NZ514794A - Pharmaceuticals containing nerium extracts and processes for extraction - Google Patents

Abstract

A method of preparing a sterile water-soluble extract from Nerium orleander containing oleandrin, and other digoxin-like glysosides possessing active glycosides for use in treating several types of tumors comprising the steps of: (i) soaking dried powder (20g) obtained from the species in 100ml of sterile distilled water overnight. (ii) filtering the water solution under sterile conditions. (iii) adjusting the volume of the resultant solution to 30 - 40ml followed by: (iv) millipore filtration under sterile condition, (v) freeze drying of the filtrate under sterile conditions and wherein the active products are extracted from the leaves (16 - 19cm long) of the Nerium oleander harvested during August - December.

Description

FEELD OF INVENTION The present invention relates to a cancer treatment based on a sterile extract from the leaves of a plant of the genus Nerium, family Apocynaceae.

BACKGROUND In Arab folk medicine a variety of plants have been used in powder and decoction for treating a number of diseases in humans including cancers and other cell-proliferative immune deficient diseases. Among these plants are Peganum harmala, Nerium oleander, Arum spp, Capparis spinosa, Ecballium elatrum etc. However, the popularity of these plants decreased because of their extreme toxicity both in experimental animals and in humans.

The plant N. oleander (Apocyanaceae) is an ancient plant; it has been claimed to possess a potent anti-inflammatory and antitumour properties. Further, it has been used for centuries in some Mediterranean areas in human therapy as a digital-like cardio tonic drug and also endowed with good diuretic properties.

However, the popularity of the plant decreased because of its extreme toxicity both in human 15 and animals.

In addition no single process is known which leads to standard extracts characterised by a constant, reproducible physico-chemical print and a consistent biological activity.

There is a need for a process of extracting the beneficial agents from N. Oleander independently of the toxic agents.

OBJECT It is an object of the present invention to provide an improved method of extracting. STATEMENT OF INVENTION In one aspect the invention relates to a simple, reproducible and one-step procedure for preparing a sterile water-soluble extract from the leaves of Nerium oleander containing 9 oleandrin, and other digoxin-like glycosides possessing active glycosides useful in treating several types of tumours like breast, lung, stomach, some colorectal, prostate adenocarcinoma, squamous-cell carcinoma.

Preferably the extract includes oleandrins, oleandrigenin as major glycosides with trace 5 amounts of digitoxin, gitoxigenin and the like with sugar and amino acid moieties as achieved by some qualitative and quantitative procedures.

Preferably wild Nerium oleander plants are harvested in a selected area close to running water.

Preferably the active products are extracted from the leaves (16-19 cm long) of the plant N. 10 oleander harvested during August - December.

More preferably the, extract is effective in the treatment of some tumours as mentioned above and is obtained by: a. Rinsing the fine ground powder of N. oleander in sterile distilled water (1/5 W/V) for 4-24 h at temperature ranging between 0-50°C. b. Filtering the aqueous suspension of (a) several times and adjusting the pH to between 5.7-6.0. c. The filtered suspension of step (b) is filtered using Millipore filtration system and under laminar flow conditions. d. The concentration of endotoxins, of the suspension, of step (c) is testing and adjusted to 20 the range of 30 units / ml. e. No bact< rial growth m the suspension of step (d) after 48 h incubation.

Preferably the extract produced by the method above shows batch to batch consistency when tested for physico-chemical properties.

More preferably in addition to the physico-chemical properties, the extract has several defined biological characteristics both in vitro and in vivo.

Preferably the in vitro characteristics include: a. The extract possesses cytotoxic activity against Hela, K Raji, KB, Hep-2 (IC50 from 1-5 5 M-g/ml) and a moderate cytotoxicity against P388, Lmo> T47D, RD, As49> L20B (IC50 6-10 (ig/ml). b. The extract causes a block at the S-phase of the cell life cycle at conc. 1 ng/ml for K562 and for P388 and KB at 10 jag/ml. c. The extract expresses strongly some oncogens indicating anti-oncogenic activity at low 10 concentrations. d. The extract demonstrates a significant ribosome-inactivating protein activity (RIP) at concentrations less than 1 jj.g/ml. e. The extract is not mutagenic against two bacterial strains; S. typhimurium TA1530 and S. typhimurium 1537 at concentrations up to 50 jig/ml.

Preferably the in vivo characteristics include: a. The extract of the invention inhibits tumour growth of B16-Melanoma in BDFi mice significantly (T/C = 135 at the dose of0.025 ml). b. The extract of the invention inhibits tumour growth of Lewis lung carcinoma in BDFi mice significantly (T/C = 127 and 137 at the doses of0.025 and 0.05 ml respectively). c. The extract of the invention is not active against leukaemia models P388 and L1210 (T/C = 109 and 104 respectively for the dose 0.05 ml). d. The, .extract of the invention possesses a significant anti-inflammatory activity superior to thfe^eference standards phenylbutazone and ibuprofen. e. The LD50 of the extract of the invention in mice is 0.35 ml/25 gm. IM and 0.3 ml and 0.33 ml/25 gm IP and Sc respectively with apparent side effects, in high doses, like tachycardia, myorelaxation and motor in co-ordination. f. The extract of the invention shows no apparent side effects at doses up to 0.1 ml/25 gm in single or repeated doses. g. The extract of the invention has no effect on body weight, hemoglobin concentration, erythrocyte count and blood indices like PCV, MCV, HCH and MCHC subchronically for a period of 8 weeks in mice. h. The extract of the invention when studied subchronically for 8 weeks causes significant increase in leukocytes count (mostly in the lymphocytes) and a decrease in the ALT, AST and urea activities at the dose of 0.05 ml. i. The extract of the invention has neither cutanous sensitisation effects on guinea pigs skin nor any inflammatory changes in the eyes of rabbits at the doses of 0.5 and 0.1 ml respectively.

Preferably the extract is effective in the treatment of some human tumours wherein the sterile aqueous extract can be used for administration by oral, rectal, intramuscularly and by intravenous routes.

In another related aspect the invention relates to an extract, which is effective for certain human tumours and is obtained according to the following steps: 1. Soaking the dried powder (20g) obtained from the leaves of Nerium in 100 ml sterile distilled water for overnight. 2. Filtration under sterile conditions. 3. Adjustment of the volume of the resultant solution. 4. Millipore filtration under sterile conditions. <» . Freeze drying of the filtrate under sterile conditions.

Preferably the Nerium species is Nerium oleander.

Preferably step (i) is conducted at room temperature and under sterile conditions.

More preferably in step (ii) the volume of the final solution is adjusted to 30-40 ml.

More preferably in step (iii) Millipore filtration system (0.45 and 0.22 fim) are used.

Preferably the method resulted in a sterile extract with endotoxins concentrations of less than 30 units/ml.

In a further related aspect the invention relates to a pharmaceutical composition of the sterile extract of Nerium oleander, combined with at least one pharmaceutical^ acceptable 10 excipient.

Preferably the pharmaceutical compositions are suitable for injection.

More preferably the pharmaceutical composition is effective in treating some human tumours wherein 0.2-0.4 ml is injected by intramuscularly route on daily basis.

More preferably the administration of the composition is followed by a rise in temperature 15 - (38°C to 40°C) in most of the patients.

Alternatively the pharmaceutical composition may be in the form of a topical preparation, oral formulation or rectal formulation.

To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves 20 without departing from the scope of the invention as defined in the appended claims. The disclosures and the description herein are purely illustrative and are not intended to be in any sense limiting.

DETAILED DESCRIPTION The treatment of oleander leaves with water, water-ethanol mixture or ethanol gives a full range of extracts having both antitumour and cardio-tonic activities. The extracts, upon 5 evaporation to dryness, provide a solid residue, characterised by a complex physico-chemical fingerprint (TLC, HPLC, and NMR). The balance of antitumour / cardiotoxicity can be modified by handling the extraction conditions.

The following examples are given by way of illustration only and shall not be taken as being in any way limiting as to the spirit or scope of the invention.

Example: A. Preparation of the Extract: 200 g of sterile dried, finely ground leaves obtained from oleander are suspended in 1000 ml of distilled water and is stirred at room temperature and under laminar flow conditions for twelve hours. The solution was filtered several times using Whatmann 15 no.l filter paper and Millipore filtrations. The volume of the clear, dark brown filtrate is adjusted to 350ml and the pH of the solution to 5.9. The filtrate was divided into two equal portions. The first portion was aliquoted under laminar flow conditions into sterile vials and some were stored at 4 °C in the refrigerators and others were left at room temperature conditions. The second portion of the extract 20 was lyophilized (freeze-dried) under sterile conditions and the dry powder was stored in sterile containers.

B. Sterility Tests: One aspect of the present invention was to prepare a sterile aqueous extract and lyophilized powder that meets the requirements described in the Pharmacopoeias. 25 Therefore, three criteria were followed to meet the above requirements and these were: 1. Direct swab culture using different media and direct staining smear technique after 48h of incubation. 2. Determination of the concentration of bacterial endotoxins in the extracts and the powder using the conventional Limulus Amoebocyte Lysate (LAL) test. 3. The pyrogen test of the extract and the lyophilized powder using rabbits.

No bacterial growth was observed after 48h incubation of the extract and the lyophilized powder with different growth media. Furthermore, the concentration of bacterial endotoxins was approximately or less than 30 units / ml. However, the 10 pyrogen tests in rabbits showed an increase in the temperature at about 2-2.30h after intravenous administration.

C. Physico-Chemical Characterisation 100ml of the aqueous extract are evaporated to dryness at 25 °C under vacuum. The solid extract is kept under vacuum in a desiccator overnight. Aliquots from both the 15 solid residue and the lyophilized fraction are used for sugars and amino acids investigations as well as for TLC and HPLC. 1) Sugar Test: A positive test was obtained on using Fehling's solution reagent, as follows: Procedure: 50 mg of the solid and the lyophilized fractions were boiled with 0.1 N 20 HC1 (5 ml) for about lh. The acidic solution was then neutralised by addition of sodium hydroxide (0.5 N). The total volume was adjusted to about 3 ml by heat evaporation. To this was added a solution of Fehling 1 and 2 (3ml each) and distilled water (18 ml) and the mixture was heated to the boiling point for 10 minutes. The colour turned olive-green with the precipitation of a red solid indicating the presence 25 of sugar moiety in both fractions, due to the formation of CU2O. 2) Amino Acids Test: A positive test was obtained for amino acids on using the Ninhydrin solution reagent (0.25 g) in acetone (20 ml), as follows: Procedure A) Filter Paper Method: The solid and lyophilized fractions were dissolved in a mixture of water and ethanol (1:2) and one drop of the resulting solution was placed onto filter papers and one drop of the reagent was added. The filter papers were dried oven over a stream of hot air. A violet colour was immediately appeared indicating the presence of amino acids in both fractions.

B) Solution Method: The solid (50 mg) from both fractions was dissolved in 5 ml water and 3 ml of the reagent was added. The reaction mixture was heated with continuous stirring for about 15 minutes. A deep violet colour solution was observed from both fractions indicating the presence of amino acid moieties. 3) Cardiac Glycosides: The presence of glycosides in the room temperature oleander extract was examined by the two methods TLC and HPLC, which showed positive results and were compared with the standard glycosides.

A) TLC Technique: i. Plate: Silica gel G, 250 nm thick, activated at 120 °C for 45 min. ii. Mobile phase: Benzene: ethanol (7:3). iii. Location Reagent: P-anisaldehyde reagent prepared by mixing 10 ml of a nisaldehyde, 90 ml of ethanol and 10 ml of concentrated H2 SO4.

Sample: The solid (100 mg) was suspended in 1 ml of a mixture of CHCI3 and methanol 25 (1:1) with gentle heating on water bath, and then centrifiiged.

The clear pale yellow solution was placed on the silica gel plate. Comparative samples of the standards (0.2 mg/ml) were prepared using the same solvent and 100 ul of the resulting solution was placed onto the plate.

Procedure and Results The usual procedure for TLC was carried out. After spraying the reagent, the plate was heated in an oven at 100 °C for 4 min. The spots appeared were varied in colour from yellow, blue, violet, green and olive green and with the following Rf values: 0.16, 0.23,0.29, 0.40,0.45,0.51,0.55, 0.60, 0.61, 0.66, 0.70, 0.73.

The Rf values obtained from the standards are as follows: Standards Rf Values Gleandrin 0.69 Oleandrigenin 0.65 Digitoxin 0.59 Gitoxigeriin 0.56 Digoxin 0.51 Stigmasterol 0.77 Therefore, generally speaking, the Rf values of the glycosides present in the extract are well comparable with those of the standards. A quantitative comparison could be achieved by using the HPLC techniques.

B) HPLC Techniques: i. Column: Silica (Lichrosorb SI60, 5*m, and 25 cm X 4mm internal diameter). ii. Wave Length (p.): 254 nm. iii. Flow Rate: lml/min. Range: 0.02. iv. Attenuation: 8.chart speed: 0.25 cm/rain. v. Mobile phase: cyclohexane: ethanol: acetic acid (60:9:1). vi. Sample: The solid (125 mg) was suspended in (5 ml) of a mixture of CHCI3 and methanol (1:1) and gently heated. The suspension was filtered through a filter paper, and the filtrate was filtered once more through Millipore filter (0.22 fim) before use. Authentic standards were prepared for comparison.

Procedure and Results The usual procedure for HPLC was carried out. The results obtained with regards to the retention time values (Rt) and intensity as area under peak in (parenthesis) is follows: 3.46(393,531), 5.01 (166, 5681, 5.53 (157,139), 5.99(780, 527), 6.81 (207, 438), 10.25 (891, 259), 11.67(347, 948), 15.65 (197, 817), 16.17 (161,237), 25.32(534,109), 28.68(107,804), 31.53(452,784), 35.81(888, 755).

The Rt values together with area under peak values for a certain concentrations of the standards are as follows: Standards Concentration Rt values (mg/mD (Mini Area Oleandrin 0.75 7.2* 335,542* Oleandrigenin 0.75 8.7 147,564 Digitoxin 2.0 14.9 198,308 Gitoxigenin 0.6 .9 355,044 Digoxin 0.2 12.04** 240,370 - 12 D cc "O "C dJ H t: ci' 0 s? 1 C'5 N- '!T K) « fi. ™ C 03 73 N § ® a: Stigmasterol 0.6 4.3 605,862 * This value represents the average of two Rt values at 6.92 and 7.43 min observed for oleandrin and the area also represents the average of both areas 346, 937 and 324, 146 respectively.

** The standard (digoxin) showed one peak at Rt value of 12.04 with a shoulder at 14.25.

The Rt values 7.65 and 8.93 min, from the extract, are considered to be due to the presence of oleandrin and oleandrigenin in the extract. Therefore, they are present in 125 mg of the dry extract per 5 ml of CHCI3: methanol of at least 0.23 and 0.19 mg, respectively. The HPLC chart showed also trace quantities of digitoxin, gitoxigenin and digoxin (not recorded by the instrument) (see Fig. 1), together with many related compounds appeared at retention times between 18-43 min.

We found that by changing the preparation conditions by boiling the leaves in water or using alcohol as a solvent for extraction, the Rf and Rt values obtained from both TLC and HPLC were significantly varied from one extract to another. This reflects on the behaviour of each extract on its biological activity see Fig.2 and Tables 1 and 2.

Table 1: The Rf values obtained from TLC for solids 1,1' and 2 Rf. Solid 1 Rf, Solid 1' Rf. Solid 2 0.17 0.16 0.17 0.23 0.23 0.24 — — 0.25 0.29 0.29 — — — 0.35 0.41 0.40 0.41 0.45 0.45 0.45 0.51 0.51 0.52 0.55 0.55 0.56 0.59 — — 0.60 0.60 — 0.62 0.61 — 0.64 0.66 0.66 0.68 0.70 0.70 0.71 0.73 0.75 — — 0.96 Table 2: The Rt and area values obtained from HPLC for solids 1,1' and 2 Solid 1 Solid 1' Solid 2 Rt Aiea Rt Area Rt Area 3.46 3.46 3.46 508,751 393,531 309,259 .08 .01 .07 367,184 166,568 256,575 .27 .53 .65 332,054 157,139 387,029 6.00 .99 6.00 866,736 780,527 665,125 6.87 6.81 6.84 954,246 207,438 225,405 (798,642)* — — 8.34 — — 037,754 9.28 — — 366,636 — — 9.89 — .05 290,829 — 903,079 .27 .25 .37 675,050 891,259 014,195 11.72 11.67 — 458,093 347,948 — .65 .65 .46 108,073 197,817 063,941 16.84 16.71 — 114,777 161,237 — 19.27 — — 030,721 — — .41 .32 .25 292,342 534,109 305,670 28.71 28.68 28.68 028,953 107,804 083,710 31.65 31.53 31.56 248,361 452,784 424,195 .91 .81 — 534,553 888,755 — * After repetition For example, the boiled extract and the extraction with alcohol showed (in its TLC plate) a spot with a very high Rf value of 0.96.

The spot correlated to a compound detected and identified by others to be: a dyrenigenin (3/3 - hydroxy - 8 - 14jS - epoxy - 5 jS — carda 20:22 - enolid).

This compound, however, was not detected at all in the room temperature extract. Similarly, the boiled and extraction in alcohol showed some peaks in their HPLC analysis that had not previously been detected in the room temperature extract.

Nuclear Magnetic Resonance (NMR) Studies: Although, the NMR spectroscopy of a mixture of compounds such as plant extracts does not help very much to solve a problem, nevertheless, it provides an indication of the functional groups present in the mixture by comparison with related standard samples, as well as a fingerprint for the type of extraction.

However, the 'H and 13C NMR spectra for the solid obtained by room temperature evaporation were recorded (Fig.3). The oleander extract contains many types of compounds such as sugars, amino acids, flavenoids, steroids, alkaloids, cardiac glycosides and the like and each type of these are consistent with more than one compound. It is impossible to differentiate a single compound from this mixture. Nevertheless, the NMR spectra can be used as a fingerprint for each extract.

STUDIES OF PHYSICO-CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITIES The extract used for the study of physico-chemical properties and biological activities was prepared using the following method: Leaves of the Nerium oleander plant were collected. The leaves collected were 16-19 cm long and were collected during August-December and preferably during October. They were immediately subjected to heat treatment (20 - 50 °C), preferably 40 °C in a drying oven and allowed to stand for 48 - 96h and preferably for 72h until dried. The dried leaves are 5 directly ground in an electrical grinder to get a fine powder. Therefore, 200g of the dried ground leaves are rinsed in 1000ml of sterile distilled water and stirred at room temperature for 12h under laminar-flow conditions. The solution was filtered several times to remove the large particulate matters using many filtration systems. The yielded volume of the solution is adjusted to 350ml with sterile distilled water and filtered several times using Millipore 10 filtration systems. The filtered solution is brown in colour with a pH 5.9. The filtrate was divided into two equal portions. The first portion was aliquoted into appropriate containers under laminar-flow conditions, whereas, the second portion was freeze-dried under sterile conditions and the dry powder was stored in sterile containers.

Several criteria were followed to review the activity of the extract of the inventions, 15 including the physico-chemical properties and a consistent biological activities both in vitro and in vivo.

I. In vitro Studies a. In vitro Cytotoxic Activity The extract of the present invention was tested using the MTT-assay32 against 20 many tumour-cell lines like P38g3 Lnio, K562, Raji, T47D, SW94s, KB, L929, RD, Hela, Hep_2, A549, L20B. The IC50 (ug/ml) values were calculated and the data are presented in Table.3.

TABLE 3 The IC50 values against cell-lines Cell-line Description IC™ Cue/ml) P 388 Lymphocytic leukaemia >10 Ll210 Lymphocytic leukaemia >10 KS62 Myelogenous leukaemia <10 Raji Burkitt's lymphoma <10 T47D Breast carcinoma <10 SW948 Colon carcinoma <10 KB Oral epidermoid <10 L929 Fibrosarcoma > 10 RD Embryonal rhabdomyosarcoma >10 Hela Cervix carcinoma <10 Hep.2 Larynx carcinoma <10 L20B Mouse L-cells containing human polio-virus receptors >10 A549 Lung adenocarcinoma <1 K562» P388 and KB cell-lines were exposed to the extract of the invention at certain concentrations and then analysed for the percentage of cells present in GO, S, G2 and Gi phases of the cell-life cycle. It appears that the extract of the invention caused a block at the S- phase in the three cell-lines as shown in Table.4.

Table.4 The effect of the extract on cell-life cycle Cell-lines Concentrations fug/ml) Results KB Block at S-phase P388 Block at S-phase K.562 1 Block at S-phase b. In vitro Antioncogenic activity The extract of the invention is tested for its antioncogenic activity using the liposarcoma cell line (LSA) in vitro.

Oncogens, antioncogens and growth factors produced by LSA cells are differently expressed when the LSA cells grow in the presence or absence of the extract. The present results showed that the extract of the invention express some oncogens as showed in Table.5.

Table 5. Immunocytochemical review of statement of different oncogens, oncogene suppressor gene and growth factors in LSA cell grown in the presence of the extract.

Bio-active protein review Aaueous extract c-neu (Ab-2) + v-src (Ab-1) + c-fos (Ab-1) +++ c-myc (Ab-1) - c-jun / AJP (Ab-1) + human rasp2i - c-erb-B2 - Suppressor oncogene proteins - Human P53 Growth factors TGF- cc + TGF-P - TNF - a + C- In vitro Ribosome-lnactivatingprotein (RIP) The extract of the invention was also tested for its activity as a ribosome-inactivating protein. The extract demonstrated a significant RIP activity at concentrations less than 1 ug/ml.

D -In vitro Mutagenic Effect The mutagenic effect of the extract of the invention was also tested using two bacterial strains; Salmonella typhimurium TA1530 and S. typhimurium TA1537 in vitro. The results indicate the extract of the present invention show no mutagenic effect on the two bacterial strains at concentrations 10, 25 and 50 ug/ml.

H. In vivo Studies La. In vivo Antitumour activity The extract of the invention was tested in vivo in mouse models following standard protocols for this testing as set by the National Cancer Institute.

TEST EXAMPLE 1 P-388 mouse leukaemia was inoculated at lxl06 into the abdominal cavity of CD2F1 male mice. The extract of the invention (two dose levels; 0.025 and 0.05 ml) was introduced once (e.g., after 24h of implantation) and continued for 9 days. The results obtained according to the following: ILS = T"CC x 100 ILS — increase in the life span T — the medium survival time of the treated mice.

C = the medium survival time of the untreated mice (control group) TEST EXAMPLE 2 L1210 mouse leukaemia was inoculated at lxlO6 into the abdominal cavity of 2CDF[ male mice. The extract of the invention (two dose levels; 0.025 and 0.05 ml) was introduced once (e.g., after 24h of implantation) and continued for 9 days.

The results obtained according to the following: ILS= T'CC xlOO The results indicated that the extract of the invention is not active against leukaemia models P388 andLnio and the T = 109 and 104 respectively for the 0.05ml dose of the extract.

C TEST EXAMPLE 3 Tumour homogenate of Melanoma-B16 was implanted subcutanously into BDFi male mice. The extract of the invention was introduced intraperitoneally for 9 days at two dose levels - 0.028 and 0.05ml. The extract of the invention showed a significant increase in the life span. The T/C for the dose 0.025ml was 135.

TEST EXAMPLE 4 Lewis lung carcinoma was inoculated at 2xl06 intramuscularly into 2BDFj male mice in the test. The extract of the invention (0.025 and 0.05ml) was introduced intraperitoneally for 11 days. Two dose rates were used - 0.025 and 0.05ml. The extract of the invention displayed a significant increase in the life span. 1.b Anti-inflammatory Activity The anti-inflammatory activity of the extract of the invention was assessed following the standard protocols. Two experiments were used to study this activity in vivo: i. The percentage reduction in the volume of asciatic fluid. ii. The Freund's adjuvant In both models albino rats were used. Two reference standards were used for comparison purposes (phenylbutazone and ibuprofen). The results showed the extract of the invention possesses a superior anti-inflammatory activity at the dose 0.05ml when compared to a reference standards. b. Toxicity Studies 1.b. The Acute Toxicity The LDso of the extract of the invention in albino balb/c mice using the intramuscular route is 0.35/25gm. The LD5o of the extract of the invention using the IP and Sc routes is found to be 0.3 and 0.33/25gm respectively. Some apparent side effects like tachycardia, myorelaxation and motor in co-ordination were observed with high doses. When the extract of the invention was administered in single or repeated dosage IM} IP and Sc up to 0.1ml/25gm in normal mice no side effects were apparent. 2.b. Sub-chronic Toxicity The potential lethal effect of the extract of the invention was investigated subchronically over a period of 8 weeks with an experimental design of 3 groups of 50 animals per group. Groups comprised male and female balbc/mice. Group I received 0.025 ml/daily intraperitoneally (IP); group II received 0.05ml daily/IP, whereas, the third group received distilled water daily /IP and served as a control.

Morbidity and mortality checks were made daily. Body measurements were scheduled at day (0) before injection and then on a weekly basis. A number of animals were sacrificed per week. Complete blood counts (CBC) and liver and kidney profiles were done on each sacrifice. In addition, biopsies were taken from kidney and liver during each sacrifice for pathology study.

The following observations were recorded during the entire period of the experiment: 1. Animals generally gained weight (all groups) indicating that the extract of the invention is not anorexic at the doses used. 2. The extract of the invention showed no significant changes in the haemoglobin concentration or in the erythrocyte count in all the groups at the doses used. 3. No significant changes were observed in the blood indices (PCV, MCV, HCH and MCHC). 4. The extract of the invention lead to an increase in the leukocytes count in the experimental groups. Significant increases in the lymphocyte count were recorded at both dose levels.

. The extract of the invention caused a decrease in both the ALT and AST activities I at week 6 of treatment This decrease continued to the end of treatment and was observed in the treated groups only. This decrease was more significant in group II that received 0.05ml of the extract of the invention indicating its effect on some liver functions. In addition, a slight decrease in urea activity was noticed in both the treated groups indicating effects of the extracts on some kidney functions. 6. Four of the animals in group II died during the last three weeks of treatments. 7. Pathologically, three biopsies from kidney taken from group II at week 8 showed some oedematous interstitial tissues with individual cell necrosis in the distal tubules. Further, three biopsies from the liver taken from group II at week 8, showed multiple focal inflammatory cells infiltered mainly around the central veins with histiocytes. However, some of these changes both in the kidney and liver were also observed in the control biopsies. 2.C. Special Toxicity Test: The effect of the extract of the invention on the eye in rabbits and on the cutanous sensitisation in guinea pigs was studied using standard protocols. Again two dose levels were used in both tests that is, 0.05 and 0.1ml. The results indicated that the extract of the invention showed neither erythema nor any other skin changes nor any inflammatory changes in the eyes of the tested animals as compared to the controls.

ANTICANCER PROPERTIES Clinical studies.

During the period between March 1988 to January 1990, the sterile extract of the invention was clinically tested for phase 1/n . A total of 35 patients; 25 consecutive eligible patients with advanced breast cancers and 10 patients with resected colon cancers (2 had Duke's C and 8 had Duke's D), were selected for this study. All patients had received palliative chemotherapy and or radiotherapy in recognised medical centres. The main purpose of this study was to review the toxicity and determine the appropriate doses of the intramuscular injection and the oral drops of the extract of the invention.

Two routes of administration were followed: the intramuscular and oral. The intramuscular dose (IM) was adjusted during the course of treatment, which continued for two to four months. For the IM routes, a single daily injection of about 60-80mg of the active ingredients of the extract of the invention was administered (about 0.4-0.6mI). The dose was later adjusted and it was found that 5Qmg of the active ingredients (which is equivalent to 0.3ml) of the extract of the invention was an appropriate dose. Oral administration was used as an adjuvant to the IM routes. The oral administration was initially between about 40-50 drops / day and preferably about 30-40 drops ( day one hour after meals. The oral administration was adjusted later to be 20 drops / day.

The above study confirms the potency of the sterile extract of the invention. Following four months of treatment with the extract of the invention 5 patients with advanced breast cancer achieved complete response and the other 20 patients showed a partial response. The 2 patients with Dukes C showed complete response after 6 months of treatment with the extract of the invention with no peritoneal recurrences (a median duration of follow-up of 18 months). The other 8 Dukes D patients achieved partial response. However, four patients had recurrence of 18 months.

The limiting non-cumulative toxicity of the extract of the invention was a sharp increase in the body temperature (up to 41°C), which was later, adjusted to 38.5°C with the dose (dose-limiting). This increase in the body temperature was observed 2-4 h after the IM injections. Vomiting and nausea developed in 30% of the patients. There were no treatment related deaths.

Due to the significant results obtained during the early phase I/H study in both advanced breast cancers (ABC) and resected colon cancer (RCC), a late phase II study was conducted between February 1990 to April 1992.

The extract of the invention was tested on 70 patients, of which 55 patients were reviewed for efficacy. Of this 55 involving 40 patients had advanced breast cancers and 15 had resected colon cancers.

The patients were treated by both IM and orally for a period of 6-8 months regularly. They were put on follow-up treatment for another 6 months and their progress was monitored for a period of 8-12 months. In advanced breast cancers (ABC), 12 cases of complete response (CR) and 28 cases of partial responses (PR) were observed with a response rate of 70% and CR of 30%. In resected colon cancers, 5 with Duke'sB of complete response (CR) and 4 with Duke's C of complete response (CR) with a response rate (26%) and 6 with Duke'sD of partial response (PR), with a response rate of 40% and a CR rate of 60 %. Nevertheless, two patients had recurrence within 12 months.

Major toxicities included a tolerable rise in body temperature (38.5 °C) and nausea in some patients (10%). In addition, some patients experienced diarrhoea (10%). This was especially observed in patients with colon cancers. These results suggested that the herbal extract of the invention was a promising treatment for the advanced breast and colon cancers with acceptable toxicity.

Additional clinical studies were performed between April 1993 to March 1994. Sixty-five cancer patients enrolled in this study. These patients were diagnosed at recognised medical centres in Jordan and were treated with the extract of the invention. The staging and typing of the cancers is recorded in Table 6. Again, two routes of administration were used the intramuscular and the oral routes.

Table 6 Tvce of cancer No. of Patients % Responses Breast (adenocarcinoma) 71 Colorectal (adenocarcinoma) 52 Prostate (adenocarcinoma) 6 66 Skin (basal-cell carcinoma) 70 Stomach (adenocarcinoma) 2 62 Lung (adenocarcinoma) 4 54 Larynx (squanibUs-cell) 3 72 Chronic lymphocytic leukaemia (CLL) 3 'Lymphoma (MALT) 2 Regular treatment with the extract of the invention continued for 3-6 months depending on the case. Very promising results were obtained from some cancer types (see Table 6). Regular follow-up has been performed using different scans such as CT, MRI, bone and liver tomography, X-rays and the like. It appears from this study the efficacy of the extract of the invention is as high as 72%.

Further, some of the advanced cancers showed complete response including breast, larynx, colorectal, skin, lung cancers (see case reports below). ^Therefore the extract of the invention can be used to treat other types of cancers. However, some side effects were recorded in - some patients in this trial were not observed during the first trials. Among these side effects, was a general weakness or fatigue, which appeared in more than 40% of the patients. Furthermore, some male patients experienced a decrease in the sexual power which persisted for nearly two months following the treatment. In addition, 90% of the patient's demonstrated pains at the site of metastasis, which appeared within about 1-2 weeks following the treatment.

Between June 1996 and October 2000, a total of 179 patients with various cancers were treated with the extract of the invention in a multi-international centres as a collaborative work. These studies on the extract of the invention were carried out in Mexico, Armenia, Italy and the United States of America.

Table 7 Tvne of cancer No. Of patient % Responses Breast 55 70 Lung 21 70 Prostate 16 68 Colorectal 22 56 Stomach 12 61 "Thyroid 8 4 71 Skin a. Basal cell 61 ^TbVMelanoma 4 ■'iranjn-r--.; C— I©3i6Mastoma multiforme 45 ^Osteosarcoma:"" 51 -Nasopharynx 6 -62 * Chronic lymphocytic leukaemia (CLL) Non-Hodgkin's lymphoma 2 The results of the above studies suggest the extract of the invention has potential activity against various cancer types. The formulation of the sterile extract was modified by the introduction of some pharmaceutically accepted excipients. These modifications were introduced into the extract of the invention due to some undesirable secondary side effects reported by the Italian team. Among the important side effects that reported was the general weakness (fatigue) in women with lung adenocarcinoma. The side effects appeared 2-3 weeks after the treatment.

The excipients added to the extract of the invention depended on the desired route of administration. The present pharmaceutical composition may be formulated as oral, rectal, injectable and topical. Therefore, the sterile extracts obtained from N. oleander were combined with more than one phannaceutically accepted excipients in order to reduce some of the undesirable side effects. The addition of such excipients was intended allow improved administration of the formulation rather than to change the active itself. Suitable drug delivery technologies for the administration of the extract of the invention are: 1. Sustained release tablet/capsule system to provide continuous release of the active ingredients over a period of time. 2. A cream-based formulation for topical treatment in the form of liposome systems and the like. 3. Coupling the sterile extract with soluble polymers as targetable drug carriers and the like. 4. A suppositories-based formulation for rectal treatment.

. An injectable-based formulation by adding some surfactant excipients and the like.

The process for preparing the extract for use in the treatment of cancer is as follows: Leaves of N. oleander (16-19cm long) are collected during August-December. After washing the leaves with distilled water they were subjected to heat treatment (20-50°C) in drying oven and were ground in electrical grinder. To approximately 1kg of the ground fine powder of oleander leaves, 5L of sterile distilled water was added. The resultant liquid was held at room temperature and stirred for about 12h under laminar flow conditions. The solution was filtered several times to remove any large particles. The resultant volume was adjusted to 2L with sterile distilled water and then filtered using Millipore filtration system.

The filtrate solution was divided into two portions. The first portion was aliquoted into sterile vials under sterile conditions. Some of these vials were kept in the refrigerator and <x> cT) others were left at ambient temperature. Both sterile extracts were stable for more than 6 months. The second portion of the extract was transferred into sterile bottles (lOOml/each) for lyophilization under sterile conditions. The process was completed after 48-72h. One litre of the sterile extract yielded 30g of the lyophilized powder. The powder was kept in 5 sterile, sealed bottles and stored at - 18°C. The freeze-dried extract of oleander leaves remained stable for at least 2-3 years.

Protocol used for treating cancer patients The extract prepared above can be used for the treatment of cancer patients under the following protocols: 1. Regular treatment: A: Daily intramuscular injections of 0.2-0.4ml for a period from 4-12 months depending on staging.

B: Daily one tablet and/or one suppository as adjuvant to the IM route. Again the period of treatment will vary depending on the staging and typing. 2. Follow-up treatment: Follow-up treatment is used to prevent relapse or recurrence. During this period the patient is advised to receive one course of IM i.e. 0.3ml every second day for the first month. This is followed by another course for another month where the dose is one injection every three days and so on.

Case Reports Case Report 1: Breast Cancer with Bone Metastasis A 48 year old women presented with painless lump on the left breast. The lump had been discovered by self examination during a shower. The women had had a hysterectomy 6 years before. On examination, it was found that the right breast and axilla were free. The left 25 breast and axilla showed a painless lump about 2.5x2.5cm in the lower outer quadrant. There were no skin or nipple involvements. The left axilla showed solitary mobile painless axillary lymph node. On 29 April 1992 a core biopsy of the left breast lump was done and >: % b fi c; r. m '•"T' u:- '£ P. 7c a. revealed the lump as an infiltrating undifferentiated adenocarcinoma. On 30 April 1992 a modified radical mastectomy was performed. Early discharge was defined by the fourth postoperative day. On 5 May 1992 the pathology report diagnosis showed infiltrating ductal carcinoma with multicentric intraductal carcinoma and two proximal axillary lymph node metastasis. On 1 June 1992 a bone scan showed patchy radionuclide uptake at the lower lumber spine consistent with degenerative changes and no evidence of bony secondaries could be seen. On 11 February 1993 a bone scan showed an area of moderate tracer uptake at the fourth lumber spine, another smaller area in the lower cervical spine.

On 29 April 1993 the patient started to take the extract of the invention using both the 10 intramuscular and oral routes. Two weeks later, the patient was pain-free at the lumber spine and she put on weight (17kg). And no other side effects were reported. The patient demonstrated a rise in temperature to 38°C for the first week following treatment with the extract of the invention. On 3 July 1993 a bone scan was performed. The bone scan showed minimal abnormal increase in tracer uptake in the fourth lumber spine in comparison with 15 previous scan. No significant changes in the fourth lumber spine was detected and no "g evidence of progression of the disease. On 5 January 1994 another bone scan was performed which showed a slight irregular increase, tracer uptake in the fourth vertebra and the rest of | the skeleton showed good, uniform and symmetrical tracer uptake. An X-ray was also performed at the same time. The x-ray which showed the abnormality in the fourth lumber 20 vertebra was due to degenerative changes, and no new bony lesions were detected. Further, no evidence of bony metastasis was detected.

Regular follow-up was performed to the patient every 6 months including CBC, liver and kidney profiles and a bone scan. The last follow-up was in 1998 and the bone scan revealed that the entire skeleton showed good, uniform and symmetrical uptake. No evidence of bony 25 metastasis was detected.

Case Report 2 Laryngeal Epidermoid Cancer witfi Stomach Metastasis.

A 65 year old male with a laryngeal epidermoid (squamous type) tumour, with increase in the thickness of the stomach wall treated at its primary site with a combination of surgery and radiation. He was referred to a nuclear medicine department on 2 May 1993 and 30 diagnosed as T2NOMO (no metastasis). On 6 June 1993 indirect laryngoscopy confirmed the same diagnosis. On 16 June 1993 an abdominal CT scan showed nodular filling defect centering the stomach mostly due to the lower part entering the stomach. No retroperitoneal lymphodenopathy was observed. The patient had normal liver, kidneys, spleen and adrenals.

On 23 June 1993 the patient started treatment with the extract of the invention both intramuscularly and orally on regular basis. On 28 July 1993 a barium meal showed normal oesophagus, stomach and duodenum. A biopsy of the left vocal cord done after 2 months of treatment with the extract of the invention revealed intensive proliferation of abnormal squamous cells with mitosis (squamous-cell carcinoma grade II). He continued the treatment with the extract of the invention and another biopsy was performed on 28 November 1993 from the left vocal cord and the result of the biopsy showed multiple fragments of a papillary verrucous squamous papilloma. No obvious frank malignancy was observed. The patient continued the treatment on maintenance dose for another 8 months.

Case Report 3: Stomach Adencarcinoma A 65 year old female patient was referred for a symptomatic gallstone. She underwent surgery on 2 April 1991. The routine exploration of the upper abdomen showed some lymph nodes enlargement at the lower part of the greater curvature. The pathological examination of the lymph node showed it to be a poorly differentiated adenocarcinoma. Three weeks later, a G.I. fibroscopy was performed and revealed an external compression of the stomach at the level of the greater curvature, which was confirmed doing a CT. scan. Another CT scan was performed on 9 April 1992 showed increased thickness in the stomach mainly at the small curvature. The patient started the treatment with the extract of the invention on 3 August 1993. At the time of presentation the patient was complaining of recurrent vomiting, loss of appetite and severe abdominal pain. These complaints disappeared within one month of commencing treatment with the extract of the invention. A CT scan was performed on 12 September 1993 showed normal stomach wall with the disappearance of thickening.

On 18 October 1993 an oesophageal and gastric biopsy through endoscopy showed mild non-specific esophagitis and mild chronic gastritis with helicobactor pylori were identified. The patient continued the maintenance therapy with the extract of the invention for another three months.

Case Report 4: Colon Adenocarcinoma (Duke's B) with reactive hyperplasia of lymph node. o. ^r o <N CO CD g A 60 year old male with a history of cancer of transverse colon. The patient was C'i c' complaining from abdominal pain and diarrhoea. The patient was operated as a transverse £ colon cancer, which was resected, and an end-end anastomosis was performed. The 'c pathological report showed moderately differentiated adenocarcinoma of transverse colon (Duke's B) with reactive hyperplasia of the lymph nodes. The body weight was 54kg. £ Neither radiotherapy nor chemotherapy was given. Two weeks after the operation, the « patient was complaining of abdominal pain and discomfort. The carcino-embryonic antigen •I (CEA) was elevated to 13.5ng. The patient started to take the extract of the invention on 27 June 1993 using the two routes; the IM and the oral. A barium enema performed on 4 July 10 1993 showed right hemicolectomy. Barium was seen to flow easily from rectum to hepatic flexture, and small bowel was seen. No intrinsic or extrinsic masses were observed. The patient was still complaining from abdominal pain, loss of appetite. A CEA was performed 9 on 18 July 1993 and it was found to be 5.6ng. During this period the patient was free of ?? abdominal pain and had an increased appetite and sexual desire. There was a significant to ■g 15 increase in the body weight (63g). A liver isotopes scan performed on 11 August 1993 j? showed multiple areas of poor to mild selective tracer uptake mainly in the right lobe. These | finding were most likely consistent with liver metastasis. Another CEA measurement was ,Sj done on 4 September 1993 and was found to be within normal range of 5.4ng and the patient J was feeling free from most of the complaints. A liver-spleen tomographic study performed on 31 October 1993 showed no evidence of new lesions and further improvements in the size of existing lesions. Tracer uptake in mid portion of the right lobe of the liver also improved. An abdominal CT scan performed on 12 December 1993 showed normal pancreas, liver, gall bladder and kidney.

The patient was put on a maintenance therapy treatment from 1 January 1994.

Another abdominal CT-scan was performed on 10 June 1994 indicating normal findings. An additional CT- scan was performed on 12 December 1994. This indicated normal liver, pancreas, spleen, gall bladder and both kidneys. In addition no periaortic or pelvic lymph node enlargement was observed. The patient was monitored until 1998 and all findings were normal indicating complete response (CR).

.Case Report 5: Osteogenic Sarcoma with Lung Metastasis A 20 years old male patient with osteogenic sarcoma of the left tibia. On 13 March 1996 a bone scan of the upper third of the left tibia showed diffuse increase tracer uptake. No other abnormality was detected. He was treated with both chemotherapy and radiotherapy until 2 August 1998. A surgical amputation of the left leg was performed on 23 September 1998. A 5 CT scan of the chest showed rounded mass in the lower aspect of the left lung could be metastasis. He received 9 courses of chemotherapy later and the last cycle was in August 1998.

The patient started to take the extract of the invention as injectable and tablet-dosage form on 19 July 1999. Two months later a CT scan showed no evidence of lung metastasis. This was 10 followed by aspiration from the mass of the left lung and pleural effusion on 29 September 1999, which revealed blood and dense neutrophilic infiltration with scanty macrophages, and no evidence of malignancy was observed. Another aspiration was done on 20 December 1999 from the same mass of the left lung and pleural effusion, which again revealed a cellular aspirate in a hemorrhagic stroma, and no evidence of malignancy was observed. The patient was put on follow-up therapy for another 6 months.

Case Report 6: Breast Adenocarmoma with Multiple Bone Metastasis A 55 year old female patient with cancer in the right breast. A true cut needle biopsy of the right breast mass was performed and the pathological examination revealed invasive carcinoma of the breast (probably lobular). A bone scan showed multiple areas of increased 20 activity of the lower thoracic vertebrae, upper lumber vertebrae, skull and multiple ribs. On 27 November 1996 a modified radical mastectomy was performed. This was followed by chemotherapy and radiotherapy treatments.

The patient started to take the extract of the invention both IM and orally (tablet-dosage form) on 18 July 1997. A liver CT scan performed on 26 August 1997 showed multiple 25 space-occupying lesions in the right lobe most likely due to liver metastasis. Two months later, a bone scan showed good, uniform and symmetrical tracer uptake throughout the skeleton. On 7 November 1997 a liver / spleen tomography study in comparison with previous showed no more deterioration with some evidence of slight improvements i.e. more liver functioning tissue was observed in the upper part of the right lobe. Another liver / 30 spleen tomography was performed on 12 April 1998 and showed more improvements in the upper part of the right lobe in comparison with previous. The patient was improved clinically as well especially in appetite and no more pains were experienced in the spine region. The patient was put on follow-up therapy since April 1998.

Case Report 7: Prostate Adenocarcinoma with Bone Metastasis A 62 year old man with prostate cancer and having metastasis in the pelvic region for the last 7 years. The patient had chemotherapy for the past three years. He began to take the extract of the invention both IM and rectally on 5 May 1997. The patient experienced no change in temperature. The chemotherapy treatment was suspended after one week of taking the extract of the invention. Upon the 5th day of treatment, the patient had pain in the pelvic area for 4-6h. After 2 weeks the patient was able to sleep well and no more pain was experienced. The prostatic antigene (PSA) at the time of presentation was l,200ng/ml, which reduced significantly after one week of treatment with the extract to 528ng/ml. Accompanied by improvement in the appetite and bowel movement. Two months later the PSA was found to be 22Gng/ml. A bone scan was performed after nearly four months of treatment with extract indicated a normal tracer uptake in the pelvic area. Another bone scan was done on May 1998 showed a good, uniform and symmetrical tracer uptake throughout the skeleton. The patient was put on follow-up therapy since May 1998 up to now.

Case&Regort 8: Nasopharyngeal Carcinoma with Pleural Effusion A 16 year old woman diagnosed with nasopharyngeal lymphepithelioma stage II. She was given 3 cycles of chemotherapy followed by external bean radiotherapy in 1997 after which she showed complete response. Four months later she presented with pale, tender masses below the angle of jaw (lymph node). A fine needle aspiration (FNA) performed on both the left and right lymph node. The left lymph node showed metastatic malignant cells with features of non-small cell carcinoma probably squamous. On 29 March 1998 a plain chest X-ray showed right pleural effusion. A CT-scan performed on 2 May 1998 showed right lamellar pleural effusion reaching the apical pleural space and no definite of hilar or mediastinal masses. A plain chest X-ray done on June 1998 showed scoliosis of the dorso-lumbar spine convex to the left. Lamellar pleural effusion was observed in the right side reaching the apex, basal effusion was noted and a non-homogenous opacity was seen in the right lower lung zone.

She started to take the extract of the invention on 8 May 1998 both IM and rectally. The first three weeks were crucial since the patient was suffering from dyspnea, nausea, chest pain vomiting, fatigue and general weakness (body weight was 37 kg). A longside with the extract, she was taken bronchodilator, pethidine lOOmg (TV) and oxygen treatment.

A bone scan was performed on 5 June 1998 showed two focal area of abnormal tracer accumulation, one in the right pubic bone and the other in the left acetabulum. A CT-scan on 6 June 1998 showed right pleural effusion with thickening, infiltration and a telectasis of the right basal segment. A small nodule involving right pleura also suggestive metastasis. Three months later following the treatment with the extract of the invention, the patient showed improvement in respiration and a decrease in the acute pains. A chest CT scan performed during this period i.e. on 5 August 1998, found the pleural effusion and the thickening were diminished in size. A bone scan was done also on 10 August 1998 showed that the two bone lesions seen in the pervious scan done on June 1998 with less tracer uptake especially the focal lesion in the left acetabulum. No evidence of new bony lesions and no evidence of disease progression were noted. The patient showed weight gain (4 kg), no chest pains, normal respiration and good appetite and no requirement for analgesic drugs.

Another chest CT-scan was done on 10 December 1998 and in comparison with the previous CT scan done on August 1998, no more pleural effusion was seen. On other hand, a bone-scan done on 15 December 1998 and compared with previous scan performed on August 1998 showed symmetrical and uniform tracer uptake throughout the entire skeleton. Further clinical improvements were observed on the patient including weight gain, good appetite and normal activities. The patient was put on a maintenance therapy for a period of 8 months.

OFFICE OF UJ 1 k AUG 2003 -36 RECEIVED

Claims (9)

1. A method of preparing a sterile water-soluble extract from Nerium orleander containing oleandrin, and other digoxin-like glysosides possessing active glycosides for use in treating several types of tumors comprising the steps of: (i) soaking dried powder (20g) obtained from the species in 100ml of sterile distilled water overnight. (ii) filtering the water solution under sterile conditions. (iii) adjusting the volume of the resultant solution to 30-40ml followed by: (iv) millipore filtration under sterile conditions, (v) freeze drying of the filtrate under sterile conditions and wherein the active products are extracted from the leaves (16 - 19cm long) of the Nerium oleander harvested during August - December.

2. An extract as claimed in claim 1 wherein step (i) is conducted at room temperature and under sterile conditions.

3. An extract as claimed in claim 1 or claim 2, wherein a Millipore filtration system "(0.45 and 0.22|om) is used.

4. An extract as claimed in any preceding claim, wherein the method results in a sterile extract with endotoxin concentrations of less than 30 units/ml.

5. A method as claimed in any preceding claim, wherein the extract includes oleandrins, oleandrigenin as major glycosides with trace amounts of digitoxin, gitoxigenin, sugar and amino acid moieties.

6. A method as claimed in any preceding claim, wherein wild Nerium oleander plants are harvested in a selected area close to running water.

7. A method as claimed in any preceding claim, wherein the extract is obtained by: C)0U897NZBc1aims300603/ca OFFICE OF N.Z 1 k AUG 2003 37 | RECEIVED (a) rinsing the fine ground powder of Nerium oleander in sterile distilled water (1/5 W/V) for 4 - 24 h at temperature ranging between 0 -50°C. (b) filtering the aqueous suspension of (a) several times and adjusting the pH to between 5.7 - 6.0 under laminar flow conditions using a Millipore filtration system.

8. A method as claimed in any preceding claim, wherein the extract has both in vitro and in vivo biological characteristics and wherein the in vitro characteristics include: (a) cytotoxic activity against Hela, Kraji, Hep-2 (IC50 from 1-5 (J-g/ml) and a moderate cytotoxicity against P388, L1210, T47D, RD, A549, L20B (IC50 6-10 g/ml). (b) a block at the S-phase of the cell life cycle at conc. 1 g/ml for K562 and for P388 and KB at 10 fig/ml. (c) a significant ribosome-inactivating protein activity (RIP) at concentrations less than 1 ng/ml. (d) non mutagenic effects against two bacterial strains; S.typhimurium TA1530 and S. typhimurium 1537 at concentrations up to 50 ng/ml.

9. A method as claimed in claim 8, wherein the in vivo characteristics include: (a) an inhibition towards tumour growth of B16-Melanoma in BDFi mice significantly (T/C = 135 at the dose of 0.025ml). (b) an inhibition towards tumour growth of Lewis lung carcinoma in DBFi mice significantly (T/C = 127 and 137 at the doses of 0.025ml and 0.05ml respectively). (c) no cutaneous effects 011 guinea pig skin nor any inflammatory changes in the eyes of rabbits at the does of 0.5 and 0.1ml respectively. 00U897NZBciaims3(XX503/ca INTELLECTUAL PROPERTY OFFICE OF N2 1 h AUG 2003 -38- RECEIVED (d) significant anti-inflammatory activity superior to the reference standards phenylbutazone and ibuprofen. (e) an LD50 in mice is 0.35 ml/25gm. IM and 0.3ml and 0.33ml/25gm IP and Sc respectively with apparent side effects, in high doses, like tachycardia, myorelaxation and motor in-co-ordination. (f) no apparent side effects at doses up to 0.1 ml/25gm in single or repeated doses. (g) no effect on body weight, hemoglobin concentration, erythrocyte count and blood indices like PCV, MCV, HCH and MCHC subchmically for a period of 8 weeks in mince. (h) a significant increase in leukocytes count (mostly in lymphocytes) and a decrease in the ALT, AST and urea activities at the dose of 0.05ml. (i) no cutaneous sensitisation effects on guinea pig skins nor any inflammatory changes in the eys of rabbits at the doses of 0.05 and 0.1ml respectively. 14 A method as claimed in any preceding claim, wherein the sterile aqueous extract can be used for administration by oral, rectal, intramuscularly and by intravenous routes. 15 A pharmaceutical composition of the sterile extract of Nerium oleander formed from any of claims 2 to 14, wherein the composition is combined with at least one pharmaceutically acceptable excipient. 16 A pharmaceutical composition as claimed in claim 15, wherein the pharmaceutical composition is suitable for injection. 17 A pharmaceutical composition as claimed in claim 15 or claim 16, wherein the pharmaceutical composition is in the form of a topical preparation, oral formulation or rectal formulation. PIPERS Attorneys for 1) RASHAN, Luay Jamil 2) AL-SHAKARCBQ, A. Munem Mohammad Daoud OOUS97NZBclaim5300603/ca