Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/785—Polymers containing nitrogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/02—Antidotes

Definitions

• This invention relates to inhibition of toxins and other toxic materials or substances, and in particular it relates to the use of dendrimers as binding agents for toxic peptides, proteins, or polyamines and other toxic materials or substances.
• Dendrimers are 3-dimensional polymeric materials of low polydispersity which are characterised by a large number of surface terminal groups. In addition the manner in which these materials are prepared allows tight control over the size, shape, and number and type of surface groups. Dendritic materials have several features that are useful for use as therapeutic materials: fixed shape which presents a large and defined surface with which to interact with biological surfaces and receptors; and the large number of terminal groups allow for multiple interactions with the biological targets.
• the present invention provides the use of dendritic polymers in the inhibition of toxic materials or substances, including but not limited to toxins or toxic peptides such as snake, scorpion, spider and bee venoms, as well as toxic peptides or other toxic materials or substances released during bacterial or viral infection.
• a method of prophylactic or therapeutic inhibition of a toxic material or substance in a human or non-human animal patient which comprises administration to the patient of an effective amount of a dendrimer having a plurality of terminal groups wherein at least one of said terminal groups has an anionic- or cationic-containing moiety bonded or linked thereto.
• Particularly preferred compounds for use in the method of the present invention are dendrimers having sulfonic acid-containing moieties, carboxylic acid-containing moieties, phosphoric or phosphonic acid-containing moieties, boronic acid-containing moieties, neuraminic or sialic acid-containing moieties or moieties containing modified neuraminic or sialic acid; primary, secondary, tertiary or quaternary amino-containing moieties, pyridinium-containing moieties; guanidinium-containing moieties; amidinium- containing moieties; phenol-containing moieties; heterocycles possessing acidic or basic hydrogens; zwitterionic-containing moieties; or mixtures of the above moieties, linked to terminal groups thereof.
• poly ionic dendrimers The compounds used in the method of this invention are referred to herein as poly ionic dendrimers, and this term is used throughout this specification and the claims which follow to include not only the dendrimers per se, but also their pharmaceutically or veterinarily acceptable salts, for example the alkaline metal or alkaline earth metal salts such as the sodium, potassium or calcium salts, as well as pharmaceutically acceptable anions such as fluoride, chloride, bromide, iodide, citrate, acetate, p-toluene sulfonate, and the like.
• pharmaceutically or veterinarily acceptable salts for example the alkaline metal or alkaline earth metal salts such as the sodium, potassium or calcium salts, as well as pharmaceutically acceptable anions such as fluoride, chloride, bromide, iodide, citrate, acetate, p-toluene sulfonate, and the like.
• Preferred compounds used in accordance with the present invention include poly ionic dendrimers of the general formula I:
• I is an initiator core
• Z is an interior branching unit
• n is an integer which represents the number of generations of the dendrimer
• A is an anionic- or catiomc-containing moiety which may be linked to interior branching unit Z through an optional linking group X.
• Dendrimers are macromolecular highly branched compounds formed by reiterative reaction sequences starting from an initial, core molecule with successive layers or stages being added in successive "generations" to build up a three-dimensional, highly ordered polymeric compound.
• Dendrimers are characterised by the following features: I an initiator core(I) which may have one or more reactive sites and be point-like or of significant size so as to effect the final topology of the dendrimer; ii layers of branched repeating units (Z) attached to the initiator core; iii functional terminal groups (such as moieties A) attached to the surface of the dendrimer, optionally through linking groups (such as linking groups X).
• the present invention uses dendritic structures as frameworks for the attachment of ionic moieties; the invention is not limited to the spherical dendrimers described in detail herein but can be based on any dendritic structure.
• the variety of dendrimers in both shape and constitution are well known to persons skilled in the art.
• dendrimers The preparation of dendrimers is well known, and is described by way of example in U.S. Patents Nos. 4,289,872 and 4,410,688 (describing dendrimers based on layers of lysine units), as well as U.S. Patents Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329 (describing dendrimers based on other units including polyamidoamine or PAMAM dendrimers).
• the dendrimers disclosed in these US patents are described as being suitable for uses such as surface modifying agents, as metal chelating agents, as demulsifiers or oil/water emulsions, wet strength agents in the manufacture of paper, and as agents for modifying viscosity in aqueous formulations such as paints. It is also suggested in U.S. Patents Nos. 4,289,872 and 4,410,688 that the dendrimers based on lysine units can be used as substrates for the preparation of pharmaceutical dosages.
• 88/01180 disclose conjugates in which a dendrimer is conjugated or associated with another material such as a carried pharmaceutical or agricultural material.
• International Patent Publication No. WO 95/24221 discloses dendritic polymer conjugates composed of at least one dendrimer in association with a carrier material which can be a biological response modifier, and optionally a target director.
• dendrimer as used herein is to be understood in its broadest sense, and to include within its scope all forms and compositions of these dendrimers as disclosed in Patent Publications Nos. WO 88/01178, WO 88/01179 and WO 88/01180. The term also includes linked or bridged dendrimers as disclosed in these patent publications.
• the preferred dendrimers of the present invention comprise a polyvalent core covalently bonded to at least two dendritic branches, and preferably extend through at least two generations.
• Particularly preferred dendrimers are polyamidoamine (PAMAM) dendrimers, PAMAM (EDA) dendrimers, poly(propyleneimine) (PPI) dendrimers and polylysine dendrimers.
• At least one, and preferably a substantial number, of the terminal groups on the surface of the dendrimer has an anionic- or cationic- containing moiety covalently bonded thereto.
• the branches of the dendrimer may terminate in amino groups or other functional reactive groups such as OH, SH, or the like, which subsequently can be reacted with the anionic or cationic moieties.
• the terminal groups of the dendrimer are amine groups
• the anionic- or cationic-containing moiety may be linked to the dendrimer by a variety of functional groups including amide and thiourea linkages.
• Preferred anionic- or cationic-containing moieties which may be bonded to the terminal groups of the dendrimer include sulfonic acid-containing moieties, carboxylic acid- containing moieties (including neuraminic and sialic acid-containing moieties and modified neuraminic and sialic acid-containing moieties), boronic acid-containing moieties, phosphoric and phosphonic acid-containing moieties (including esterified phosphoric and phosphonic acid-containing moieties) and primary, secondary, tertiary or quaternary amino-containing moieties, pyridinium-containing moieties; guanidinium-containing moieties; amidinium-containing moieties; phenol-containing moieties; heterocycles possessing acidic or basic hydrogens; zwitterionic-containing moieties; or mixtures of the above moieties.
• Suitable anionic- and cationic-containing moieties which may be bonded or linked to the amino or other terminal groups include, by way of example, the following groups (in which n is zero or a positive integer, more particularly n is zero or an integer of from 1 to 20): -NH(CH2) n S0 3 ' -(CH j JnSOj- -Ar(S0 3 ' )n
• R 1 alkyl or arylalkyl
• R 2 , R 3 alkyl or arylalkyl
• various neuraminic or sialic acid-containing moieties or modified neuraminic or sialic acid-containing moieties may be bonded or linked to the dendrimers in accordance with this invention.
• These moieties include the various N- and O- substituted derivatives of neuraminic acid, particularly N- and O-acyl derivatives such as N- acetyl, O-acetyl and N-glycolyl derivatives, as well as moieties in which the neuraminic acid group is modified.
• Suitable modified neuraminic groups include groups which are substituted in the 4-position with an amino, amido, cyano, azide or guanidino group, as well as unsaturated neuramine acid groups. These moieties may be linked to the dendrimers through the 2-, 7-, 9- or 5-NAc positions.
• n is an integer of from 1 to 20 or more, more preferably from 1 to 10.
• the dendrimers include at least three or more terminal groups.
• the optional linking groups which may be present to act as a spacer between the dendrimer and the moiety A may consist of an alkyl chain (optionally substituted or branched), an alkoxy, polyalkoxy, alkylthio or polyalkylthio chain (optionally substituted), or an alkenyl, multiple alkenyl, alkynyl or multiple alkynyl chain (optionally substituted).
• anionic or cationic dendrimers of this invention may be prepared by standard chemical methods which are well known to persons skilled in this art. Suitable methods are described by way of the example in Examples below.
• the anionic or cationic dendrimers of the present invention have been found to inhibit toxic materials or substances.
• toxic materials or substances as used herein is intended to refer in particular to toxins of biological (animal, plant, microbial or viral) origin, including but not limited to animal toxins or toxic peptides such as snake, scorpion, spider and bee venoms, toxic polyamines, and toxic peptides or other materials or substances released during bacterial infection (such as bacterial endotoxins and exotoxins), or during protozoal, fungal or viral infection.
• inhibitor is used herein in its broadest sense to include either full or partial inhibition or suppression of the toxic effect of the toxic material or substance in a human or non-human animal patient.
• the term is also used to encompass both prophylactic and therapeutic treatment.
• the present invention provides a pharmaceutical or veterinary composition for prophylactic or therapeutic inhibition of a toxic material or substance in a human or non-human animal patient, which comprises a dendrimer as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent.
• Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
• the use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent.
• the specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
• this invention provides the use of an effective amount of a dendrimer as broadly described above in the prophylactic or therapeutic treatment of, or in the manufacture of a medicament for prophylactic or therapeutic treatment of a human or non-human animal patient by inhibition of a toxic material or substance.
• a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy.
• the methods of this invention may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects.
• modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes.
• Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like.
• compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
• compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
• compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient.
• This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol.
• the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono-or di-glycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• the active component may also be formulated for delivery in a system designed to administer the active component intranasally or by inhalation, for example as a finely dispersed aerosol spray containing the active component.
• sustained release delivery systems can include sustained release delivery systems.
• Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules.
• Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.
• a pump-based hardware delivery system can be used, some of which are adapted for implantation.
• the active component is administered in prophylactically or therapeutically effective amounts.
• a prophylactically or therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.
• daily oral doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day.
• Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day.
• higher doses or effective higher doses by a different, more localised delivery route
• Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
• the active component according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art.
• veterinary compositions include those adapted for:
• oral administration external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
• drenches e.g. aqueous or non-aqueous solutions or suspensions
• tablets or boluses e.g. aqueous or non-aqueous solutions or suspensions
• pastes for application to the tongue for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
• parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;
• topical application e.g. as a cream, ointment or spray applied to the skin;
• Figures 1 to 4 show the effects of various concentrations of BRI 2923 in inhibition of the HIV toxic Vpr peptide fraction P3.
• PAMAM dendrimers refer to polyamidoamine dendrimers based on an ammonia core as detailed in US Patents Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329;
• PAMAM (EDA) dendrimers refer to polyamidoamine dendrimers based on an ethylene diamine core;
• BHAlys x lys y lys z dendrimers refer to polylysine unsymmetrical dendrimers based on a benzhydrylamine core and lysine branching units as described in US Patents Nos. 4,289,872 and 4,410,688.
• PAMAM 2.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as described above.
• the crude product was purified by gel filtration (Sephadex G10; water) and then freeze dried to give an off white solid.
• 1 H and 13 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 2.0 dendrimer ( ca. 65:35).
• PAMAM 3.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as above except that a slight excess of sodium carbonate was used and the benzyltrimethylammonium hydroxide was omitted.
• 1 H and 13 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 3.0 dendrimer ( ca. 50:50).
• the above reaction sequence could be carried out without isolating the bromoacetylated dendrimer by simply adding the sodium sulfite solution to the crude aqueous extract obtained from the first reaction.
• succinimidyl acetylthioacetate (67mg; 0.33mmol) was added to a solution of PAMAM 2.0 (52mg; 0.05mmol) in dry DMF (2ml) and the resulting solution stirred at room temperature for two days. The mixture was then concentrated (30 /10 " mmHg) to give an oily residue. The residue was partitioned between water and chloroform, and the water layer separated and concentrated to give a viscous oil
• Solid maleic anhydride 60mg; O. ⁇ mmol was added to a stirred solution of PAMAM 4.0 (51mg; O.Olmmol) in dry DMF (2ml). The mixture initially became cloudy but soon gave a clear solution which was stirred overnight at room temperature. The solution was then concentrated (35°/10 "4 mmHg) to give a viscous oil. 'H and C nmr (D 2 O) showed complete conversion of the PAMAM 4.0 to the polyamide together with some maleic acid. The crude polyamide was then dissolved in water (2ml) and a solution of sodium sulfite (126mg; l.Ommol) in water (2ml) added.
• PAMAM 1.0 and PAMAM 3.0 (BRI2785) dendrimers terminated with sodium N-(2-sulfoethyl)succinamide groups were similarly prepared.
• Trifluoroacetic acid (lml) was added to a suspension of BHAlyslys 2 lys 4 lys 8 DBL ⁇ 6 (36.5mg; 5.0 ⁇ mol) in dry dichloromethane (lml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in dry DMSO (2ml) and the pH adjusted to 8.5 with triethylamine. A solution of the crude tetrabutylammonium 4-nitrophenyl N-(2- sulfoethyl)succinamate (ca. 0.2mmol) in DMSO (lml) was then added dropwise and the mixture stirred overnight at room temperature.
• BHAlyslys 2 , BHAlyslys 2 lys 4 (BRI2787) and BHAlyslys 2 lys 4 lys 8 (BRI2788) terminated with sodium N-(2- sulfoethyl)succinamide groups were similarly prepared.
• Solid sodium 4-sulfophenylisothiocyanate monohydrate (500mg; 1.96mmol) was added to a solution of PAMAM 4.0 (300mg; 0.0582mmol) in water (10ml) and the resulting solution heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated and the yellow solid residue purified by gel filtration (Sephadex LH20; water). The pure fractions were combined and freeze dried to give the sodium 4-sulfophenylthiourea terminated PAMAM 4.0 dendrimer as a fluffy white solid (370mg).
• 13 C nmr (D 2 O) ⁇ 36.9; 41.1; 43.1; 48.3; 53.6; 55.8; 129.0; 131.1; 144.4; 178.5; 179.1; 184.4.
• PAMAM 1.0, PAMAM 2.0 (BRI2790), PAMAM 3.0, and PAMAM 5.0 (BRI2991) dendrimers terminated with 3, 6, 12, and 48 sodium 4- sulfophenylthiourea groups respectively were similarly prepared.
• Solid sodium 4-sulfophenylisothiocyanate monohydrate (130mg; 0.5mmol) was added to a solution of PAMAM 4.0 (EDA) (69mg; 0.01 mmol) in water (4ml) and the resulting solution heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated and the solid residue purified by gel filtration (Sephadex LH20; water). The pure fractions were combined and freeze dried to give PAMAM
• BHAlyslys lys lys lys 8 BHAlyslys 2 lys 4 lys 8 lys ⁇ 6 lys 32 (BRI2992), and BHAlyslys 2 lys 4 lys 8 lys 16 lys 32 lys 64 (BRI2993) dendrimers terminated with 16, 64, and 128 sodium 4-sulfophenylthiourea groups respectively were similarly prepared.
• Solid sodium 3,6-disulfonapthylisothiocyanate 160mg; 0.41mmol was added to a solution of PAMAM 4.0 (51mg; 0.01 mmol) in water (3ml) and the resulting solution heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated and the brown solid residue purified by gel filtration (Sephadex LH20; water). The pure fractions were combined and concentrated to give the sodium 3,6- disulfonapthylthiourea terminated PAMAM 4.0 dendrimer as a brownish solid (73mg).
• H nmr (D 2 O) : ⁇ 2.30; 2.60; 2.74; 3.20; 3.57; 7.75; 7.86; 8.28. 13
• C nmr (D 2 O) ⁇ 35.0; 39.9; 43.1; 48.1; 53.8; 56.1; 128.4; 128.6; 129.3; 131.0; 131.3; 136.0;
• Solid sodium 3,6-disulfonapthylisothiocyanate (220mg; 0.57mmol) was added to a solution of PAMAM 4.0 (EDA) (74mg; 0.01 mmol) in water (4ml) and the resulting solution heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated and the brownish solid residue purified by gel filtration (Sephadex
• Trifluoroacetic acid (2ml) was added to a suspension of BHAlyslys 2 lys 4 lys 8 DBL 16 (73mg; 0.01 mmol) in dry dichloromethane (2ml) under nitrogen. A vigorous evolution of gas was observed for a short time and the resulting solution was stirred at room temperature for two hours and then concentrated. The residual syrup was dissolved in water (5ml), the solution passed through a column of Amberlite IRA- 401 (OH) and the filtrate concentrated to give BHAlyslys 2 lys 4 lys 8 lys 1 6 as a viscous oil.
• Solid sodium 4-sulfonapthylisothiocyanate (180mg; 0.5mmol) was added to a solution of PAMAM 4.0 (51mg; O.Olmmol) in water (5ml) and the mixture heated under nitrogen at 53° for two hours and then cooled. The water was distilled under reduced pressure from the resulting suspension and the off white solid residue purified by gel filtration (Sephadex LH20; water). The pure fractions were combined and freeze dried to give the sodium 4-sulfonapthylthiourea terminated PAMAM 4.0 dendrimer as a fluffy white solid (60mg).
• 13 C nmr (D 2 O) ⁇ 35.8; 40.5; 43.1; 48.4; 53.6; 55.9; 127.6; 128.6; 130.3; 131.9; 132.5; 133.5; 134.7; 140.5; 142.7; 177.8; 178.0; 185.4.
• Solid sodium 3, 6, 8-trisulfonaphthylisothiocyanate (250mg; 0.5mmol) was added to a solution of PAMAM 4.0 (51mg; 0.01 mmol) and N,N-dimethyl-N-allylamine buffer (pH 9.5; lml) in water (2ml) and the mixture heated under nitrogen at 53° for two hours and then cooled. The mixture was concentrated under reduced pressure to give an orange solid. The residual solid was dissolved in water (2ml) and passed through a short column of Amberlite IR-120(Na). The filtrate was then concentrated and the residue purified by gel filtration (Sephadex LH20; water).
• H nmr (D 2 O) ⁇ 2.25; 2.66; 3.08; 3.20; 3.33; 3.38; 4.01; 7.40 (br d); 7.62 (br d).
• 13 C nmr (D 2 O) ⁇ 36.7; 40.9; 43.0; 43.6; 53.5; 55.5; 61.0; 131.6; 135.0; 137.2; 140.4; 174.5; 178.6; 179.2.
• Solid potassium N-hydroxysuccinimidyl 4-sulfobenzoate (lOOmg; 0.3mmol) was added to a solution of PAMAM 4.0 (EDA) (35mg; 0.005mmol) in 0.1M pH 8.5 borate buffer (5ml) and the solution stirred at room temperature for two hours. The resulting milky solution at this stage had a pH of 4.5. 1M Sodium carbonate solution (lml) was then added to give a clear solution which was concentrated to give the crude product as a white solid. The crude product was purified by gel filtration (Sephadex G25; water) to give PAMAM 4.0 (EDA) terminated with 32 sodium 4-sulfobenzamide groups (47mg).
• BHAlyslys 2 lys 4 lys 8 lys 16 BRI2922 Trifluoroacetic acid (4ml) was added to a suspension of BHAlyslys 2 lys lys 8 DBL 16
• PAMAM 4.0 BRI6043 l,l'-Carbonyldiimidazole 85mg; 0.52mmol was added to a solution of 4- trimethylammoniumbenzoic acid iodide (154mg; 0.5mmol) in dry DMF (4ml) and the mixture stirred at room temperature under argon for two hours. During this time a white solid separated from the solution.
• a solution of PAMAM 4.0 (58mg; 0.01 lmmol) in dry DMF (2ml) was then added and the mixture stirred overnight at room temperature. After this time most of the precipitate had dissolved and a ninhydrin test of the solution was negative.
• PAMAM 4.0 BRI6044 Solid 4-nitrophenyl 4-(chloromethyl)benzoate (150mg; 0.5mmol) was added to a stirred solution of PAMAM 4.0 (52mg; 0.0 lmmol) in dry DMSO (3ml). The resulting yellow solution was stirred at room temperature for 20 hours, when a ninhydrin test was negative (pH ca.8.5). The solution was then concentrated (10 mmHg; 40 ) and the residue shaken with a mixture of water and dichloromethane (1:1). The insoluble gel-like material was collected by filtration, washed with water (2X) and dichloromethane (2X), and then air dried.
• the crude 4-(chloromethyl)- benzamide terminated dendrimer was dissolved in 25% aq. trimethylamine (20ml) and the yellow solution left to stand overnight. The solution was then concentrated, the residue dissolved in water (5ml) and the solution passed through a column of Amberlite IRA-401 (OH). The colourless filtrate was concentrated to give a viscous oil which was purified by gel filtration (Sephadex G10; 10% AcOH) to give PAMAM 4.0 terminated with 24 4-(trimethylammoniummethyl)benzamide groups (90mg).
• 13 C nmr (D 2 O) ⁇ 26.6; 33.4; 38.8; 43.2; 43.5; 53.6; 53.6; 54.1; 56.8; 62.8; 73.0; 132.1; 135.3; 137.5; 140.0; 176.4; 176.9; 183.6.
• H nmr (D 2 O) ⁇ 2.17; 2.36; 2.50; 2.78; 2.85; 3.25; 3.40;
• PAMAM 4.0 Sodium cyanoborohydride (32mg; 0.5mmol) was added to a mixture of PAMAM 4.0 (EDA) (69mg; 0.0 lmmol), 4-formyl-2-hydroxybenzoic acid (83mg; 0 0.5mmol), and sodium hydrogen carbonate (42mg; 0.5mmol) in water (4ml).
• EDA Sodium cyanoborohydride
• the inhomogeneous orange mixture was stirred for four hours at room temperature, during which time it became homogeneous.
• the orange solution was then concentrated and the residue purified by gel filtration (Sephadex LH20; water) to give PAMAM 4.0 (EDA) terminated with ca. 32 4-carboxy-3-hydroxybenzylamine
• Solid sodium ethyl 4-(phosphonomethyl)phenylisothiocyanate (109mg) was added to a solution of PAMAM 4.0 (EDA) (69mg; O.Olmmol) in DMF (30ml). The resulting solution was stirred for 17 hours at room temperature under nitrogen, maintaining the pH at 8 with periodic addition of saturated NaHCO 3 solution. The reaction mixture was then concentrated to give a white solid residue, which was purified by gel filtration (Sephadex LH20; water) and then freeze dried to give the product as a fluffy white solid (30mg).
• EDA PAMAM 4.0
• Methyl [(8-octanoic acid N-hydroxysuccinimide ester) 5-acetamido-4,7,8,9-tetra-O- acetyl-3 ,5-dideoxy-2-thio-D-glycero- -D-galacto-2-nonulopyranosid]onate was prepared by the following procedure.
• a PAMAM [EDA] 4.0 [(8-octanamido)- 5-acetamido-3,5-dideoxy-2-thio-D- glycero- -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6112
• PAMAM [EDA] 4.0 [(8-octanamido)- 5-acetamido-3,5-dideoxy-2-fhio-D- glycero- ⁇ -D-galacto-2-nonulopyranosidoic acid] 32 was obtained as a pale lemon powder llOmg. 77%
• PAMAM [EDA] 4.0 [(ll-undecanamido)-5-acetamido-3,5-dideoxy-2-thio- D-glycero- ⁇ -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6147
• PAMAM [EDA] 4.0 [ (acetamido)- 5-acetamido-3,5-dideoxy-2-thio-D- glycero- ⁇ -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6121
• PAMAM [EDA] 4.0 [(4-methylbenzamido)- 5-acetamido-3,5-dideoxy-2- thio-D-glycero- ⁇ -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6120
• Methyl 4-azido-5-acetamido-7,8,9-tri-O-acetyl-2-S-acetyl-3,4,5-trideoxy-2-fhio-D- glycero- ⁇ -D-galacto-2-nonulopyranosonate was prepared by the following procedure.
• Methyl [(8-octanoic acid N-hydroxysuccinimide ester) 4-azido-5-acetamido-7,8,9- tri-O-acetyl-3,4,5-trideoxy-2-thio-D-glycero- -D-galacto-2-nonulopyranosid]onate was prepared by the following procedure.
• a PAMAM [EDA] 4.0 (8-octanamido)- 4-azido-5-acetamido-3,4,5-trideoxy- 2-thio-D-glycero- ⁇ -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6146
• the free sialoside was obtained by the following method:
• PAMAM [EDA] 4.0 [(8-octanamido)- 4-amino-5-acetamido-3,4,5-trideoxy- 2-thio-D-glycero- -D-galacto-2-nonulopyranosidoic acid] 32 BRI 6149
• PAMAM [EDA] 4.0 [4-benzamidoboronic acid] 32 BRI 6160
• PAMAM [EDA] 4.0 [4-benzamidoboronic acid] 32 BRI 6160
• 4-carboxyphenylboronic acid N-hydroxysuccinimide ester 130mg.
• 1M sodium carbonate solution lml.
• the solvent was removed under vacuum and the residue was dissolved in 10% ammonia solution (5ml.). This solution was subjected to size exclusion chromatography on Sephadex LH20 eluting with 10% ammonia solution .
• the product, PAMAM [EDA] 4.0 4-carboxyphenylboronic acid N-hydroxysuccinimide ester
• Trifluoroacetic acid (2ml) was added to a stirred suspension of BHAlyslys 2 lys 4 lys g lys 16 DBL 32 (147mg) in dry dichloromethane (2ml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in N,N-dimethyl-N-allylamine buffer (pH 9.5; 5ml) and then solid 3,6-disulfonaphfhyl isothiocyanate (400mg) added. The pH of the mixture was then adjusted to 9.5 by the addition of 1M sodium carbonate and the solution heated at 53 °C for three hours under nitrogen.
• the reaction mixture was concentrated and the residue redissolved in water and the solution passed through a column of Amberlite IR 120 (Na).
• the filtrate was concentrate was concentrated to give the crude product, which was purified by gel filtration (Sephadex LH20; water) to give BHAlyslys 2 lys 4 lys 8 lys 16 lys 32 with 64 sodium 3,6-disulfonaphthylurea groups as a white fluffy solid (175mg).
• Trifluoroacetic acid (3ml) was added to a stirred suspension of BHAlyslys 2 lys 4 lys 8 lys 16 DBL 32 (300mg; 0.02mmol) in dry dichloromethane (3ml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in water and the solution passed through a column of Amberlite IRA 401 (OH) and the filtrate concentrated to give a viscous oil (187mg). The oil was dissolved in a 1:1 mixture of pyridine/water (8ml) and solid sodium 3,5-disulfophenyl isothiocyanate (680mg; 2mmol) added. The resulting solution was heated at 53° C for three hours under nitrogen.
• Trifluoroacetic acid (3ml) was added to a stirred suspension of BHAlyslys 2 lys 4 lys 8 lys 16 DBL 32 (300mg; 0.02mmol) in dry dichloromethane (3ml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in water and the solution passed through a column of Amberlite IRA 401 (OH) and the filtrate concentrated to give a viscous oil (186mg). The oil was dissolved in a 1: 1 mixture of pyridine/water (8ml) and sodium 3,5-dicarboxyphenyl isothiocyanate (450mg; 2mmol) added. The resulting solution was heated at 53 °C for 13 hours under nitrogen.
• Trifluoroacetic acid (2ml) was added to a stirred suspension of BHAlyslys 2 lys 4 lys g lys 16 DBL 32 (147mg; 0.01 mmol) in dry dichloromethane (2ml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated to give a viscous oil.
• the oil was dissolved in N,N- dimethyl-N-allylamine buffer (pH 9.5; 5ml) and solid 4-phosphonooxyphenyl isothiocyanate (250mg) added.
• the pH of the resulting solution was adjusted to 10 with 1M sodium carbonate and the mixture heated at 53 °C for three hours under nitrogen. The solution was then concentrated to give a white solid residue.
• Trifluoroacetic acid (2ml) was added to a stirred suspension of BHAlyslys 2 lys 4 lys 8 lys 16 DBL 32 (147mg; 0.01 mmol) in dry dichloromethane (2ml) and the resulting solution stirred at room temperature under nitrogen for two hours and then concentrated to give a viscous oil.
• the oil was dissolved in N,N- dimethyl-N-allylamine buffer (pH 9.5; 5ml) and solid 4-phosphonophenyl isothiocyanate (250mg) added.
• the pH of the resulting solution was adjusted to 9 with saturated sodium bicarbonate solution and the mixture heated at 53 °C for three hours under nitrogen. The solution was then concentrated to give a white solid residue.
• PAMAM 4.0 terminated with 24 sodium 4,6-diphosphononaphthylthiourea groups as a brown solid (81mg) after freeze drying.
• Solid fluorescein isothiocyanate (188mg) was added to a solution of PAMAM 4.0 (EDA) (74mg; O.Olmmol) in water (3ml). Saturated sodium bicarbonate solution was added to adjust the pH to 9 and the resulting homogenous solution stirred overnight at room temperature and then concentrated. The orange residue was purified by gel filtration (Sephadex LH20; water) to give PAMAM 4.0 (EDA) terminated with 21 fluoresceinthiourea groups as a fluffy orange solid (193mg) after freeze drying.
• Solid (phenyl-3-boronic acid) isothiocyanate (lOOmg; 0.5mmol) was added to a solution of PAMAM 4.0 (EDA) (69mg; O.Olmmol) in water (5ml). 1M sodium carbonate was added to the isothiocyanate dissolved (pH ca.10). The mixture was then heated at 53 °C for two hours under nitrogen, and then filtered and the filtrate concentrated to give a brownish solid residue. The crude product was purified by gel filtration (Sephadex LH20; water) to give PAMAM 4.0 (EDA) terminated with 32 (phenyl-3-boronic acid)thiourea groups as a white fluffy solid (87mg) after freeze drying.
• PAMAM generation 2.0 core (0.0479mmol; 50mg) was evaporated from a 0.5ml solution in MeOH and then re-dissolved in 10 ml of water.
• 1-N- pyridinium 12-dodecanoic acid bromide (0.14g; 0.384mmol), N-hydroxybenzotriazole hydrate [HOBT] (52mg; 0.384mmol) ; triethylamine (53 ⁇ l 0.384mmol) and l-(3- diethylaminopropyl-3-ethyl) carbodiimide .HCl [EDC] (74mg; 0.384mmol), were added to the solution. This reaction mixture was stirred overnight at room temperature.
• ⁇ nmr (D 2 O) ⁇ 1.15, 1.45, 1.9, 2.15, 2.75, 2.8, 3.15, 3.35, 3.5, 4.55, 8.05,
• PAMAM 4.0 dendrimer BRI-6809.
• PAMAM generation 4.0 core (0.05 mmol; 69mg) was evaporated from a
• 12-dodecanoic acid bromide (0.143g; 0. 4mmol), N-hydroxybenzotriazole hydrate [HOBT] (77mg; 0.4mmol) ; triethylamine (56 ⁇ l 0. 4mmol) and l-(3- diethylaminopropyl-3-ethyl carbodiimide .HCl [EDC] (77mg; 0.4mmol) were added to the solution.
• the Cytosensor Microphysiometer (Molecular Devices Inc., CA) is a light addressable potentiometric sensor-based device that can be used to indirectly measure the metabolic rate of cells in vitro (Parce et al. , 1989; McConnell et al. , 1992). Metabolism is determined by measuring the rate of acid metabolite production from cells immobilised inside a micro volume flow chamber.
• Modified medium Human CEM cells were centrifuged and resuspended in low-buffered serum- free/bicarbonate-free RPMI 1640 medium (Molecular Devices; hereafter referred to as "modified medium"). The cells were seeded at a density of 60,000-75,000 cells/capsule onto the polycarbonate membrane (3 ⁇ m porosity) of cell capsule cups (Molecular Devices). Cells were immobilised using an agarose entrapment medium (Molecular Devices). The seeded capsule cups were transferred to sensor chambers containing the silicon sensor which detects changes in pH (and thus cellular metabolism). The Cytosensor system used for this set of experiments contained eight separate chambers for the measurement of acidification rates. Modified media was pumped across the cells at a rate of 100-120 ⁇ l/min. Each cell chamber was served by fluid from either of two reservoirs.
• Basal acidification rates were monitored (in the absence of any treatment) for at least 30 min. After this time, the venoms/peptides were exposed to the cells at a range of concentrations for periods of up to 4hrs. A concentration of toxin showing a pronounced effect on the cells, but less than maximal, was selected for testing of inhibition of this toxicity by a range of concentrations of BRI2923. In all experiments, at least one chamber was not exposed to any of the compounds, providing a negative control.
• BRI 2923 was dissolved in water and the solution was pH adjusted to 7.2. 5 Concentrations ranging from 100 ⁇ M to 1 nM were added to the venom/media solutions and incubated for periods ranging from 6 min (the minimum incubation period possible using this equipment) to 1 hr and then introduced to the cells. All experiments were repeated in triplicate.
• Snake venoms consist of many toxic components each of which have different modes of toxicity.
• Cobra venoms contain a cytotoxic peptide which causes cell lysis in a way similar to bee venom toxin, melittin.
• the amino acid sequence of the cytotoxin isolated from Naja malenoleuca indicates that this toxin is highly basic (cationic) and would thus be readily inactivated by polyanionic compounds such as BRI2923. This electrostatic inactivation as a basis for reduced toxicity is supported by the experimental finding that an incubation period of 6 minutes gives the same result as the longer incubation periods of 30-60 mins used.
• Vpr is an Human Immunodeficiency Virus (HIV-1) accessory gene product, 14- kDa, 96-amino-acid protein.
• HIV-1 Human Immunodeficiency Virus
• HIV-2 HIV-2
• Vpr gene product optimises HIV infection and disease progression. (reviewed by Cullen, 1998; Emerman & Malim, 1998).
• Vpr induces apoptosis and cytopathic effects in both human cells and yeast (Stewart et al, 1997; Zhao et al, 1996; Macreadie et al, 1996).
• Vpr protein has been fractionated and the peptide sequence which causes apoptosis has been isolated and designated Vpr P3.
• Vpr P3 has been found to permeabilise CD4 T lymphocytes (such as CEM cells) and causes their death by apoptosis. The following experiments use this toxic Vpr peptide fraction P3 (Arunagiri et al, 1997).
• Human CEM cells were centrifuged at 1400 RPM for 7 minutes and resuspended in modified RPMI 1640 medium. Cells were then immobilised using an agarose entrapment medium and spotted onto the centre of the polycarbonate membrane of the capsule cup (Molecular Devices Ltd., CA.). The cells were seeded at a density of approximately 43,000 - 75,000 cells per capsule cup. The seeded capsule cups were transferred to sensor chambers containing the silicon sensor and positioned on to the microphysiometer. Modified media was pumped across the cells at a rate of 120 ⁇ l/min. Each cell chamber was served by fluid from either of two reservoirs, which could be alternated using a software command.
• CEM cells were used at 1.3 x 10 to 1.3 x 10 cells/ml which corresponds to early to mid log phase growth of these cells.
• the cells were set up on the microphysiometer and the Vpr P3 peptide was perfused through the cell chambers after an equilibration period of at least 30 minutes.
• the concentration range used for Vpr P3 was 10 M - 2 x l0 M .
• Vpr P3 needed to be in contact with the cells for upwards of 2 hours for the full effect of the toxicity to be apparent.
• Vpr P3 peptides were made to the appropriate concentration and the BRI2923 was added, the solution mixed, and left to equilibrate for 20-30 minutes prior to placement on the cytosensor.
• a positive control channel containing the same concentration of the Vpr P3 used to test BRI2923 inhibition was also run for the same time period as well as a time control channel.
• the Vpr P3 peptide solution and the Vpr P3/BRI2923 solution were in contact with the cells for a minimum period of 2hr 30min to a maximum period of 4hr. Also, after washout of the compounds, the metabolic rate of the cells was monitored for a short time period (minimum of 6 min).
• the VPR P3 peptide tested on its own causes an initial increase in metabolic rate followed by a decline and subsequent cell death due to apoptosis of the CEM cells.
• BRI 2923 at 10 M also demonstrated inhibition of 2x10 M Vpr P3 -induced apoptosis (experiment in triplicate).
• BRI2923 10 M showed a significant reduction in the effects produced by 2x10 " M Vpr P3 (triplicate experiment).
• the cells in the chamber containing BRI2923 did not show any time related decline in metabolic rate.
• Previous cytosensor experiments using BRI2923 have also suggested that the dendrimer may confer a cytoprotective effect in addition to the inhibition of apoptosis caused by VPR P3.
• Toxin • Cholera Toxin (Sigma Cat. No. C8052) - A subunit surrounded by five B subunits.
• HCP-8 (adherent human ileocecal carcinoma epithelial cell line) in log phase growth were trypsinised (trypsin EDTA) in 2ml/75cm 2 flasks after 2 PBS (phosphate buffered saline) washes.
• a C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4 lymphocytes. Apoptosis, 2, 69-76
• Emerman, M., and Malim, M.H. (1998) HIV-1 regulatory/accessory genes: keys to unravelling viral and host cell biology. Science, 280, 1880-1883.
• Macreadie I.G., Arunagiri, C.K., Hewish, D.R., White, J.F., and Azad, A.A. (1996) Extracellular addition of a domain of HIV-1 Vpr containing the aminoacid sequence motif H(S/F)RIG causes cell membrane permeabilization and death. Mol. Microbiol, 19(6), 1185-1192.

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