidrug resistant tumor, and a haematological malignancy.

23. The method according to claim 22, wherein said cancer is chosen from a breast cancer, a colon cancer, a prostate cancer, a malignant glyoma and a thyroid cancer,

24. The method according to any one of claims 18 - 19 and 21 , wherein said disease is a metabolic defect in a urea-cycle enzyme.

25. The method according to any one of claims 18 - 21 , wherein said disease is chosen from multiple sclerosis, sickle cell anemia, amyotrophic lateral sclerosis, and Huntington's disease.

26. A method of producing an extract from camel urine having an NMR spectrum as set forth in figure 1 comprising:

• removing a fraction of at least 10 kDa from a camel urine sample to produce an extract having at least one molecule of molecular weight lower than 10 kDa.

27. The method according to claim 26, wherein said removing is by filtration of said camel urine sample.

28. The method according to claim 27, wherein said filtration is performed with a filter membrane.

29. The method according to claim 28, wherein said filter membrane has pores preventing passage of molecules having at least 10 kDa.

30. The method according to any one of claims 28 - 29, wherein said filter membrane is treated with water prior to filtration of said camel urine sample.