WO1999038520A1

WO1999038520A1 - Blood cell composition as a vaccine for hiv and other pathogenic organisms

Info

Publication number: WO1999038520A1
Application number: PCT/US1999/001922
Authority: WO
Inventors: Yash Sharma
Original assignee: Yash Sharma
Priority date: 1998-01-29
Filing date: 1999-01-29
Publication date: 1999-08-05
Also published as: AU2482099A; WO1999038520A9

Abstract

The invention describes a naturally occurring non-toxic and safe composition and a method of preparation of the same containing potent anti-viral factors, protease inhibitory factors, reverse transcriptase inhibitory factors, gp-120 blocking factors and immune modulating factors. The composition is compatible with human blood and can be used as therapeutic agent for treatment and cure for immune deficiency diseases (e.g., AIDS), immunologically mediated diseases and neoplastic diseases. The composition can be used as a source of vaccine for human against HIV and other unknown organisms.

Description

BLOOD CELL COMPOSITION AS A VACCINE FOR HIV AND OTHER PATHOGENIC ORGANISMS

Field of the Invention

This invention relates to soluble factors having anti-HIV activity which are derived from leukocytes and immune cells of animals and humans, their method of extraction and preparation and pharmaceutical preparations comprising the anti-HTV factors.

Background of the Invention The spread of human immunodeficiency virus infection (HTV), the cause of the incurable disease Acquired Immunodeficiency Syndrome (AIDS), has reached epidemic proportions around the world and is the chief cause of death among young people in the United States. Over 25 million people have been reported to be infected with HTV world-wide, and this number is expected to rise to over 100 million by the year 2000.

HIV-1, a retrovirus, has been identified as an etiologic cause of the AIDS disease. The virus interacts with the human immune system causing serious and oftentimes fatal immunosuppressions in infected patients. HTV infection is widespread throughout out society. The groups which run the highest risk of HIV-1 infection include homosexual and bisexual males and intravenous drug users. Other high-risk groups include recipients of blood transfusions and sexual partners in the HTV-infection risk groups. The HIV-infection may also be transmitted merely be heterosexual intercourse. Several cases of HTV infection are known through heterosexual intercourse even where a condom was used.

A cure for HTV-1 infection has not yet been found. In view of its rapid spread, an urgent need exists for drugs and methods for treating the disease. Recent advances in HIV/AIDS research have provided valuable information which has led to the discovery of new accurate HTV diagnostic systems which detect HTV RNA measurement or "viral load", new pharmaceuticals such as protease inhibitors, and combination therapies, in addition to education and counseling. All of these efforts have contributed to better care of HTV-infected patients and disease management

One drug which has been approved by the Food and Drug Administration (FDA) for treatment of HTV infection is AZT (S-azido- ^'-dideoxy-thymidine) which is believed to function by slowing or inhibiting viral replication. AZT, however, dees not cure the disease nor does it prevent infection. Further, AZT is known to be responsible for serious side effects, such as bone marrow suppression, and it is poorly tolerated in a high proportion of patients. Its beneficial effects have also been reported to diminish in 12-18 months.

Other examples of anti-viral pharmaceuticals include alpha interferon, gamma interferon, ampliquen, azimexon and isopinosine. As with AZT, such drugs and treatments may add a few months to a few years to the lives of HTV-infected patients who have developed AIDS, but do not cure patients of the disease. These drugs are also highly toxic and may have severe side effects. Additionally, in similar manner to AZT, the HTV virus becomes resistant to these drugs and a strict dosage regimen must be maintained, with a patient taking 20 or more pills a day being a common occurrence. The cost of conventional drugs is also prohibitive (over

$20,000-$30,000 a year), and are not within the reach of many desperate patients, particularly those in third world countries such as the African Continent, where the HTV infection epidemic is at its worst.

In view of the above, it is a goal of the present inventors to conduct research in the areas of naturally occurring compounds and to extract and isolate substances which have anti-HTV activity, and which are without side effects, are safe and non-toxic, inexpensive and can lead to a cure for HTV infected patients.

As is known, many pharmaceutically active substances and compounds are naturally present in biological samples and vegetation. For example, it is known in the medical arts that certain primates such as baboons are not infected by the invasion of the viruses, including HTV, bacteria, and certain other disease-causing organisms, apparently due to some unidentified peculiar immune response systems which is not present in human beings.

As reported in Ricordi et al., Transplantation proceedings, 26(30:1302-1303 (1994), a late stage ATDS patient was the recipient of a xenographic bone marrow transplant from a baboon. It was hoped that the baboon's immunity to the development of ATDS could be transferred to the human body by at least complimenting the patient's failing immune systems. As an experiment, the procedure was not considered successful, as the baboon's cells did not survive in the subject. Thus, the failure of engraftment of transplanted xenogenic cells has so far precluded the theory that bone marrow or blood cells derived from a species immune or resistant to HTV infection and/or the development of AIDS or other viral resistance could transfer such resistance to humans.

It is therefore an object of the present invention to provide in isolated form from mammals a substance or factor and/or extract of an immune response system which is anti-HTV active and anti- viral active in general, and which is effective to treat HTV-infected patients and patients infected with other viruses. It is also an object of this invention to isolate and provide a mammalian immune response factor which is effective as a vaccine in humans against HTV infection, and other immune system targeted viruses Summary of the Invention

In accordance with those goals and objectives set forth above, the present invention now provides a soluble composition produced from lysate of leukocytes and immune cells from whole blood, bone marrow and blood and fetal cells of primates and human beings which is therapeutically effective in treating HTV-l infection, and is also effective as a vaccine in the prevention of HTV-l infection, as well as in the prevention of infection from several other viruses.

The inventive composition is effective in the therapeutic reduction of viral load in infected patients, and is also effective in the treatment and prevention of other known human viruses including hepatitis virus, herpes virus, protein enveloped virus, non-enveloped virus, RNA viruses and DNA viruses; the inventive compositions are safe, non-toxic and display no adverse side reactions.

Additionally, the anti- viral composition of this invention has properties of inhibition of protease activity, inhibition of reverse transcriptase activity and blockage of gpl20. This invention is more fully described by the following, detailed discussion with preferred embodiments and embodiments.

Brief Description of the Drawings

FIGs. and 1 and 2 are HPLC spectra of an active factor of the inventive composition. FIG. 3 is a positive ion electrospray-MS spectrum of an active factor of the inventive composition.

FIG.4 is an EI-MS spectrum of an active factor of the present composition.

FIG. 5 is a "best-fit" library matter for the spectrum shown in FIG. 4.

FIG. 6 is a UV chromatogram of an active factor of the inventive composition. FIGS. 7-12 are ESI-MS spectra of several major components detected within the chromatographic profile.

FIG. 13 is a graph summarizing hematologic evaluation of whole blood after one hour contract with the inventive composition.

FIG. 14 is a graph illustrating inhibition of HTV-l protease by composition. FIG. 15 is a graph illustrating inhibition of HTV-l reverse transcriptase activity by composition.

FIG 16 is a graph illustrating inhibition of tumor necrosis. Factor Alpha UI is a cell line used for analysis. FIG. 17 is an electrophoresis gel study of the composition of showing the difference in electropheresis pattern between human and baboon cell preparation of the composition.

FIG. 18 is a graph showing effect of baboon PBMC extract composition on different strains of HTV-l, chronically infected H9/UIB cells and STV (Simian Immune Deficiency Virus).

FIG. 19 is a graph showing effect of baboon PBMC extract composition on pre- and post-treated human cells and inhibition of viruses with the composition, which indicates the inventive composition's efficacy as an HTV vaccine.

FIG. 20 is a graph showing effect of baboon PBMC extract on % inhibition of HTV-l up to 12 days of anti-viral activity at 37°C indicating high degree of stability of the composition at elevated temperatures.

FIG. 21 is a graph illustration results of baboon tissue culture grown cell extract composition of the present invention on reverse transcriptase activity using cell line CEM-TART cells. FIG. 22 is a graph illustrating results of baboon PBMC extract composition of the present invention as reverse transcriptase activity.

Detailed Description of Preferred Embodiments

It is known that the initial pathogenic event in HTV infection is the binding of HTV-l to the CD-4 receptor on a subset of T cells. CD-4 is the cell protein to which the HTV-l virus binds. The protein gpl20 is the virus protein which is responsible for the binding. The virus interacts with the human immune system, with resulting immunosuppression from depletion and functional abnormalities of CD4+T cells.

In treatment of immune deficiency diseases caused by viruses such as HTV, an effective method is to reduce the viral burden in the circulating blood, thereby boosting the immune system and providing essential nutritional factors to stimulate production of non- infected healthy blood cells, particularly CD4+T lymphocytes.

As discussed hereinabove, certain primates, such as for example baboons, are not infected by the invasion of viruses, bacteria and other disease-causing organisms, apparently due to some heretofore unidentified peculiar immune response system/ substance not present in humans.

In accordance with the present invention, there is now provided a composition comprising a soluble lysate product from leukocytes and immune cells of primates, such as baboons, and humans who are immune to or otherwise resist infections by viruses, microorganisms or other disease-causing agents, which is therapeutically effective in treating HTV infections and other viral infections as well as acting as a vaccine in the prevention of HTV infection and other viral infections.

Blood, bone marrow, cord blood, stem cells and progenitor cells from primates, mammalians and human species may be different from other primates, mammalians and human species in genetic, enzymatic make-up, or may contain certain peculiar natural components that bestow resistance and immunity to certain viral or bacterial infections. These factors may be present in the immune cells or other blood cells or tissue that produce blood cells, and may be in the form of protein, peptides, amino acids, carbohydrates, lipids or other biochemical nature that are present naturally in the living body of these resistant-to-infections species. Blood is composed of red blood cells, platelets, stem cells, macrophages, leukocytes as cellular components. Leukocytes commonly known as white blood cells (WBC) consist of neutrophils, granulocyte, lymphocytes, monocytes, eosinophils, and basophils. Lymphocytes, in particular T cells, participate in regulating the immune system of the body, and are themselves regulated by certain peptide hormones. T cells, which control and regulate the immune system are classified as CD4+T lymphocytes and CD8+T lymphocytes. The role of other lymphocytes is not well understood, though they are known to act as phagocytes and ingest foreign particles.

Eosinophils are known to exert potent parasiticidal effect through their cationic proteins and enzymes. Primate stem cells and granulocytes, bone marrow, and cord blood all secrete Viral Suppressive Factors and include Regulated Upon Activation Normal T Cells Expressed and Secreted (RANTES) Chemokines, Macrophage Inflammatory Proteins (MTP-1 Alpha and MIP-I Beta), and Beta chemokine microphage derived chemokine (MDC). Immunomodulators include polypeptide thymic hormone Thymosine Alpha- 1, subsets of CD4+T cells thl responsible for cellular immunity and th2 responsible for humoral immunity.

Without wishing to limit this invention to any particular theory, it is thought that the present inventive soluble lysate product may include by way of example, but without limitation, immunopotentiating polypeptide hormone, extracts of CD8+T lymphocytes containing viral suppressive factors, CD4+T lymphocytes containing factors that regulate cellular hormonal and immunity, hemopoietic factors, and other cell nutrient species such as calcium and ascorbic acid. The composition of the present invention provides an active, stable and safe solution that can be sterilized and used at a physiologically active pH suitable for injection or transformation into the human body for treatment and prevention of immunosuppression diseases by immunoresponsive therapeutically active substances such as described above, and a variety of other diseases such as hepatitis B, hepatitis C, cytomegalic virus (CMV), herpes virus and other viral diseases.

In one preferred embodiment of the invention, the inventive therapeutic composition can be prepared by first providing a leukocyte fraction of whole or cord blood derived from a primate such as a baboon, chimpanzee, monkey or gabon, which species is immune to infection from HTV and other currently known human-to-human transmissible viruses. This leukocyte fraction may comprise one or more of stem cell, progenitor cell, lymphocytes, monocytes, and granulocytes, which contain suppressive factors and immune modulating factors. Peripheral blood mononuclear cells and buffy coat cells can then be separated from the leukocyte fraction using any conventional method such as the Ficol-Hypaque technique, centrifugal separation or any other method suitable for the separation of leukocytes. The cells are then lysed, stabilized, the pH adjusted accordingly as desired and the solution is then sterilized for therapeutic use in humans. The following example is illustrative of this broadly described preferred embodiment. EXAMPLE I

Nucleated cells are selectively concentrated by using procedures such as density gradient separation with hydroxyethyl starch, Ficol-Hypaque, leukocyte removal filtration, direct lysis or any other method including the use of antibody-coated bead non-erythrocyte cells from blood.

1.1 The following protocol illustrates a preferred procedure that may be employed for isolating primate bone marrow cells from whole blood/buffy coat:

Baboon blood is processed for obtaining buffy coat using standard protocol for isolation of PBMCs from buffy coat by Ficol-Hypaque (Histopaque) as follows:

1. Transfer 10 ml blood aseptically to 50 ml tube;

2. Layer 10 ml blood over 3 ml of Histopaque;

3. Centrifuge at 550g (2000rpm) for 30 minutes, remove the band containing PBMC, resuspend in phosphate buffered saline (PBS)

4. Centrifuge at 400 g (1500 rpm) for 10 minutes at room temperature, remove supernatant, gently resuspend the pellet first by finger tapping and then by pipetting in the 10 ml PBS;

5. Centrifuge at 400 g (1600 rpm) for 10 minutes at 10°C, remove supernatant;

6. Resuspend pellet in RPMI (10 ml), count the cells and examine viability.

The purified nucleated cells are then washed 3 times with HBSS and stabilizer solution containing excess calcium ions. The washing is performed in a refrigerated centrifuge at 4500x g for 10 minutes at 18-20 °C. Following the last wash, the deposit is resuspended in HBSS without Pana Sera Plus to contain 50-500 million cells/ml. The cells are then ruptured by sonification under ice. The sonicate is repeatedly frozen and thawed by alternate immersion in liquid nitrogen and water at 37 °C in five rapid and successive cycles. After the last thaw cycle, the slurry is centrifuged in a refrigerated centrifuge at 4500x g for 10 minutes to obtain a clear supernatant. The supernatant is sterilized by passing through 0.2 micron membrane filter and mixed 1:1 by volume with Sterile Secondary Ingredients (SSI) under aseptic conditions. SSI comprises sterile intrinsic factor, cyanocobalamin, folic acid, sodium ascorbate, calcium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium chloride and sucrose. The final composition containing cell lysate and SSI, has a pH of 6.8-7.4. The composition containing the fortified lysate of stem cells, and granulocytes is then aseptically dispensed in 1-10 ml aliquots in sterile ampules for storage in liquid nitrogen until used. Each 1 ml contains the lysate obtained from 10-50 million cells.

1.2 The following flow diagram summarizes the steps which may be employed in preparing the inventive composition of this preferred embodiment:

FLOW DIAGRAM STEPS

1. OBTAIN BABOON WHOLE BLOOD IN CITRATE PHOSPHATE DEXTROSE

(CPD, an anti-coagulant) 60 ml

2. ISOLATE PBMC FRACTION USING FICOL-HYPAQUE

(Ficol-Hypaque 1:3 pipette at the bottom of the tube or layer blood on F.H. gives 1 million cells/ml)

3. LYSE TN STERILE DISTILLED WATER

(1 ml water for 10 million cells)

4. REFRIGERATE 4°C 1-96 HOURS

(to complete lysis)

5. THAW AND ADD STABILIZING SOLUTION (Use 10% v/v, contains Ca, and KC1 in PBS at pH 7.4)

6. INCUBATE AT ROOM TEMPERATURE 15 MINUTES (provides precipitate which settles in 15 minutes

7. CENTRIFUGE 1500 G FOR 15 MINUTES (to remove the precipitate)

8. FILTER USING 0.22 MICRON FILTER (to obtain clear solution and sterilize)

9. ALIQUOT AND FREEZE-18 °C UNTIL USED

(to avoid repeated freezing and thawing) 10. DILUTE IN STERILE SALINE FOR INJECTION FOR USE

11. STERILIZE BY GAMMA RADIATION

12. TEST FOR BACTERIAL AND VIRAL CONTAMINATION

(Nutrient Agar culture-72hr, co-culture with uninfected human PBMC 6 days)

As shown by the following examples, upon repeated injection the inventive composition exerts a direct and indirect action on viral particles to adversely affect the replication of the infective agents. The composition thus lowers the rate of virus replication and virus load in the host body. Direct lysis of the cell-free infectious agents in the host blood is thought to be attributable to any of several viral and enzyme inhibitory factors present in the inventive lysate composition. Following the completion of the therapeutic regimen, transfusions with homologous blood may be necessary to restore the innate leukocyte population to its normal level in a subject host.

As also shown by the following examples, the present invention also circumvents a failure of mass immunization. For example, normal human BMCs (bone marrow cells) have been shown to resist infection by HTV if pre-treated by the inventive composition for given period of time. Conventionally, routine vaccines are prepared from the antigenic fragments of infectious agents. Injections of these vaccines stimulate the host to produce specific antibodies that bind to the antigen sites of the infecting organ. However, the infective organism often mutates to alter its antigenic sites so that these antibodies have no specific binding sites on the antigen molecule; under these circumstances, the humoral antibody that protected the host earlier against the parent strain of the agent becomes ineffective. Administration of a non-specific composition such as the one described herein, is therefore a more powerful therapeutic tool. As illustrated in the following examples the inventive composition causes a reduction in viral load which is thought to potentially lead to a complete or substantially complete elimination of HTV infection in a patient. The inventive composition also boosts the immune system by increasing the CD4 T lymphocyte count, improves conditions for new virus- free white blood cells and is non-toxic, side-effect free and completely compatible with human red blood cells. EXAMPLE 2 PREPARATION OF PREFERRED COMPOSITIONS 2.1 Composition (A)

Composition (A) described in this invention consists of:

1. Baboon stem cells/mononuclear cells/leukocytes (from 75 million cells or 10 ml whole blood) dissolved in D. water or other hypotonic solution: 1.0 ml

2. calcium chloride 10 ug

3. potassium chloride 10 ug

4. Sodium phosphate 10 ug

5. potassium phosphate 10 ug

6. sodium chloride ug

7. sucrose 10 ug

METHOD OF PREPARATION FOR COMPOSITION (A): 1. Draw 50 ml of baboon blood/bone marrow/cord blood into a sterile multiport bag containing CPD anticoagulant solution.

2. Separate peripheral blood mononuclear cells using Ficoll-Hypaque technique.

3. Check cell count using cytometer.

Add 1.0 ml deionized water mix well using vortex and store at -20 °C for 24-96 hours till a clear solution is obtained. Check the cell count as above. All cells should be lysed completely.

4. Filter using 0.2 micron filter.

5. Add 100 microliter of stabilizing solution, mix well be vortex.

6. Filter again using 0.2 micron filter, store at -20°C. 7. Dilute in sterile saline solution.

8. Sterile using gamma radiation standard procedure.

9. Store at 4°C till used. 2.2 Composition (B)

Composition (B) described in this invention consists of:

1. Human stem cells/leukocytes/cord blood/bone marrow from 75 million cells

(10 ml whole blood) lysed in D. water or hypotonic solution (1.0 ml) 2. Other salts as described in Composition (A).

METHOD OF PREPARATION FOR COMPOSITION (B): Follow the steps as described for Composition (A).

2.3 Composition (C)

1. Baboon PBMCs from 10 ml whole blood grown in tissue culture media using Phytine Heam agglutinin Interleukin-2 (TL-2) stimulation in RPMI medium lysed in D. water (1.0 ml)

2. Other ingredients as in Composition (A).

METHOD OF PREPARATION OF COMPOSITION (C) Follow the steps for Composition (A).

2.4 Composition (D)

1. Baboon tissue culture cells grown from three different baboons, pooled and lysed in 1.0 ml D. water (1.0 ml)

2. Other ingredients as in Composition (A).

METHOD OF PREPARATION OF COMPOSITION (D) Follow the steps for Composition (A). 2.5 Method of Isolation of PBMC from Whole Blood/Buffy Coat

1. Transfer 10 ml blood aseptically to 50 ml tube.

2. Layer 10 ml blood over 10 ml of Histopaque.

3. Centrifuge at 550 g (2000rpm) for 30 minutes, remove the band containing. PBMC, resuspend in PBS.

4. Centrifuge at 400 g (1500rpm)

5. Centrifuge at 400 g (1600 rpm) for 10 minutes at 10°C, remove supernatant.

6. Resuspend pellet in RPMI (10 ml), count the cells and examine viability. (Hanks buffer, Histopaque, PBS should be kept at room temperature at all times.)

EXAMPLE 3 3.1 Composition Analysis.

At least one active fraction of the present inventive composition is thought to be a relatively small molecule of molecular weight 200-500, which is referred to herein Natural Killing Agent ("ANKA"). Amino acid analysis has shown that ANKA is not a protein or polypeptide type of cytokine, but is a carbohydrate. Further, Because of its small size, ANKA may be given orally, it may effectively distribute beyond the intravascular compartment and would not evoke an antibody response in humans.

3.1.1 Scaled-Up Preparation of Lysate of Baboon PBMCs for Analysis

60 ml of baboon whole blood in CPD as coagulant was obtained from Naval Blood Research Lab (NBRL) under sterile conditions. Using Advanced Bio Sciences ABS protocol for isolating PBMCs by Ficol-Hypaque technique, blood was divided into two equal parts of 30 ml in sterile centrifuge tubes. 10 ml of histopaque was added at the bottom of the tubes.

Both tubes were centrifuged at 1500G for 30 minutes, and the primate bone marrow cell (PBMC) fraction was separated from the top with very few RBCs. The PBMC fraction thus obtained was washed with PBS one time. One tube containing PBMC fraction was mixed with 30 ml RPMI culture media containing equal part of PHA and IL-2 stimulation factors and incubated at 37 °C for continuous cultures.

The remaining PBMC fraction in the other tube was used to prepare lysate by adding 5 ml sterile water, mixing well by vortex and freezing at -20 °C for 4 days.

Human PBMCs were provided by ABS and were used to prepare lysate simultaneously as control following same procedure as for baboon cell and lysed in 5 ml of sterile distilled water and frozen for 4 days.

Frozen lysates were thawed and examined under microscope for cell debris, DNA and other substances. Both human and baboon cells were completely lysed leaving no intact cells in the solution, cell debris and DNA strands were seen in both preparations.

0.5 ml stabilizing solution was added to both lysates, mixed well by vortex and allowed to incubate for 10 minutes at room temperature. Both tubes showed precipitation, and were again examined under microscope. Precipitation, and other substances, including DNA strands were seen and precipitate was settling at the base of both tubes.

Both tubes were centrifuged at 1500 G for 10 minutes, and supernatant was filtered using 0.22 micron filter. Both tubes gave clear supernatant and were frozen at -20 °C.

Next, both tubes were thawed, examined for turbidity, centrifuged at 1500G for 15 minutes; no precipitate was seen and the solutions were aliquoted in 100 ul fractions and frozen. 0.3 ml baboon preparation was diluted in 30 ml sterile saline and saved.

Tissue culture was terminated with cell count over 200 million, the cells washed twice with PBS and lysed with 14 ml distilled water, complete lysis was observed after few minutes. The lysate was stored at -18 °C.

An aliquot was used to test for protein level by biosynic acid, in a biosyn assay, a conventional method for protein analysis. Human cell preparation contained 2.6 mg total protein per ml.

Baboon cell preparation contained 1.4 gm total protein per ml.

Gel electrophoresis showed several bands in both preparations. Human pattern was different than the baboon preparation. Analytical results are as follows. 3.1.2 HPLC analysis.

FIGs 1 and 2 are HPLC spectra of an apparent active factor of the inventive composition.

As shown, one major peak and two minor peaks of material are detected. In this essay, a Water's column (Delta Pak 18 15 urn, 300 A, 0.39 x 30 cm) with a flow of 1 ml/min was employed, and the contents of peaks absorbing at 215 nm were collected and freeze-dried. Injections of 200 to 500 ul produced a very large peak eluting at 30% acetonitrile and two minor peaks eluting at approximately 50% acetonitrile in a gradient of 0 to 100% acetonitrile in 60 minutes. The solvent system employed was 0.1% aqueous trifluoroacetic acid/0.1% TFA:acetonitrile.

The large peak with an O.D. of 2.0, upon elution had a distinct aromatic ring absorbance with a peak maximum at 258 nm. The compound is thought to be of a relatively small molecular weight containing a benzene ring, possibly with an alkyl substituent. The spectra is characteristic of phenylalanine, with the compound possibly being a metabolite or antibiotic that is in cells.

3.1.3 Electrospray — MS Analysis

Electrospray ionization spectrometry was performed on the isolated substance having a peak at 215 nm in HPLC analysis using a VG Biotech BIO-Q instrument with a quadruple analyzer. Myoglobin was used to calibrate the instrument. Sample aliquots of 50 ul were injected into the instrument source. Elution was carried out using a mixture of 50% acetonitrile containing 0.1% TFA, at a flow rate of 1 Oul/min.

As shown in FIG. 3, this sample gave a positive ion electrospray-MS spectrum showing notable possible sample-related signals at m/z 86, 194 and 288. The appearance of this spectrum with the absence of no readily assignable pseudomolecular ions(s) may suggest this sample to not be peptide/protein in nature, or a non-nitrogen containing analyte with a lipid-like structure. 3.1.4 MAXIMA 820 Concentration Report AMINO ACID ANALYSIS RAW DATA USING AMINO ACID ANALYZER RESULTS

TABLE 1

5 SAMPLE: AASO8273 # 1 In Method: AA METHOD Type: STND

Acquired: 27-AUG-1996 12:56 Instrument: A A Analyzer

Rate: 2.0 points/sec Filename: AASO8273 Duration: 38.000 minutes Index: Disk 0 Operator: ?.T Injection Volume: 10.0

DETECTOR: PLUGEOMETER

PK# E># Retention Time Type Peak Height Peak Area Area Percent Code Base Solution Component

(^'minutes') (microvolt) Cmicrovolt-secl Cost Name

1 1 12.392 BB 28944.496 384660.19 2.58 EXT AREA 200.00000 ASP 5 2 2 13.733 BP 32783.035 514441.94 3.45 EXT AREA 200.00000 SBR

3 3 14.433 PB 28333.787 455977.10 3.06 EXT AREA 200.00000 GLO

4 4 15.353 BP 29857.281 468454.98 3.15 EXT AREA 200.00000 CLT

5 5 15.841 PP 42794.352 706717.02 4.75 EXT AREA 200.00000 SIS

6 6 16.600 PS 30264.739 632134.69 4.25 NONE AREA AMM 0 7 7 19.117 BB 43904.310 681096.79 4.57 EXT AREA 200.00000 ARG

8 8 20.042 BB 38475.041 627056.05 4.21 EXT AREA 200.00000 TBR

9 21.042 BP 6012.0828 91589.765 0.62

10 9 21.456 PB 33054.356 583811.98 3.92 EXT AREA 200.00000 AGA

11 10 24.450 PB 24962.667 342468.92 2.30 EXT AREA 200.00000 PRO

25 12 11 27.600 BB 60972.698 880246.31 5.91 EXT AREA 200.00000 TYR

13 12 28.867 BP 88987.957 1275834.7 8.57 EXT AREA 200.00000 VAG

14 13 29.300 PB 77494.869 1163061.5 7.81 EXT AREA 200.00000 MET

15 31.475 BB 10576.845 137614.51 0.92

16 14 32.025 BP 42031.412 557056.16 3.74 EXT AREA 200.00000 GFS

3 0 17 15 32.725 PP 104782.12 1647272.4 11.06 EXT AREA 200.00000 ILE

18 16 33.267 PB 100684.42 1710485.6 11.49 EXT AREA 200.00000 LEO

19 17 34.283 BB 121501.46 2030303.3 13.64 EXT AREA 200.00000 PBE

TOTAL 946417.92 14890286 3200.0000 3.1.5 Amino Acid Analysis

Approximately 0.2 ml of HPLC material of O.D. 2.0 was hydrolyzed and submitted to amino acid composition analysis with results, summarized below in Table 2, showing no amino acids present.

TABLE 2

Purity (HPLC): 100% Animo Acid Analysis p moles molar ratio A or H calc found

Asp

Ser

Glu

Gly

His

Arg No amino acid found

Thr

Ala

Pro

Tyr

Val

Met

Lys

Lie

Leu

Phe

0 3.1.6 Additional Analytical Analysis

This study demonstrates continuing analysis of the test sample, including the direct probe EI-MS with the goal of providing a characteristic fragmentation pattern of the sample. Sample Preparation

Prior to analysis the sample was dissolved in distilled water and used as supplied.

IE-MS

Electron Impact Mass Spectrometry was carried out on M-Scans VG analytical ZAG 2-SE high field mass spectrometer with reference to SOP# MS-003, MS-002 and MS-007. The source temperature was maintained at 200 °C and spectra were acquired with an electron voltage of 70eV. The sample was inserted into the instrument via the solids probe and heated until an appropriate spectrum obtained. Calibration was performed using PFK.

LC-MS

CHROMATOGRAPHY The following chromatographic conditions were used:

Pumps: Hewlett Packard Model 1100 Binary system.

Solvent A: 0.1% aqueous TFA.

Solvent B: Acetonitrile : water (9:1) v/v containing 0.1% TFA.

Gradient: Start at 0% B and hold for 5 min.

Raise to 80% B after 40 min. Flow Rate: 0.25 ml/min.

Stationary Phase: C 18 Vydac: 2.1 mm X 25 cm.

(S/N : 950308-40-1)

DETECTION

UV

UV detection was achieved using an HP variable wavelength detector operating at 215 nm. MASS SPECTROMETRY

MS detection was achieved using a VG BioQ triple quadruple mass spectrometer operating in the positive ion electrospray ionization mode, with the following parameters:

1. Scan Range m/z 35-700

2. Cone Voltage : 57V

3. Source Temp. : 100°C

4. Acquisition Delay : 0 min. UV.

0 min. MS.

Calibration was performed with direct injection analysis of Csl prior to LC-MS. RESULTS EI-MS

FIG. 4 illustrates a typical EI-MS spectrum obtained during analysis of the sample. This shows a number of major possible sample-related signals including those at m/z 69, 87, 114, and 129.

FIG. 5 illustrates a "best-fit" library match for this spectrum and although not identical, is similar to that of dimethyl decane.

LC-MS

FIG. 6 illustrates the UV chromatogram obtained following sample injection. In contrast to previous analyses, this shows a major component eluting at 26.74 min.^"together with numerous additional components eluting throughout the gradient. FIGS. 7-12 illustrate the ESI-MS spectra obtained for a number of the major components detected within the chromatographic profile. Most of these appear to show distinct, strong possible (M+H)⁺ at pseudomolecular ions which are summarized in Table 3 below. TABLE 3: Retention time and possible m/z value for the pseudomolecular ions detected during LC-MS

Retention Time (min) Possible (M+KY

22.17 331.6

25.24 481.8

26.74 383.6

28.66 225.5

32.42 244.6

288.6

36.93 485.5 529.6 573.5 590.6 634.6 661.6

The major peak (26.74 min.) shows a possible (M+H)⁺ at mz 383.6 which has not been detected in previous analyses.

Peaks at 32.42 and 36.93 min. show several major signals with a 44amu differences. This is indicative of the present of an ethoxylate polymer.

Further analysis of these data by Selective Ion chromatography (data not shown) failed to reveal any significant component with signals at m/z 92, 151, and 133 detected with signals at m/z 130, 114, and 69 (detected by probe EI-MS). A major signal at m/z 86 (previously detected by direct ESI-MS analysis; Report 35300961181) was detected in a component eluting at 5.36 min. A signal at m/z 288 (previously detected by direct ESI-MS) was observed in a number of components eluting between 31-33 min. However, these appear to relate to the ethoxylate polymer.

It has been hypothesized that bone marrow-derived cells from species resistant to AIDS could be transplanted in patients with AIDS to transfer resistance to the disease. As discussed above, failure of engraftment of the transplanted xenogenic cells has so far precluded the validation of this hypothesis. To determine whether a soluble factor could convey resistance to AIDS or exert an antiviral effect, a sample of the present inventive composition was prepared as a sterile lysate of baboon white cells, using the PBMC separation technique of Ficoll-Hypaque, to which neutral stabilizers were added. This solution demonstrated remarkable in vitro properties against HIV, such as a greater than log-6 killing even in very high dilutions, a tenfold boosting of CD-4 lymphocytes, and no evidence of cytopathic effects. Rat toxicity studies showed no clinical or laboratory evidence of toxicity even in dosages 100 times the calculated human dosage. Analysis set forth above indicates that an apparent "active" fraction of the present inventive composition is relatively small (molecular weight 200-500) molecule (ANKA)and is not a protein or polypeptide type of cytokine, and therefore might be given orally, could distribute beyond the intravascular compartment, and would not evoke an antibody response in humans. Examples follow.

EXAMPLE 4

EFFECT OF INVENTIVE COMPOSITION ON HUMAN BMC COUNTS

Composition Dose Calculation Method A BABOON BLOOD

Mean RBC count 5,000,000 cmm

Mean WBC count 5,000 cmm

Mean HEMATOCRIT 35%

Calculations based on weight of whole blood:

1.0 ml whole blood weights 1.2 grams

Hematocrit 35% = 0.42 grams while cells being 1/1000 of the red cells = 0.00042 grams = 0.42 mg PBMC fraction being 40% of total WBC = 0.168 mg

10.0 ml blood will have 1.68 mg PBMC weight 12.5 ml blood will have 2.1 mg PBMC weight or 2100 microgram

1/10 dilution has 210 ug 1/100 dilution has 21 ug 1/500 dilution has 4.25 ug 1/1000 dilution has 2.1 ug 1/2000 dilution has 1.0 ug

Method B

One ml of whole blood gives one million PBMCs when Ficol-hypaque method is used. Total protein concentration of composition extract from 30 ml whole blood contained 1.4 mg/ml proteins. Procedure: BMCs from 10.0 ml freshly drawn heparinized human whole blood were isolated using the standard BBI protocol. BMCs were diluted in 10 ml of RPMI- 1640 tissue culture media (see SIGMA catalog, pp. 1473-94), mixed well by gentle inversion and divided into two equal parts in 15 ml tubes. 0.1 ml of 1/10 composition was added to one of the tubes and labelled as test (treated), and the other untreated tube was used as control.

Cell counts were made using a hemocytometer immediately after treatment with the composition and at 24 hours, 3 days, and 7 days.

TABLE 4: Results (BMCs/mm3)

0-hr 24-hr 3 days 7 days Control 2030 1850 1430 1090

Test 1980 2100 2700 2760

EXAMPLE 5

EFFECT OF INVENTIVE COMPOSITION ON HUMAN RED BLOOD CELLS

Procedure: 1.0 mL freshly drawn heparinized whole blood was diluted in 4 mL of PBS pH 7.4. 0.1 mL of baboon PBMC lysate was added and mixed by gentle inversion and marked as a composition treated sample. 1.0 mL of the same whole blood diluted in 4 mL of PBS was used as control untreated sample. An aliquot of the same blood was tested for reference values. Values are reported as corrected for the dilution factor.

TABLE 5: RESULTS

Day-0 Day-21 Day-42

Untrtd Composition Untrtd Composition Untrtd Composition

RBC 5.3 5.3 4.2 5.1 3.0 5.1

Hb 12.8 12.9 8.4 12.8 6.3 12.6

Hct 32.3 32.3 20.2 33.0 15.8 32.0

Lysis 0% 0% 10% 0% 40% 0%

45% 0% 0% 92% 0% 58.4% 0% saline

Reference Values

RBC — 5.4 Hgb — 12.7

Hct — 33.3

EXAMPLE 6

AMERICAN RED CROSS STUDY: EFFECT OF COMPOSITION ON FACTOR VIII AND FACTOR IX CLOTTING ACTIVITY

Composition was first added to plasma at 1 : 10 dilution, and samples were incubated for 1 , 2, 5, and 23 hours at 37°C, after which they were frozen and assayed for Factor Vπi and Factor IX clotting activity by one stage APTT method. 0.15M sodium chloride was used as control. Samples were also incubated at room temperature and at 4°C.

The Factor VIII and Factor TX activity of the samples treated with composition (0.076 and 0.389 units/ml respectively) compared well with saline control as indicated in Graph 3.

SUMMARY OF ACTIVITY DATA IN PLASMA INCUBATED WITH SALINE OR COMPOSITION ADDITIONS

Time % Activity Remaining (hours FVm Saline FVπi/Comp FTXSaline FIX/Comp 0 91.81 111.11 91.13 93.01

1 93.92 114.29 98.77 103.06

2 80.11 120.91 96.95 93.83

5 93.5 114.29 93.51 93.68

23 67.95 87.45 40.14 62.03

Activity in Plasma Incubated with Composition

10 15 20 25

Hours at 37C EXAMPLE 7

7.1.1 HIV Infectivity Assays

Protocol for preparation of infected cells in HTV-l inhibition test can be summarized below as follows. Normal PBMCs (from Red Cross) were infected with 6-log HTV-l MN strain

(AIDS reagent Program CAT# 317).

PMBCs 2x10 -5 were incubated with HTV-l MN at 35°C for 3 hours in a 15ml tube.

The excess free virus was removed by washing the cells with lx PBS 2 times. The infected PBMCs were cultured in RPMI + 10% bovine calf serum + PS at 37°C and 5% CO₂.

12x15 ml tubes each containing 2x10 -5 cells in 1 ml media were infected in duplicated with serial dilutions of HTV-l MN..

After 3 hours the cells were spun down to remove free virus. They were then washed with PBS. One set was resuspended in 750 ul ofmedia.

Serial dilutions of composition were added 750 ul per well containing 750 ul media and infected 2x10 -5 cells as described below. Four control wells did not have composition and 750 ul media was added to make up the volume.

Dupont P-24 antigen assay kits were used to determine P-24 output of all samples.

7.1.2 Dav l

1. 50 ml baboon blood was obtained as described above.

2. The blood was divided in four aliquots 12 ml each.

3. PBMCs were isolated using BBI protocol for Ficol-Hypaque isolation of PBMCs (standard procedure).

4. Lysed the PBMCs from one aliquot in 1 ml. Kept at 4°C (4 days total lysis).

5. Add 100 ul of stabilizing solution to lysed PBMCs. Others store at 20 °C.

The content of each tube was added to one infected set. Then they were transferred to 3 rows of a 24 well plate.

1 2 3 4 5 6 uninfected - - - - PBMCs > > > > >

MN - - - - infected > > > > >

PBMCs undiluted 1:10 1:100 1:1000 1:10000 1:1000000 uninfected - - - - and - - - - treated - - - -

2x10⁵ -> -> -> -> -> 1:1 1:4 1:16 1:64 1:256 1:1024

Empty > > > > >

The plate was incubated for 7 days at 37 °C with 596 CO₂. Fresh blood was added (196) to 10 ml PBS and treated with 100 ul of baboon lysate at RT. No hemolysis was observed after 2 weeks.

Dav 4 Use 100 ul of stabilizing solution to add to 1 ml PBMCs lysate.

Normal blood was drawn and PBMCs were isolated using standard protocol. The PBMCs were counted and cultured in RPMI.

Dav 5

The following day, twelve 15 ml tubes each containing 2 x 10 ⁵ cells in 1 ml media were infected in duplicate with a series of dilution of HTV- 1 MN of 1 ml.

After 3 hours, the cells were spun down to remove free virus. Then they were washed with PBS. One set was resuspended in 750 ul of media. The other set received baboon lysate as follows. Dav 10

Dupont P-24 Kit was used to determine the P-24 output of all samples, except for the negative control where only 2 tubes were used.

The ODs at 490 nm were

NC Infected Infected and treated

Plate NC 0.099 0.469 0.341

0.117 0.417 0.121

Uninfected 0.055 0.235 0.017

PBMCs 0.137 0.043

0.076 0.057

0.075 0.061

SUMMARY

Experiment #1: Determination of Composition Antiviral Activity

Materials and Methods:

Dupont P-24 assay kit for determination of P-24 output Normal PBMCs from the American Red Cross

HTV-l MN (AIDS reagent program CAD # 137) RPMI media (Difco) 24 well cell culture plates PBMCs isolated as per BBI standard protocol and incubated with HIV-1 MN at 37 C for 3 hours in 15 mL tube. The excess (free viruses) were removed by washing the cells with PBS one time.

RESULTS

Well # OP P-24 Svncvtia CPE

1— UNinf-PBMCs 0.099 70 NEG NEG

2— UNinf-PBMCs 0.117 89 NEG NEG 3— UNinf-PBMCs 0.055 135 NEG NEG

4— Uninf-PBMCs 0.012 90 NEG NEG

5— Inf-PMBCs-No-Comp 0.469 14.7M POS 4+

6~Inf-PBMCs-No-Comp 0.417 10.9M POS 4+

7-Inf-PBMCs-No-Comp 0.435 123M POS 4+ 8-Inf-PBMCs-No-Comp 0.235 6.66M POS 4+

9— Inf-PBMCs-No-Comp 0.389 8.82M POS 4+ zlO-Inf-PBMCs-No-Comp 0.249 7.3M POS 4+ ll-Inf-PBMCs-Comp-l:2 0.017 320 NEG NEG

12-Inf-PBMCs-Comp-l :4 0.043 922 NEG NEG 13-Inf-PBMCs-Comp-l:8 0.057 871 NEG NEG

14-Inf-PBMCs-Comp-l:16 0.061 824 NEG NEG

15-Inf-PBMCs-Comp-l :32 0.012 265 NEG NEG

16-Inf-PBMCs-Comp-l :64 0.041 865 NEG NEG

UnRx Rx

MeanP-24 9.68M 624 SD +/- 3.24M 326 CV 0.19 0.25

% of Control 100 0.04 P-24 as picogms-HIV-1/ml EXPERIMENT #3: Determination of Compositions's Antiviral Activity Materials and Methods:

Dupont P-24 assay kit for determination of P-24 output

Normal PBMCs Baboon PBMCs from 10.0 mL whole blood, from two other baboons #266 and #267 and tissue culture cells from #266, #267 and pooled PBMCs from tissue culture cells of #266 and #267 (5 PBMC samples lysed and tested).

HTV-l MN (AIDS reagent program CAT # 137)

RPMI media (Difco) 24 well cell culture plates

PBMCs isolated as per BBI standard protocol and incubated with HTV-l MN at 37 C for 6 hours in 15mL tube. The excess (free viruses) were removed by washing the cells PBS one time.

RESULTS (5 Lysates were tested) Well# Svncvtia CPE l-Unlnf-PBMCs NEG NEG

2-UnInf-PBMCs NEG NEG

3-UnInf-PBMCs NEG NEG

4-UnInf-PBMCs NEG NEG

5-Inf—PBMCs— NO-Comp POS 4+ 6-Inf~PBMCs— NO-Comp POS 4+

7-Inf— PBMCs— NO-Comp POS 4+

9-Inf— PBMCs— NO-Comp POS 4+

9-Inf— PBMCs— NO-Comp POS 4+ lO-mf-PBMCs— NO-Comp POS 4+ Lysate-1 (#266 cells)

Inf-PBMCS-Comp-TREATED

11 500 NEG NEG 12 1000 NEG NEG 13 2000 NEG NEG 14 4000 NEG NEG 15 8000 NEG NEG 16 16000 NEG NEG

EXAMPLE 8

Test for Virucidal Activity, Cell Viability Toxicity to Red Cells, Etc. as Follows: a. Raw 10 ml fresh human blood in heparinized syringe and transfer to 50 ml centrifuge tube. b. Isolate PBMC as above in step 3 and use these human PBMC as indicator cells. c. Set up 4 wells as controls, add 100 ul indicator cells and 6 log HTV virus in tissue culture media. d. Set up remaining wells for testing the drug sensitivity to viruses, add 100 ul indicator cells and 6 log HTV virus in tissue culture media. e. Incubate 37 °C for 7 days. Check syncytia formation after 24 hours, 3 days, 7 days. f. Determine P-24 by ELIS A Assay in each well. g. Make 2% solution of fresh red blood cells to make up the volume to 5 ml, add 100 ul of drug stored at 4°C. Examine for hemolysis at 1.2, 7, and 14-day intervals. Bring to room temperature before examination of clumping or hemolysis. RESULTS

1. Plaque and syncytia formation seen in all control wells.

2. Almost equal quantity of P-24 determined in control wells.

3. No syncytia formation seen in any of the test wells.

4. Approximately 100% inactivation as percent of control P-24 antigen level assayed in all test wells including 1 :64 dilution indicating that the drug was equally potent at 1:64 dilution as at 1:2 dilution.

5. No homolysis or clumping was observed in red cell tube with the drug for up to 14 days.

6. PBMC count in lower dilutions 1/2 was more than in 1 :64 dilution indicating improved CD-4 cell in wells with more drug. CONCLUSIONS

1. All lysates were active against HIV up to 1/800 dilutions indicating that drug is active even in nanogram range.

2. Tissue culture grown cells are equally potent as viral inactive agent as fresh baboon cells.

3. The lysate from the first experiment indicated that the results re reproducible and the lysate is stable at 4°C for 6 weeks.

4. Higher CD-4 T lymphocytes in lower dilutions indicated that the immune modulation property of lysates is does dependent in in vitro tests. EXPERIMENT #2: Determination of Antiviral Activity of Composition

20 ml of fresh blood in CPD from two more baboons #266 and 267 was obtained from NBRL Boston and processed immediately to isolate PBMC as in step 3 of Experiment 1. Fresh human blood was also drawn at the same and processed to obtain PBMC. PBMC samples from both human and two baboons were transferred to tissue culture mediate to see the ability of the PBMC of baboons to grow in tissue culture media.

Materials and methods:

Dupont P-24 assay kit for determination of P-24 output

Normal PBMCs from American Red Cross

Baboon PBMCs from 10.0ml whole blood, from two other baboons #266 and #267 HTV-l MN (ATDS reagent program CAT #137

RPMI Media (Difco)

24 well cell culture plates PMBCs isolated as per BBI standard protocol and anubated with HTV-l MN at 37°C for 3 hours in 15 ml tube. The excess (free viruses) were removed by washing the cells with PBS one time.

EXPERIMENT # 2: RESULTS Baboon # 267

Well # OD P-24 Syncytia CPE

1 (uninfected PBMCs) 0.099 68 Neg Neg

2 0.117 82 Neg Neg

3 0.055 146 Neg Neg

4 0.012 96 Neg Neg

5 (infected PBMCs) 0.469 16000000 Pos 4 + (no composition)

6 0.417 12600000 Pos 4 +

7 0.435 121000000 Pos 4 +

8 0.235 7720000 Pos 4 +

9 0.389 8000000 Pos 4 +

10 0.249 75000000 Pos 4 +

11 (infected PBMCs) 0.012 125 Neg- Neg (composition treated) 1:10

12 1:50 0.028 580 Neg Neg

13 1:100 0.046 850 Neg Neg

14 1:200 0.050 660 Neg Neg

15 1:400 0.018 180 Neg Neg

16 1:800 0.044 618 Neg Neg

Untreated Treated

Mean P-24 9410000 475

+/- SD 3450000 290

CV 0.26 0.22

% of Control 100.0 0.02

P-24 results are expressed in picograms of HTV-l P-24/mL Five Lysate Samples Were Tested Simultaneously. a. Lysate obtained from baboon #266 PBMC b. Lysate obtained from tissue culture grown cells of baboon #266. c. Lysate obtained from baboon #267 PBMC. d. Lysate obtained from tissue culture grown cells of baboon #267. e. Lysate obtained from Experiment 1 (6 weeks old) stored at 4°C.

EXPERIMENT #3:

1. Fresh whole blood from two baboons #266 and #267 was obtained and processed to isolate PBMC fraction as above. Fresh human blood was also drawn and processed simultaneously to obtain PBMC for use as indicator cells. PBMC fraction from both baboons as well as human PBMC fractions were added to the culture media for growing PBMC fractions in tissue culture and cells were examined under microscope on daily basis for 7 days.

2. PBMC lysates were made by adding cells to 1.0 ml distilled water and storing the cells at 4°C overnight. 100 ul stabilizing solution was added to all lysates and lysates were tested for virus inactivation as above.

3. Each lysate was tested at dilutions 1/10, 1/50, 1/100, 1/200, 1/400, and 1/800. 750ul of each dilution of the lysate was added to the test wells containing HTV (6 log) in 50 ul media, human PBMC in 200 ul culture media.

Control wells contained no drug. 750 ul culture media was added to make the volume.

4. Plates were incubated at 37°C for 4 days and each well was examined under microscope growth.

5. P-24 antigen ELISA assay was performed on samples from all wells. Experiment #3: Determination of Antiviral Activity of Composition Materials and methods:

Dupont P-24 assay kit for determination of P-24 output

Normal PBMCs Baboon PBMCs fro 10.0 ml whole blood, from two other baboons #266 and #267 and tissue culture cells from #266 and #267 and pooled PBMCs from tissue culture cells of #266 and

#267 (5 PBMCs samples lysed and tested.

HTV-l MN (AIDS reagent program CAT #137

RPMI Media (Difco) 24 well cell culture plates

PMBCs isolated as per BBI standard protocol and anubated with HTV-l MN at 37 °C for 6 hours in 15 ml tubes. The excess (free viruses) were removed by washing the cells with PBS one time.

Experiment #3: Results

5 lysates were tested.

Well # OD P-24 Syncytia CPE

1 (uninfected PBMCs) Neg Neg 2 Neg Neg 3 Neg Neg 4 Neg Neg 5 (infected PBMCs) Pos 4 + (no composition)

6 Pos 4 + 7 Pos 4 + 8 Pos 4 + 9 Pos 4 + 10 Pos 4 +

Lysate 1 (# 266 cells)

(infected PBMCs) (composition treated)

11 1:500 Neg Neg

12 1:1000 Neg Neg

13 1:2000 Neg Neg 14 1:4000 Neg Neg

15 1:8000 Neg Neg

16 1:16000 Neg Neg Lysate 2 (# 267 cells)

(infected PBMCs) (composition treated)

11 1:500 Neg Neg 12 1 :1000 Neg Neg

13 1:2000 Neg Neg

14 1:4000 Neg Neg

15 1:8000 Neg Neg

16 1:16000 Neg Neg

Lysate 3 (# 266 & 267 tissue culture grown cells pooled)

(infected PBMCs) (Composition treated)

11 1:500 Neg Neg

12 1:1000 Neg Neg 13 1:2000 Neg Neg

14 1:4000 Neg Neg

15 1:8000 Neg Neg

16 1:16000 Neg Neg

Lysate 4 (# 267 tissue culture grown cells)

(infected PBMCs) (Composition treated)

11 1:500 Neg Neg 12 1:1000 Neg Neg

13 1:2000 Neg Neg

14 1:4000 Neg Neg

15 1:8000 Neg Neg

16 1:16000 Neg Neg

Lysate 5 (Lysate from baboon # 1-3/29/96 tested for stability and reproducibility)

(infected PBMCs) (composition treated)

11 1:500 Neg Neg

12 1:1000 Neg Neg 13 1:2000 Neg Neg

14 1:4000 Neg Neg

15 1:8000 Neg Neg

16 1:16000 Neg Neg

EXAMPLE 9: PREPARATION OF HUMAN AND BABOON LEUKOCYTE LYSATES

Freshly obtained human and baboon blood (15 ml each) were diluted with equal volume of Hanks balanced salt solution (final 30-ml) and carefully laid over 10 ml of Ficoll- Paque (Sigma). Samples were centrifuged at 550 x g at 20°C for 40 min. Top layers were carefully discarded and middle layers containing PBMC were collected. The cells were washed twice with PBS (10 ml each) followed by centrifugation at 400 x g at 20 and 10°C in the first and second wash respectively. Cells were then re-suspended in 10 ml RPMI- 1640 (serum-free) for cell count and viability (3 x 10⁷ PBMC/10 ml). Cells were then centrifuged at 400 x g for 10 min at 10°C. Cell pellets were lysed by resuspension in 1 ml of distilled water and kept at 4°C for 4 days. Lysed cells demonstrate turbidity. After 4 days, 0.1 ml of stabilizer was added per 1 ml of leukocyte lysates and samples were kept at -20°C for 24 hrs to further lyse cells. Samples were defrosted at room temperature followed by addition of 49 ml of saline solution. Diluted samples were filter sterilized using Corning 150 ml sterile filtration unit (0.2 micron cellulose acetate membrane) and stored at 4°C for future assays. PROTOCOL FOR HTV INHIBITION ASSAY

HTV reverse transcriptase assay was performed in duplicate by incorporation of 32P-dttp into oligo-A template primed with oligo-dt. In this experiment CEM-TART cells were treated overnight with composition. Followed by 6 days infection with HTV-l after-rev clone. Reverse transcriptase activity was measured after detergent treatment of the cell supernatants according to the established methods. RESULTS

Composition at a concentration of 1:500 consistently resulted in 60-70% inhibition of HTV growth as measured by reverse transcriptase assay. See FIG. 21. Inhibitory activity of composition at a higher than 1 :500 dilution was not significant. 3H-thymidine incorporate assay (indicative of cell growth) was also inhibited up to 30% by 1 :500 diluted composition. Results are summarized below in Tables 6 and 7.

CONCLUSION

Here, it should be noted that our assay system performed on CEM-TART cells (established T cell line) is not identical to that by BB Labs which used human PBMC. It is clearly demonstrated that various cells have different responses to the anti-HTV activity of chemokines (Science 274, 1393-1395 (1996). Therefore, it is possible that CEM-TART cells are not as responsive to anti-HTV activity of composition.

et

ZZ6lO/66Sfl/IDd 0ZS8C/66 OΛV ANTI-VIRAL ASSAY 1) ENTER DATA AND IFN STD CONC Logy = aLogx - =11 Babooa sup=l_

2) PRESS ALTF3 AND TYPE DETECT (ENTER) TO CALCULATE DATE: 13-Sep-96

BY: 3) CHOOSE THE COLUMNS WΠΉ R HeU-VSV

VC STDI STDI SAMl SAMl SAM2 SAM2 SAM3 SAM3 SAM4 SAM4 CC WELL F TIER NUMBER ( )

0.07 2.04 2.12 0.06 0.06 0.07 0.06 0.09 0.06 0.07 0.06 2.20 1.00

0.07 1.92 2.11 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 , 1.96 2.00 14.00

0.06 2.00 2.03 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 1.99 3.00 7.00

0.06 2.06 2.21 0.07 0.07 0.07 0.08 0.07 0.07 0.07 0.06 ' 2.07 4.00 3.50

0.06 0.98 1.48 0.07 0.07 0.07 0.08 0.07 0.07 0.07 0.06 2.06 5.00 1.75

0.04 0.25 0.39 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.06 1.98 6.00 0.88

0.06 0.08 0.08 0.06 0.07 0.06 0.07 0.07 0.06 0.06 0.06 1.90 7.00 0.44

0.05 0.07 0.08 0.07 0.07 0.08 0.08 0.07 0.06 0.06 0.07 1.83 8.00 0.22

STD SAMl SAM2 SAM3 SAM4 COLUMN # INITIAL TIE

Log=Log 100.00

50% CPE = 5.05 0.00 0.00 0.00 0.00 (A - H) % of STD 100.0C ********** ********** ********* ********** R 2= 0.77 0.26 0.24 0.07 0.22

50% CPE = 3.71 0.00 0.00 0.00 0.00 Log-Log % of STD 100.00 ********** ********** ********** ********** (B - H) R 2= 0.82 0.07 0.03 0.49 0.21

50% CPE= 2.72 0.00 0.00 0.00 0.00 Log-Log % of STD 100.00 ********** ********** ********** ********** (C - H) R 2= 0.88 0.01 0.01 0.43 0.20

F #l F #2 F #3 F #4

Used RLS-004 as the Ref Std (1.50)

Baboon Cell culture supernatant HPLC fraction #1-4 (1.100)

Tocicity - CPE 0.00 see

Docket No. D3550-00Q01 PHI/367740

EXAMPLE 10: L97-85 BABOON CELL EXTRACT ANTI-HIV ACTIVITY

Cell extract was diluted and used in an equal volume of HIV-1 IIIB dilution to infect activated human PBMC overnight at 37 °C. Cells were then washed and placed in culture in media containing the indicated dilution of lysate. 1/2 media changes with media containing the indicated dilutions were made at 3 day intervals. Conditioned media was tested by HIV-l p27 antigen capture. This assay is summarized below in Table 8.

TABLE 8

DAY Dilution

IA 1:20 1:100 1:500 1:2500 Control

Np ml (% Inhibition^

6 116 60 12 206 427 212

(45) (72) (94) (3) (-100)

9 256 76 61 368 378 361

(29) (79) (83) (-2) (-5)

1 :6500 dilution used for assay of day 6 and day 9 samples.

Results are summarized below in Table 9.

TABLE 9

EVALUATION OF COMPOSITION BABOON CELL EXTRACT ANTI-HIV ACTIVITY

30 ml baboon blood was collected in CPD. 30 million PBMC were obtained by using ABL Ficol - Hypaque protocol. Cells were lysed in 1 ml sterile distilled water, stored at - 20C for 4 days. 0.1 ml stabilizing solution was added to the cell lysate, mixed well by vortex and centrifuged at 1500 G for 30 minutes, supernatant was filtered through 0.2 micron filter and transferred into a sterile tube.

The stabilized lysate was diluted to 50 ml in saline, and aliquot in 1ml vials for storage and future use.

Cell extract was diluted used with equal volume of HIV-1 IIIB dilution to infect human PBMC overnight. Cells were then washed and placed in culture in media containing indicated dilution of the composition. 1/2 media changes with media containing the indicated dilutions were made at 3 day intervals. Conditioned media was tested by HIV-1 p 27 antigen capture. The results are summarized below.

Dav Dilution 1:4 1:20 1:100 1:500 1:2500 Control

p-24 ng/ml (% inhibition)

116 60 12 206 427 212 (45%) (72%) (94%) (-100%)

9 256 76 61 368 378 361

12 (75%) (92%) (54%) (38%) (32%)

1 :6500 dilution was used for assay of day 6 and day 9 samples. Percent inhibition Graph 1.

Human PBMCs were pretreated with composition for 24 hours (I) and also infected cells were post-treated with composition (II) and assayed for HIV-1 IIIB. P-24 level was determined on day 8. Composition solutions (50X of 1 ml lysate from 30 million cells) 1:4, 1:20, 1:100, 1:500, 1:2500, and 1:12500 were used for inhibition. Percent inhibition is indicated FIG. 19. EXAMPLE 11

SPECIFICITY EXPERIMENTS 10/97 SAME PREPARATION OF BABOON CELL EXTRACT AS L97-100

Target cells were activated human PBMCs and were incubated with the indicated virus at 1 : 10 dil. and dilution of extract overnight. Cells were then washed and plated in the indicated dilution of extract with 1/2 fluid changes at 3-4 day intervals. Antigen capture was with HIV p25 OTC kit. This assay is summarized below in Table 11.

TABLE 11

VIRUS DAY DILUTION CONTROL

1:4 1:20 1:100 ng/ml (% inhibition) fflV-l(NSI) 10 5.3 3.7 0.96 3.3

(-60) (-12) (71)

Repeat Assay HTV-l(NSι) 10 1.9 1.4 0.468 1.2

(-58) (-16) (61)

HTV-l(Sι) 10 3.9 2.9 3.6 3.8

(-2) (40) (6)

H9/ΓΠB 10 427 457 385 442

(4) (-3) (13)

SIV-1(MAC 251) 6.5 85 54 271

(97) (69) (80)

PROTEIN ESTIMATION

Total protein measured by biosyn assay method: Concentrate (original 1.0 ml) 1/50 Saline Dilution

1.64mg/ml (1640 ug/ml) 32 ug/ml (32x50=1600ug/ml) EXAMPLE 12: DRUG MECHANISM ASSAYS RT INHIBITION ASSAY PROTOCOL

The effects of compounds on the in vitro activity of purified HIV-1 p66/p51 RT are determined by measurement of incorporation of [³H]TTP into the poly(rA):oligo(dT)(rAdT) or [³H]GTP into the poly(rC):oligo(dG)((rCdG)homopolymer template/primer systems, respectively, as previously described (see below reference). 3'-Azido-2',3'-dideoxythymidine-5'- triphosphate (AZTTP) and UC38 (NSC 629243, non-nucleoside reverse transcriptase inhibitor) serve as positive controls for inhibition of RT. See Rice W.G. et al., "The site of action of 3- nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes. Proc. Natl. Acad. Sci. USA 90, 9721-9724 (1993).

HPLC ASSAY OF HTV-l PROTEASE ACTIVITY

The object of this assay is to determine whether a drug will inhibit HTV-l protease activity. This assay is summarized below as follows: Materials: 1. ENZYME

The HTV-l Protease is received from Bachem Bioscience, Inc. (H-1256) at a concentration of 0.116 mg/ml. Thaw the stock, aliquot into 15 ul samples and refreeze at -70°C. Dilute the stock to a working concentration 1.25 ug/ml in IX buffer.

2. SUBSTRATE The sp211 substrate (VSQN YPiVQ-amide, ala-ser-Gin-Asn-Tyr-Pro-Ile-Val-Gin-

Armide) is prepared by multiple Peptide Systems, as a dry powder. Dilute the 10 mM stock in ddH₂0 (MW of peptide is 1044 and the stock is 10.44 mg/ml). Dilute the stock to a lmM working solution in IX buffer. 3. BUFFER (2X)

1MKHP0₄, pH 6.5 10% glycerol 6mM DTT lmM EDTA

1.5M ammonium sulfate

4. EXPERIMENTAL DRUGS

Prepare 4X concentration of drugs in ddH₂O.

5. QUENCH SOLUTION Prepare a 9: 1 mix of 8 M Quanidine-HCL to 10:TGA. Use 40 ul of this mix to stop the assay.

PROCEDURE:

1. Place 40 ul of the quench solution to the stop tubes.

2. Into each reaction tube, add 1) 15 ul 2X buffer

2) 15 ul H₂O or drug

3) 15 ul enzyme

4) 15 ul substrate to initiate reaction.

3. Vortex thoroughly. Quickly place 20 ul of reaction mixture into corresponding Time 0 quench tubes and vortex thoroughly.

4. Place reaction tubes in 37 °C incubator. 5. At 30 minutes, add 20 ul reaction mixture to corresponding quench tubes. Vortex thoroughly.

6. Analyze amount of sp211 substrate (by reverse phase HPLC and LNOVAPRAS program) using Waters Nova-Pak C18 (60A, 4uM) 3.9x150 mm reverse phase HPLC column (Cat. #86344, Millipore Corp., 34 Maple St., Milfor, MA 01757.

TABLE 12

?LATE JuVA ΪSIS REPORT

.::;•*€* ^?τ ;<*act taxation ^•JJ ccnn: .13 :»:

«CC :*t€ • 31-9«e-'969 19:90

S-» « SAIE 31-0-e-1969 '9:90 * '{ «ΛO OΛΓE 5-.-;«e--;₍ 6? ^•?

_22_ JU- JU- 2i_ _-Z_ _a_ _-J_ JL

3.104 3.381 0.098 0.102 0.396 0.092 0.397 0.095 0.095 0.397 0.409 3.394 0.404 3.376 :.3i] 0.753 1.045 0.954 1.109 1.185

3.075 o.xu 1.258 0.358 0.444 3.436 0.315 0.528 0.784 0.9C6 0.886 0.752 0.917 0.407 3.3U

3.572 0.343 0.378 0.471 o.sss 1.181 1.271 1.191 1.293 1.12; 3.C69 0.3M 0.301 3.343 0.41V 3.015

0.619 1.001 3.985 0.965 1 011 3.966 3.364 3.093 i.orz 3.315 '.1-0 1.121 1.031 1.003 1.021 1.033 1.134 1.175 1.149 3.090 1.144 5.017 ^•-1S3 1.144 1.123 1.036 1.105 1.068 1.145 1.122 1.141 9.014 0.093 0.074 0.0M 0.0(7 0.088 0.091 0.092 0.090 0.092 0.094 0.079 0.011

Vtll so cove oc icλit so cv r/cx tie

•(•(• tti* ly-* : Primary tertn 9T 527 961118 TABLE 13

TABLE 14

PLATE A ALYSIS REPORT -C- '769 !9:00

too -e- 1969 ^•9:90 Sl-;«e-"«49 ^aLAtE ⁾E»0 3ATE 3' ;»c-'»69

•11 ??.. 33 »Λ n 1A "" 11 ^• 1 II

A 3.103 3.380 3.095 0.990 0.088 9.386 3.:«« :.:s6 9.388 3.382 0.373 9 3.399 3.408 0.265 3.:;j 0.395 0.303 9.294 3.S20 3.233 0.240 3.216 1.045 c 3.091 3.295 0.247 3.005 0.296 0.374 0.271 0.302 9.294 0.248 0.177 1.291 0.S87 0.331 0.291 3.308

0 0.318 0.268 0.276 0.336 3.292 0.282 0.239 1.017 3.306 E 0.392 3.460 0.292 0.343 0.348 9.363 0.314 3.271 0.266 0.310 0.290

F 0.093 1.355 1.347 3.303 1.299 1.300 1.268 1.229 1.184 1.164 1.107 1.207 350 1.328 0.005

0 0.094 1. 1.283 1.318 1.321 1.297 1.302 1.272 1.243 1.234 3.008 H 0.097 0.074 0.074 0.084 0.091 0.089 0.092 9.091 0.093 0.094 0.079 0.005

WELL C*P CONC OC sβ CV T/CX NO

• ct ***• r*p« : Primary S r—n 9T 534 961118 TABLE 15

PLATE ANALYSIS REPORT .PAGE scsεε-u 91 • f nctionjtiβn Exp 10 9v961118f 132 «EPO«I OAIE : 25- CV- 1996

»IATE 8ASCOOE 0531 CONΠC 10 :52

013 ASSAY : «TT i Noc J1-0-_C- 1969 19:00 0*U6 ADO OATE : 31 -0-C- 1969 19:00

STAIN OATE 31-0«c-1969 19:00 PLATE *EA0 DATE : 31 -0ce- 1969 19:00

_£U_ J12- tt_ J-4_ _D5_ J_6_ -flZ- JU- JU- _lfi_ -11. _L2_

0.092 0.075 0.084 0.087 0.086 0.090 0.088 0.092 0.094 0.O92 0.076 0.010

0.092 0.441 0.327 0.499 0.824 1.033 1.121 1.190 1.245 1.202 0.472 0.010- 0.090 0.456 0.316 0.539 0.513 0.910 1.139 1.162 1.199 1.260 0.483 0.010 0.090 0.303 0.265 0.280 0.577 0.687 0.966 0.974 1.064 1.172 0.297 0.010 0.090 0.372 0.277 0.329 0.496 0.930 0.972 1.016 1.275 1.163 0.509 0.012 0.090 1.201 1.160 1.180 1.175 1.134 1.187 1.194 1.267 1.247 1.269 0.015 0.090 1.176 1.222 1.192 1.198 1.150 1.202 1.197 1.274 1.234 1.270 0.010 0.091 0.074 0.091 0.091 0.094 0.092 0.094 0.094 0.097 0.098 0.079 0.008

WELL GIF CONC QC MEAN CV T/CZ IN0

- tt Tost type » friββry Scroon 9T 531 961118 EFFECTS OF COMPOSITION ON THE BINDING OF HIV-1 TO CEM-SS CELLS

Dilution of Composition % Inhibition of Binding

1/10 34.1

1/100 0

1/1000 0

EXAMPLE 13

The object of this study was to investigate the potential effects of composition during intravenous injections (48) hours apart) followed by a recovery period for a total of 28 days. Five male and 5 female Sprague-Dawley CD (Crl: CD^R(SD)BR) rats (Rattus norvegicus), were received from Charles River Canada, Inc. and were housed in the Bio- Research Laboratories Ltd. spare colony from where they were transferred for use on this study. Animals were assigned to the following group:

Group No. Dose Volume Number o if Animals Identification (ml /dav) Males Females

1 Composition 1 5 5

The dose level was selected by the Sponsor according to the potential human exposure, existing toxicity data and any limitations imposed by the test article.

ADMINISTRATION OF TEST ARTICLE The test article was used as supplied by the Sponsor and administered by intravenous injection, via the tail vein on 5 occasions (48 hours apart on Days 1, 3, 5, 7 and 9) and the final dose was administered on the day of necropsy (Day 28). The dose volume administered to each animal was 1 mL/occasion. CLINICAL EXAMINATION

All animals were examined twice daily for mortality and signs of ill health or reaction to treatment. A completed detailed examination was performed weekly.

There were no treatment-related clinical signs.

BODY WEIGHT

Individual body weights were measured on each day of treatment and weekly throughout the treatment and recovery periods.

There was no treatment-related effect on food consumption.

LABORATORY INVESTIGATIONS (APPENDICES 1.2.3 AND 4) At study termination (Day 28), hematology (using EDTA as anticoagulant) evaluation was performed on all animals. Blood samples were collected, under anesthesia, for the orbital sinus 24 hours following the final dose (Day 10) and again from the abdominal aorta at necropsy (Day 28). The samples evaluated for white blood cell count (total, absolute and percent differential). There were no treatment-related effect on the white cell parameters.

TERMINAL PROCEDURES

All animals were euthanized by exsanguination from the abdominal aorta following isoflurane anesthesia and blood sample collection at the end of the recovery period. The carcasses were discarded without further examination. In conclusion, it was feasible to administer composition intravenous bolus injection for at least 6 occasions in 28 days. There were no mortalities, no treatment-related clinical observations or effects on white cell parameters.

Table 16 below summarizes this intravenous bolus toxicity study in rats. TABLE 16 PROTOCOL: INTRAVENOUS BOLUS TOXICITY STUDY IN RATS

EXAMPLE 14

Below are Day 10 hematology raw data and reference ranges for the hematology parameters. The Day 10 evaluation shows that there are no notable treatment-related effects and that the parameters evaluated are within the normal reference ranges.

REFERENCE RANGE

SPECIES: Rattus Norvegicus

STRAIN: Sprague - Dawley CD

GENDER: male SUPPLIER: Charles River

AGE: 2 - 23 months

BLOOD SOURCE: aorta, orbit, tail vein

HEMATOLOGY

TEST UNIT N MEAN S.D. RANGE

INSTRUMENT: COULTER S-PLUS IV

WBC thous/uL 138 10.6 4.1 4.7 - 19.2

RBC millions/ uL 138 7.45 0.81 5.27 - 8.70

Hgb g/dL 138 15.0 1.5 10.4 - 16.9

Hct % 138 42.0 3.9 29.5 - 47.7

MCV (um3) 137 56.6 2.8 51.2 - 64.5

MCH pg 138 20.1 1.1 18.0 - 22.5

MCHC (g/dL) 138 35.6 0.9 33.6 - 37.2

RDW % 138 13.5 1.6 11.8 - 19.2

PLT thous/uL 135 1076 154 753 - 1447

MPV (um3) 138 8.6 0.6 7.5 - 9.7

INSTRUMENT: Microscope

Seg neutrophils (seg) % 123 13 8 3 - 40

Band neutrophils % 123 0 0 0- 0

Lymphocytes % 120 86 7 68 - 96

(Lymph) .

Monocytes (mono) % 123 2 2^' 0 - 7

Eosinophils (Eosin) % 123 1 1 0 - 3

Basophils (Baso) % 123 0 0 0 - 0

Retic % 32 1.1 0.7 0.2 - 2.3

INSTRUMENT: MLA 750

PT sec 32 12.9 1.2 10.1 15.0

APTT sec 21 14.5 2.7 10.1 20.0

Tables 17 through 20 below summarize the results of the study described. TABLE 17

INDIVIDUAL HEMOGRAMS PROJECT NO 5521

DAY 10

GROUP 1 MALES AND FEMALES

WBC DIFFERENTIAL COUNT

VIAL NO WBC X 10 I N røEUUTT SEG NEUT NSEG LYMPH MONO EOSIN BASO

% % % % % %

101 13.1 7 0 89 3 1 0

102 19.3 12 0 84 3 1 0

103 9.7 17 0 81 1 1 0

104 9.9 10 0 89 1 0 0

105 12.5 15 0 83 2 0 0

151 7.7 6 0 92 1 1 0

152 7.8 6 0 93 1 0 0

153 120 12 0 86 1 1 0

154 135 6 0 92 1 1 0

155 86 11 0 89 0 0 0

TABLE 18

INDIVIDUAL HEMOGRAMS PROJECT NO 5521

DAY 10

MALES AND FEMALES

GROUP 1

WBC DIFFERENTIAL COUNT

ANIMAL NO WBC X 10 NEUT SEG NEUTNSEG LYMPH MONO EOSIN BASO

101 11.9 21 0 74 3 2 0 102 12.9 19 0 79 2 0 0 103 10.6 10 0 88 2 0 0 104 9.1 17 0 80 3 1 0 105 8.0 28 0 69 2 1 0

151 4.6 7 0 90 1 2 0 152 5.2 8 0 86 0 2 0 153 11.3 8f 0 90 4 2 0 154 7.7 6 0 88 3 1 0 155 10.0 11 0 89 1 0 0

TABLE 19

INDIVIDUAL HEMOGRAMS PROJECT NO 5521

DAY 10

MALES AND FEMALES

GROUP 1

WBC DIFFERENTIAL COUNT (ABSOLUTE) ANIMAL NO NEUT SEG NEUTNSEG LYMPH MONO EOSIN BASO

101 917 0 11659 393 131 0

102 2361 0 16212 597 193 0

103 1649 0 7857 97 97 0

104 990 0 8811 99 0 0

105 1875 0 10375 250 0 0

151 462 0 7084 77 77 0

152 462 0 7254 78 0 0

153 1440 0 10320 120 120 0

154 810 0 12420 135 135 0

155 946 0 7654 0 0 0

TABLE 20

INDΓVTDUAL HEMOGRAMS PROJECT NO 5521

DAY 10 MALES AND FEMALES

GROUP 1

101 2499 0 8806 357 238 0 102 2451 0 10191 258 0 0 103 1060 0 9328 212 0 0 104 1456 0 7280 273 91 0 105 2240 0 5520 160 80 0

151 322 0 4140 46 92 0 152 416 0 4680 0 104 0 153 904 ό 9718 452 226 0 154 462 0 6930 231 77 0

EXAMPLE 15: EFFECT OF COMPOSITION ON BLOOD PRODUCTS

THE PROTOCOL FOR THE USE OF THE INCENTIVE COMPOSITION IN THIS STUDY IS AS FOLLOWS:

Step 1 : Add composition to

RBCs Platelets

10% V/V 10% V/V

Step 2: Mix, then incubate at room temperature for 1-6 hours.

Step 3 : Centrifuge and wash three times with equal volume of phosphate buffer saline.

Step 4: Storage life:

RBCs Platelets

In Adsol In Plasma

42 days 6 days

15.1 Cell Free Viral Inactivation Study

1. Whole blood has spiked with >6LOG HTV incubated for 1 -6 hours at room temperature.

2. Then treated with 8%, 10% and 12% composition for 1 hour, 6 hours.

3. Fractionated.

4. RBCs and platelets were washed three times with PBS in equal volume.

5. Tested for viral inactivation

-- syncytia formation — P-24 antigen CELL FREE VIRAL INACTIVATION STUDY: RESULTS

TABLE

Test Samples

1 hr and 6 hrs

Control 8% 10% 12%

Syncytia Formation Pos. Neg. Neg. Neg.

P-24 Antigen Test >6LOG 0 0 0

COMPOSITION TREATMENT OF CELL ASSOCIATED (INTRACELLULAR HΓV INACTIVATION^ AND T-CELL VIABILITY STUDY

PROCEDURE:

HTV-l from infected SUPT-1 T cells was co-cultured to uninfected SUPT-1 cells 48 hours prior to composition treatment. After 1 hour of treatment with 10% or 20% composition at 37°C, composition was removed by washing. Virus replication was measured by determination of HTV-l P-24 in the supernatants after 5 days in co-cultures. Cell viability was determined by Trypan Blue Dye Exclusion of pooled cells at culture termination.

EXAMPLE 16: INACTIVATION PORCINE PARVOVIRUS

*Effect of the Composition on Cell Growth and Virus Expression Procedure:

1. Seed Vero cells in 60 mm and/or 100mm petri dishes according to SOP No. T-013.0. Label plates with appropriate code to identify assay controls.

2. Spike the composition and buffers with 1:10 dilution of the virus adjusted appropriately to provide 30-50 pfu dis in the selected dosing volume.

3. When cultures are 80 to 100%) confluent, remove medium, wash once with BSS and dose cultures with appropriate concentration of positive control (medium) spiked test article and/or spiked buffer in 0.1 to 2.0 ml.

4. After 60 minutes incubation at 36°±2°C in a humidified CO₂ incubator, remove medium containing inoculum and overlay with 3-15 ml of agarose overlay medium, allow to solidity and incubate at 30°C±2°C in a humidified CO₂ incubator.

5. Examine dishes daily until plaques begin to form in the positive control cultures. Overlay cultures with agarose containing neutral red when the plaques begin to open in the positive control cultures. Read assay when the neutral red penetrates to the cell layer and the plaques are fully formed.

EFFECT OF COMPOSITION ON VIABILITY OF SUPT-1 CELLS TREATMENT GROUP/TARGET VIRUSES

Drue Dilution No Drue 10% Comρ 20% Comp

Untreated Cell count 8.25E+05 7.50E+05 6.90E+05

SUPT-1 % Viable cells 97* 96 96

Cells

Drug Dilution No Drug 10% Comp 20% Comp

GT65 Cell count 1.80E+O5 6.00E+05 6.70E+05

Infected % Viable cells 80 93 96

Cells

Drug Dilution No Drug 10% Comp 20%) Comp

GT63 Cell count 8.25E+05 7.50E+05 6.90E+05

Infected % Viable cells 89 92 96 Cells

^♦Viability was determined by Trypan Blue Dye Exclusion culture termination.

Tables 21 through 23 below show the effects of the composition on cell viability and human red blood cell parameters. Table 21 shows cell viability, Table 22 shows hematological parameters, and Table 23 shows effect on red cell fragility.

TABLE 21

EFFECT OF COMPOSITION ON HIV INFECTIVITY

Transmission of HTV-l from infected SUPT-1 T-cells to uninfected SUPT-1 cells (in co-culture), as determined by P24 production, was depressed by pre-treatment of the infected cells for one hour with either 10% or 20% composition.

Virus Controls ( fo Drug^') HTV-l (GT63Ϊ Infected HTV- KGT65 Infected

Replicate HTV-l CGT63 HIG-lfGT65 10% Comp 10% Comp 10% Comp 20% Comp

1 14700000 3030000 425000 50462 2010 838

2 10900000 4650000 462000 3413 1454 824

3 6660000 5220000 272000 60989 1812 731

4 8820000 4440000 343000 4552 1435 871

5 7320000 5050000 263000 8559 2141 922

Mean P24* 9680000 4478000 353000 25595 1770 837

+/-S.D. 3244935 866816 89199 27822 320 70

CV 0.34 0.19 0.25 1.09 0.18 0.08

% of control 100.0 100.0 3.65 .26 .04 0.2

^♦Results are expressed in picograms of HTV-l P24/ml

TABLE 22

10% COMPOSITION TREATMENT OF RED BLOOD CELLS

DavO Dav21 Dav42 UNTRTD TRTD UNTRTD TRTD UNTRTD TRTD

White Blood Count 0.8 0.9 0.2 0.2 0.1 0.1

Red Blood Count 2.24 2.51 2.36 2.42 2.32 2.37

Hemoglobin 7.1 7.5 7.2 7.4 7.2 7.4

Hematocrit 20.2 24.0 23.2 24.3 22.4 25.4

MCV 90.4 95.5 98.4 100.4 96.6 107.0

MCH 31.7 29.9 30.5 30.6 31.0 31.2

MCHC 35.1 0.0 2.0 2.0 0.0 0.0

TABLE 23

10% COMPOSITION TREATMENT OF RED BLOOD CELLS

Dav O Dav 21 Dav 42

UNTRTD TRTD UNTRTD TRTD UNTRTD TRTD

RBC Osmotic Fragility % Hemolysis

0.00% NaCl 100.0 100.0 100.0 100.0 100.0 100.0

0.30% NaCl 97.0 92.0 98.2 98.6 100.0 90.2 0.35% NaCl 95.0 82.5 91.5 96.7 93.5 90.6 0.40% NaCl 92.0 75.8 97.2 89.1 91.3 83.2 0.45% NaCl 88.9 49.4 95.8 85.3 90.2 82.5 0.50% NaCl 82.9 28.0 94.0 64.1 87.9 81.4 0.55 %NaCl 58.4 8.9 79.2 44.4 62.6 73.3 0.60% NaCl 20.8 4.5 40.8 27.3 36.9 57.2 0.65% NaCl 2.4 3.2 17.0 20.7 17.3 45.3 0.85% NaCl 0.0 0.0 0.0 0.0 0.0 0.0

Claims

CLAIMSWhat is claimed is:

1. A composition produced from leukocytes selected from whole blood, bone marrow, cord blood and fetal cells of primates or human beings or other species immune to or at least resistant to infections by viruses, microorganisms or other disease causative agents.

2. The composition of claim 1 which comprises monocytes, mononuclear cells, lymphocytes, stem cells and progenitor blood cells from whole blood of species immune to or at least resistant to infections by viruses, microorganisms or other disease causative agents.

3. A composition of claim 1 comprising one or more of an of active soluble anti- infective agents.

4. The composition of claim 1 which is stabilized using a compound selected from the group consisting of calcium, sodium, potassium, phosphate, chloride ions, sugars, and anticoagulants, and which is present in concentrations of from about 0.001 microgram to about 5 grams for each ml of the composition.

5. The composition of claim 2 wherein cells are lysed by using hypotonic solutions, distilled water, sonication, freezing and thawing and centrifugation.

6. The composition of claim 5 which is sterilized using methods selected from microfiltration devices, and gamma radiation.

7. The composition of claim 2 wherein cells are grown using tissue culture cells and pooled for preparation of the composition.

8. The composition of claim 2 wherein cell membrane is solubilized for preparation of the composition.

9. The composition of claim 1 which has anti-viral activity.

10. The composition of claim 1 which has anti-microbial activity.

11. The composition of claim 1 which has activity against disease causative agent.

12. The composition of claim 1 which has efficacy, selected from anti-viral activity against human deficiency virus (HIV), hepatitis virus, herpes virus, cytomegalous virus, Parvo virus, protein enveloped viruses, non-enveloped viruses, RNA viruses and DNA viruses.

13. The composition of claim 1 which has anti-microbial activity against known pathogenic bacteria.

14. The composition of claim 1 which has activity against disease causative agents selected from fungi, mold and human parasites.

15. The composition of claim 1 which that is compatible with human and animal blood and blood components.

16. The composition of claim 1 wherein said composition is a protease inhibitor.

17. The composition of claim 2 wherein said composition is a reverse transcriptase inhibitor.

18. The composition of claim 1 wherein said composition inhibits binding of gp- 120 to CD+4 T lymphocytes.

19. The composition of claim 1 wherein said composition is effective to increase the amount of CD+4 T lymphocytes in a patient.

20. The composition of claim 1 wherein said composition is effective to inhibit binding of viruses to CD+4 T lymphocytes in a patient.

21. The composition of claim 1 having a molecular weight of less than 900 Daltons.

22. The composition of claim 1 which comprises as an active ingredient a compound selected from dimethyl decane, carbohydrate, carbohydrated polymer, and common sugar substitute.

23. The composition of claim 1 wherein said composition is non-toxic, does not produce adverse effects, side effects or other severe untoward reaction when administered in living body of an animal or a human.

24. The composition of claim 1 which exhibits no cytopathogenic effects when administered to patients.

25. The composition of claim 1 wherein said composition does not affect the cell viability when administered to patients.

26. A method for treatment of immunodeficiency, neoplastic and leukemic diseases, comprising adrriinistering to a patient in need thereof a therapeutic amount of the composition of claim 1.

27. A method for viral inactivating for blood and blood components for therapeutic and transfusion purposes comprising the addition thereto of a viral inactivating amount of the composition of claim 1.

28. A method for the inactivation of viruses in whole blood, red blood cells, Factor VII, Factor VIIII, Factor IX and other therapeutic agents derived from human or animal blood, organs and tissue, comprising the addition thereto of a viral inactivating amount of the composition of claim 1.

29. A method for viral inactivation for topical by administration to a patient of a cream or lotion to patient comprising a viral inactivating amount of the composition of claim 1.

30. A method for preventive therapeutic treatment against viruses comprising administration of an effective vaccine amount to a patient of the composition of claim 1.

31. A method for immune-modulating and immune regulating comprising administering an effective immune regulating amount of the composition of claim 1 to patients in need thereof.

32. Method for treatment of inflammatory conditions in patients in need thereof comprising administering an effective anti-inflammatory amount of the composition of claim 1.

33. Method for treatment of allergic conditions in patients in need thereof comprising administering an effective anti-allergic amount of the composition of claim 1.

34. Method for vaccination against human immunodeficiency virus (HTV), hepatitis virus, herpes virus, protein enveloped viruses, non-enveloped viruses, RNA viruses and DNA viruses comprising administering to a patient an effective vaccination amount of the compound of claim 1.

35. Method for preparation of composition of claim 1 comprising obtaining sample of baboon whole blood, bone marrow or cord blood; separating peripheral blood mononuclear cells, lysing said cells, stabilizing said lysed cell mixture.

36. The method of claim 35wherein said lysed cells are stabilized by a solution comprising calcium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium chloride and sucrose.

37. Method for preparation of the composition of claim 1 comprising obtaining sample of human whole blood, bone marrow or cord blood, separating peripheral blood mononuclear cells, lysing said cells, stabilizing said lysed cell mixture.

38. The method of claim 37 wherein said lysed cells are stabilized by a solution comprising calcium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium chloride and sucrose.

39. Method of preparation of composition of claim 1 comprising obtaining sample of baboon PBMC. From whole blood grown in tissue culture media, separating peripheral blood mononuclear cells, lysing said cells, stabilizing said lysed cell mixture.

40. The method of claim 39 wherein said lysed cells are stabilized by a solution comprising calcium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium chloride and sucrose.

41. Method of preparation of composition of claim 1 comprising obtaining sample of baboon tissue culture amplified cells from one or more baboons and pooled, separating peripheral blood mononuclear cells, lysing said cells, stabilizing said lysed cell mixture.

42. The method of claim 41 wherein said lysed cells are stabilized by a solution comprising calcium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium chloride and sucrose.