CA1114812A - Hapten polysaccharide conjugates - Google Patents

Hapten polysaccharide conjugates

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Publication number
CA1114812A
CA1114812A CA309,347A CA309347A CA1114812A CA 1114812 A CA1114812 A CA 1114812A CA 309347 A CA309347 A CA 309347A CA 1114812 A CA1114812 A CA 1114812A
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Prior art keywords
group
substituted
hapten
medicament
levan
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CA309,347A
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French (fr)
Inventor
Carlos Moreno
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Wellcome Foundation Ltd
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Wellcome Foundation Ltd
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Classifications

    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0051Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Fructofuranans, e.g. beta-2,6-D-fructofuranan, i.e. levan; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/35Allergens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/385Haptens or antigens, bound to carriers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6012Haptens, e.g. di- or trinitrophenyl (DNP, TNP)

Abstract

ABSTRACT
The present invention provides conjugates between levan or dextran and a medicament, processes for preparing them, pharmaceutical compositions containing them and intermediates in their preparation. The conjugates create tolerance to the administration of the medicaments in mammals.

Description

The present invention relates to conjwgates which are formed between certain medicaments, in particular penicillins, and substituted levans said conjugates being useful in providing immunological tolerance in mammals to the administration of the medicaments. The invention also relates to processes for preparing and compositions containing such conjuga~es.
Many patients suffer from allergic reactions when prescribed particular drugs by their doctor, there being a wide range of drugs which can cause such reactions. In the case of particularly sensitive patients this may prevent certain drugs being prescribed thereby severely restricting the choice of drugs open to the doctor in his ; fight to combat disease.
The penicillins are a class of drugs which have become increasîngly important in the last three decades for the treatment of bacterial infections. Unfortunately however, the penicillins can cause severe allergic reactions and anaphylaxis
- 2 ~ A5~6 on administration to animals and humans. Over the past decade several atkempts have been made to overcome these problems and to create immuno-logical tolerance to the administration of penicillins and prevent anaphylaxis. Much of this work has been carried out on benzylpenicillin which is the drug most commonly causing allergic reactions. Por example, Chiorazzi et. al (Proc.
Natl. Acad. Sci. USA, 73, 2091, 1976) reported th-at the treatment of mice with the benzylpenicilloyl derivatised synthetic co-polymer of D-glutamic acid and D-lysine resulted in the suppresion of anti-benzylpenicilloyl antibody responses and that the state of induced tolerance was highly speciic and of long duration. Similarly Borel et al (Nature, 261, 49, 1976) induced tolerance to benzylpenicillin by the administration of benzylpenicilloyl linked to several protein carriers and De Weck and Schneider ~Int. Arch. Allergy, 42, 782, 1976~ inhibited allergic reactions to benzylpenicillin in-vivo by the aclministration of benzylpenicilloylformyllysine.
It has now been found that conjugates formed by linking certain medicaments or derivatives thereof to certain substituted haptens give good tolerance and a~e poorly immunogenic, that is to say they do not stimulate the production of appreciable amounts . ' , - '.
. ~ , .

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of antibodies, after the conjugates have been administered. Furthermore, it has been found that administration of these conjugates after the induction of an allergic reaction to the medicament from which the conjugate is derived effectively counteracts the observed àllergic response.
By the term 'medicament' is meant a pharmacologically active substance useful in the field of medicine. The medicaments suitable for inclusion in the conjugates of this invention are those which cause an allergic reaction on administration to humans and animals and which contain at least one carboxy group or are modified to contain such a group. Commonly only one part of the medicament molecule or a metabolite of the medicament will cause the allergic reaction. For example, it is known that penicilloic acid and penicillamine are two metabolites of benzylpenicillin which contribute greatly to the allergic reaction observed when benzylpenicillin is administered.
Thus metabolites of medicaments which cause an allergic reaction on administration, provided they contain at least that part of the molecule which is responsible for the allergic reaction and at least one carbox~ group, are also suitable for inclusion in the conjugates of the present invention as .~i `, _ ~ _ A54 derivatives of rnedicaments.
Haptens suitable :Eor inclusion in. the conjugates of the present invention are levans and dextrans.
Levans are natura:L polymers of 2,6~ and 2,1- linked ~~=D-fructofuranose present in some bacteria and in grasses. Thus the repeating unit of levan has.the formulae (I) and ~

~CH20~ C;;20H '-HOH2Cl/ O \~o~
H2q/ ~CH20H (II) HO Ø.O ~
H

It can be seen that each furanose ring in levan has three free hydroxy groups to which substituents may be attached.
Dextran is a term applied to polysaccharides produced by bacteria growing on a sucrose substrate, containing a backbone of D-glucose units linked predominantly ~-D (1-63.

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_ 5 _ - A546 ~ ccording to the 1present invention there is provided a medicament~.substituted hapten conjugate having a molecular weight of greater than 20,000, comprising a hapten in which a number of the hydroxy groups are substituted by one or more groups -CH2-C0-NH-X-N~, wherein X is a Cl_8 alkylene group optionally substituted by hydroxy groups or a Cl 7 alkyleneamino group, wherein Y is a medicament or derivative thereof as hereinbefore defined,the carboxy group in Y
and the amino group in the substituent chain forming an amide linkage.between the two.
Particularly suitable medicaments for inclusion within the conjugates of this invention include penicillins, cephalosphorins 7 :sulphonamides, benzylpyrimidines, extracts of pollens and derivatives thereof, which contain carboxy groups.
Suitable derivatives of penicillins include penicilloic acid and penicillamine. Suitable benzylpyrimidines include those of the formula tIII):

~CH2 ~H2 ~III) ~H2 .~y ~

- 6 - ~5~.~6 wherei.n one of R, Rl, R2 is a -CH2-CO- group and the others, which may be the same or diferent, are hydrogen, hydroxy, Cl 4 alkyl or Cl 4 allcoxy.
By the term X is a Cl_7 alkyleneamino group is meant that X may be a Cl 7 alkylene group substituted by one or more amino groups or that amino groups link togcther more than one alkylene group to form a Cl_7 alkyleneamino group.
Conveniently X is a C2_s alkylene group and preferablY
a propylene group.
In a preferred aspect the present invention provides a penicilloyl-substituted hapten conjugate having a molecular weight of greater than 20,000 comprising a levan in which a plurality of the hydroxy groups are substituted by one or more groups -CH2-CO-NH-X-NHY wherein X is as hereinbeore defined and Y is a group of the formula (IV)-RN~
H 1 CH~

I ~ IH~ ~CH~ (IV) COOH

or a pharmaceuti.cally accepkable salt or esterthereof wherein R is a hydrogen a~om or an acyl si.de _ 7 A546 chain conventionally linked to the amino grol1p attached to the 6-position in naturally occurring or semi-synthetic penicillins.
Preferably R is a phenylacetyl group.
Pharmaceutically acceptable salts includ~
sodium, potassium, calcium, magnesium, aluminium, ammonium and substituted ammonium salts. The sodium and potassium salts are particularly suitable.
Bsters suitable for the purposes of this invention include those notionally derived from an alcohol ROH wherein R is an alkyl, alkenyl, alkynyl, aryl or aralkyl ~roup which may be substituted if desired. Preferably the esters are in vivo hydrolysable esters such as the pivaloyl-oxymethyl or phthalimido esters.
In a further preferred aspect of the present invention provides a sulphonamide-substituted levan conjugate having a molecular weight of greater than 20,000 comprising a hapten in which a plurality of the hydroxy groups are substituted by one or more groups -CH2-CO-NH-X-NHY wherein X is as hereinbefore defined and Y is a group -02C~ S2NH2 ~' - 8 - ~s~6 or a pharmaceutically acceptablc salt khereof.
Preferably the hapten is a levan.
It has been found that although a degree of tolerance is induced when the conjugate has a molecular weight as low as 20,000 higher molecular weight conjugates are preferred, i.e. those with a molecular weight of at least in the order of 105 and preferably at least in the order of 106- It has also been found that the degree of induced tolerance is surprisingly increased when the number of Y groups, i.e. the medicament component, attached to the hydroxy groups of the hapten is increased.
Whilst it has been found that tolerance may be - induced with a hapten substituted by only 20 Y
groups per 1,000 fructosyl rcsidues the hapten is most suitably substituted by at least 60 and preferably by at least 90 Y groups per 1,000 fructosyl residues.
The conjugates of the present in~ention do not elicit passive cutaneous anaphylaxis, particularly when they are highly substituted, and are therelore preferable to the other known tolerogens. Purthermore the conjugates suppress the immune response to the hapten and are therefore free o po~entially trouble-some side effects due to the hapten.
, _ 9 _ ~5~6 I'he present invention also provides aprocess for preparing mecLicament-substituted hapten conjugakes which process comprises the reaction of a medicament, or derivative thereof, as hereinbefore defined with a hapten in which a number of the hydroxy groups are substituted by a group of the formula (V):

-CH~,-CO-NH-X-NH2 (V) wherein X is as hereinbefore defined.
In the case of penicillins the reaction will normally be carried ou~ in an aqueous solvent, conveniently water, and at alkaline pH, for example at a pH of greater than 9. This may conveniently be achieved by buffering the solution with a conventional alkaline buffer such as carbona*e buffer. Carrying out the reaction at alkaline pH converts the penicillin to a penicilloyl moiety which reacts with the substituted levan to give a penicilloyl-substituted hapten conjugate.
The reaction will be carried out at a non-extreme temperature, for example between 0 and100C, and may conveniently be carried out at room tempera l:ure .
In the case of other medicaments or derivatives thereof hereinbefore definecl the reaction may ,L~ ,~3 ~

- 10 - t~S4G

convenicntly be carried out in the presence of a conventional condensation promoting agent such as a carbodiimide. This reaction is normally carri~d out at acid pH, suitably between 4.5 and 6.5, and at a non-extreme *emperature, i.e. between -10C and 100C, and conveniently at room temperature, in a suitable solvent, for example water or a mixture of water with a water miscible organic solvent.
Alternatively the carboxylic acid group of the medicament or derivative thereof may be converted into an N-acylating derivative by methods well known to 'chose skilled in the art. Suitable N-acylating deri~atives include acid halides and anhydrides.
15 The N-acylating derivative is then reacted with the --substituted levan under conditions well known to those skilled in preparing amides.
Haptens in which a number of the hydroxy groups are substituted by a group of the formula (V), as hereinbefore defined, are useful intermediates and as such form part of the present invention.
These intermediates may be prepared by the reaction of a hapten in which a number of the hydroxy groups are substituted by a group of the formula: -t:H2C02H, or a reactive N~acylating derivative thereof, wi*h a compound H2NXNH2, where:in X is as hereinbefore defined.
Suitable N-acylating derivatives of 'che carboxylic acid group include acid halides and anhydrides. The reaction may conveniently be carried ou'c by the reaction of the free carboxylic acid group with a compound ~I~NXNH2 in the presence of a condensation promoting reagent such as a carbodiimide, This reaction is normally carried out at acid pH, suitably between 4.5 and 6.5, and at a non~extreme temperature, i.e. between -10 and 100C and conveniently at room temperature, in a suitable solvent, for example water or a mixture of water with a water miscible polar organic solvent.
The hydroxy groups of the hapten may readily be substituted by the group: -CH2C02H, by the reaction of the hapten with a suitable reactive derivative of acetic acid, for example a monohaloacetic acid such as monochloroacetic acid. This reaction is carried out in an aqueous solvent system at alkaline pH at a non-extreme temperature. The reaction is suitably carried out in aqueous sodium hydroxide at a pH of greater than 12 at room temperature.
.~.

~ 12 - A546 In a further aspect *h;s invention provides a pharmaceutical composition for the purpose o medical or veterinary treatment which comprises a medicament substituted hapten conjugate herein-before de-fined as a substance in a form or shape, e.g. together with a pharma.ceutically acceptable carrier or in combination with a carrier, for i.nstancein a sealed or sterile state, or in a dosage orm.
The compositi.ons o the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of immune hypersensitivity in mammals including humans.
Suitable forms of the compositions of this invention include tablets, capsules, creams, syrups, suspensions, solutions and sterile forms suitable for injecti.on or infusion. Such compositions may contain conventional pharmaceutically acceptable materials such as diluents, binders, colours, flavours, preservatives, disintegrants and the like in accordance with con~entional pharmaceutical practice.
Injectable compositions containing the medicament substituted hapten conjugates, especially injcctable composi.tions suita.ble Ior intravenous a~ministration, are par~icu,larly preferred composi~ions : ~ - ,....... .

. . .

~ 13 - ~5~6 of this invention. Such compositions will be made up in a sterile form in an aqueous solvent vehicle, such as a~ueous polyethylene glycol 7 or saline. Saline, which may be buffered to physiological p~l as required, is a particularly suitable solvent vehicle.
The compositions of the present invention are prepared by conventional formulation techniques well known to those skilled in the art, or example the injectable compositions are prepared by adding the medicament-substituted conjugates to the aqueous solvent vehicle and sterilising *he resultant solution.
In a yet further aspect the present invention provides a method of treatment of immune hypersensitivity to a medicament in mammals~ including humans, which comprises the administration of an effective dose of the appropriate medicament substituted hapten conjugate hereillbefore defined.
The invention also provides a method of treatment of bacterial infections in mammals, including humans, which comprises the adminis*ration of an effective dose of a medicament substitu*ed hapten conjugate herein-before defined, wherein the medica,ment in the conjugate is an antibacterially active medicament.
~..

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Suitably between 0.01 mg/kg and 100 mg/kg and conveniently 1 mg/kg of the medicament substituted hapten conjugate will be administered daily.
The conjugates of the present invention may be administered to induce immunological tolcrance to a medicament so that the patient may then receive a course of treatment with that particular medicament or they be administered when a patient is suffering from an allergic reaction to a medicament to induce tolerance to that medicament. In the latter case the degree of substitution of the hapten needs to be higher than it is in the former. The dosage level of the conjugates of the invention ~ill also normally be higher in this case.
Immuno~enic properties o the Conjugates of the Invention The capacity o~ pen-HSA (ovalbumin and human serum albumin coupled to the potassium salt of benzylpenicillin), Pen-DAP~ CM-LE ~a penicilloyl diaminopropylcarboxymethyl levan conjugate containing 22 penicillin groups/1000 fructosyl residues), Pen-~AP~2)-CM-LE ~a penicilloyl-diaminoprop)71carboxymethyl levan conjugate containing 95 penicillin groups/1000 fructosyl residues~, and Pen-DAP-CM-LE (XM50) ~a penicilloyl-~5 diaminopropylcarboxymethyl levan conjugate of lowmolecular weight ~M.W. = 3400) to react witll goat .
. . ' ' "

.

~ 15 - A546 anti-Pen serunl (containing 6.5 mg preci.pitating antibocly per ml~ l~as measured by d:i:rect precipi.tation and by inhi.bition of preci.pitation between the same anti-Pen goat serum and Pen HSA. The results are shown in Table 1.

TA~LE 1 I unoprecip-i-tation of antibody expressed as ~ of antibocly prec~ ated by Pen~HSA
. . .
- Pen DAP~ CM-LE 53~0 Pen - DAP(2)-CM-LE 78 Pem - DAP-CM-LE~XM50) ~ 0~
The relevance of these results to allergy in mi.ce was investigated by inhibition of PCA ~passive cutaneous anaphylaxis) reactions. This reaction was measured incubating a constant dilution of antiserum tcalculated to give 10 mm PCA spot) with different amounts o inhibitor for 1 hour at room temperature.
Fifty ~50) ~1 samples from each mi.xture were injected i.nto the skin of Wistor rats that were challenged 24-48 hours later with 2.5 mg Pen IISA and 5 mg of Evans blue (i.v.). The capacity of the compounds to inhibit PCA was indicated by a clecrease in the size of the blue spot in the skin. All three compounds listed in table 1 inh;bited specific anti-Pen ant;bodies.

:

J
- 16 ~ A546 Using amounts of S0, lS a:nd 2 x 104 ng/site respectively (in the order listed in Table 1 50% inhibition was achieved.
The ca.pacity of the same compounds to elicit a direct PCA was measured. Several dilutions ~50 ~1) of anti-Pcn serum were injected i.n the skin of rats and one day later t}ley were injected in the same spot with 1 or 50 ~g or antigen right after i.v. injection o-f Evans blue.
The results are shown in Table 2.

l'ABLE 2 Direct PCA reaction with mouse a.nti-Pen and penicilloyl substi.tuted levans TABI.E 2 -. _ _ _ Antiserum Dllution Antigen (~g/site) _ . . ._ .. _ . _ 1/10 1/20 1/40 No antiserum Pen-HSA ~50) 13 9 8 5 (1) 15 10 8 5 Pen-DAP~l)-CM-LE ~50) 11 7 6 6 (1) 11 9 6 5 Pen-VAP(2)-C~-LE t50~ 55 5 55 5 Pen-DAP~C~i-LE~l50) ~50~ 5 5 5 4 .

.

-Tolerance Induction Properties of the Conjuqates -of the Invention The tolerance capacity of Pen-DAP-CM-levans was examined Eirst by injecting them into mice 2 and 3 weeks before the immunisation scheme.
CBAT6T6 and DBA/2 inbred strains of mice, bred by the Immunobiology Department of the Wellcome Foundation Limited, were immunised as shown in Figure 1. The immunogen (penicilloyl oralalbumin-Pen-OV) was mixed with aluminium sulphate containing 0.02% phenol red as indicated and precipitated with sodium hydroxide just prior to injection. Immediately after precipitation, _. pertussis vaccine was added and the volume made up with saline to give 0.2 ml/mouse.
Polysaccharides were injected i.v. (0.2 ml/mouse) diluted in phosphate-buffered saline. DBA/2 and CBAT6T6 mice respond to high and low doses of immugen respectively.
To stimulate the production of IgE antibodies 300 larvae of Nippostrongylus brasiliensis were injected subcutaenously in the back of the neck (0.2 ml/mouse).
The modified Jerre PFC (plaque forming cell) assay (Immunology 23, 843, 1972) was used to determine direct (IgM) splenic PFC specific for Penicilloyl ~.

. .

and levan determinants. Estimation of IgE titres was carried out by titrating each serum independently.
The animals were bled only once and serial dilutions of the sera (50 ~l samples) injected i.v. in the skin of Wistar rats for PCA.
Heterologous cell transfer (HCT~ was measured by the method of Kind & Scbrinho (J. Immunol. 111, 638, 1973). Duplicate 50 and 100 ~1 samples of washed spleen suspensions in 199 medium (Burroughs Wellcome) were injected into the skin of Ag~B5 HO
rats. Twenty-four hours later the animals were challenged in the same way as for PCA reactions and the diameter of the blue spots measured. The number of spleen cells secreting IgE antibodies specific for Pen or OV was assumed to be proportional to the area of the blue spots.
The results in Table 3 indicate that 1 mg doses ofPen-(l)CM-LE and Pen-DAP(2)CM-LE suppressed severely the IgE anti-Pen titre (PCA3 measured 2 and
3 weeks after boost with Pen-OV. IgM and IgG PEC~-spleen were very low in the immune controls. Both polysaccharide derivatives slightly increased the former and decreased the latter.
To demonstrate that the suppression reflected a diminished number of cells synthesising IgE

~. .

antibody, rather than peripheral neu-tralization, the immune response was measured in the spleen of the same animals (Table 3, HCT). The size of the skin reaction in this heterologous transfer is a reflection of the number of cells secreting IgE antibodies, and it is clear from the data presented that tolerance induction resulted in a substantial decrease of spleen cells forming anti-Pen antibodies of the IgE class. A similar experiment was performed using CBA mice, but this time the tolerogen was given after priming, to avoid the problems resulting from the fact that CBA mice had to be immunized several times with low doses of antigen (see Figure 1) to achieve immunity.
At the time of tolerogen injection, anti-Pen PCA
titres were 1/10. Results of such experiments are presented in Table 4. It is clear that suppression of the immune response also took place in this strain even when tolerogen was given after priming. As with DBA/2 mice the low PCA titre corresponded with smaller numbers of anti-Pen IgE cells in the spleen.
Whether the induction of tolerance could be achieved in mice already showing a high reaginic response was tested according to the following experimental scheme: DBA/2 mice primed, infected .'"' ' . ' ' .

- 2 0 - A5~6 and boosted as described in Figure 1 were tested for anti-Pen Ig~. On day 33 they were all allergic (X -- l/42). On day 47, PCA titres wers low (3 positives out of 5, PCA titre 1/23). Subsequently (day 55) mice were divided into 3 groups, the first treated wikh Pen-DAP(l)-CM-L~" the second with Pen-DAP(2)-CM-LE and the third left as control.
Two weeks later they were all boosted in the same way. PCA, HCT, IgM and IgG PFC were measured ]4 days after the boost (see Table 5). The results clearly indicate that the highly substituted Pen-DAP(2)-C~-LE provo~ed a very marked tolerance but the more weakly substitu~ed Pen-DAP(l)-CM-LE
; only gave a partial, non significallt, suppression.
As in the previous experiments, very low spleen IgM
` and IgG PFC numbers were ound. l`he lack o~
responsiveness persisted after HCT of the spleen cells.
Preparation of Penicilloyl-diaminopropyl-carboxy _ _ met_ l-levan ~1) Preparation of carbox~nethyl-levan Purified levan (3 g) from COryne'GaCte;riUm levaniformis was disâolved in water ~150 ml) and _ mixed with 10 N sodium hydroxide (12 ml) and mono-chloroacetic acid (3 g)g stirred for 1 hour at room temperature and at 60C for 3 hours, neutralised (pH 7) Wit]l 5 N hydrochloric acid and dialysed at 4C for 3 days (checking the conductivity of t~.e water~. The material was frozen, dried and analysed for sugar and carboxylic groups (156-COONa/1000 fruc~osyl residues). The yield was 3.1 g.
(2) Preparation of diaminopropyl-carbox~nethyl levan A 2~ of carboxymethyl-levan (50 ml) was mixed ~ith l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (0.4 g~ in 20~ molar excess to the carboxylic groups. The pH was adjusted to 5-6 and a 50 times molar excess of preneutralised diamino-propane ~6.4 g) was added at once. The mixture was stirred at ~oom temperature for 24 hours and for the first 3 hours the pH was maintained bet~een 5 and 6.
This material was then dialysed at 4C against 0.01 N
sodium hydroxide first and water later, analysed for ree amino groups by ninhydrin and the purity checked by gel filtration (Sephadex*G-75). The yield was 1.~ g 11~ of the carboxy groups had coupled with diamino-propane.
Repetition of the example using different relative amounts of the carbodiimide and diamino-plopane alters l:he degree of substitution of the * tr ade mark diamino-carboxymethyl-levan.

(3) Preparation of penicilloyl-diaminopropyl-carboxymethyl--levan Diamino-carboxymethyl-levan (l g) obtained from (2) was dissolved in a 10% sodium carbonate solution (50 ml) and potassium benzylpenicillin (4 g) added and dissolved. The solution was kept for two days at room temperature and dialysed at 4C, frozen and dried (l g). A full substitution of the amino groups was obtained.

Preparation of Penicilloyl-triethylenetetra_ino-carboxymethYl-levan The above conjugate was prepared by exactly the same method as penicilloyl-diaminopropyl-carboxymethyl-levan, triethylenetetramino being substituted for diaminopropane.

Pre~Paration of Penicilloyl-triethylenetetramino-carbox~methyl-dextran The above conjugate was prepared in a similar manner to the levan analogue by substituting dextran (molecular weight about 2 x 106) for levan.

Penicilloyl-triethylenetetramino-carboxymethyl-levan and penicilloyl-triethylenetetramino-carboxymethyl dextran were tested in mice infected with Nippostro~lus brasiliensis as described previously for Pen-DAP-CM-LE and found to induce tolerance.

PreLaration o~ Penicilloyl-d]`.~ ino~ ~yl~carbo_ymethyl-dextran This was prepared as described above for the lcvan equivalent but substi.tuting dextran (mol.
wt. about 2 x 106) for levan.
Preparation of 4-Sulpllonamicloben~ diami.nopr~r~ -carbox~nethyl-levan To a solution of diaminopropyl-carboxymethyl-: levan ~1 g, prepared as described above) and 4-sulphonamidobenzoic acid (1 g) was added 1 ethyl-3 ~3~_dinet-hylaminopropyl)carbodiimide hydrochloride (10 g) and the pH then adjusted to 8-9 ~ith lN NaOH.
The solution was kept at ambient ~emperature for 2 days, the pH being maintained at the aforementioned value.
The reaction mixture wa.s then dialysed against water for 4 days at 4~C and freeze-dried to give product, (SABA-DAP-CM-LE).
Mice were injected wi.th 4-sulphonamidobenzoic acid coupled to chicXen ganuna globulin (SABA-CGG).
~le allergic response to ~SABA-CGG) în the mice showed cross-reaction wi.th sulphadiazine and sulphaguanidine as measured by inhibition of PCA reactions. A number of the mice were then tolerized with SA~A-nAP-CM-LE
and sulphamethoxazolc~ sulphagllan:;dine and SABA-ova].bumin injected into separate ~roups of tolerizedand untoleri.zed mice. The tolerized mi.ce survi.ved , .

- 23 ~ .

whilst the unl:ol.erized mice cl;ed. The EIgE
ant.ibody titres o:~ the tolerizecl mice were very low compared 'co those of the untolerized mice .
Different degrees of substitution can be obtained by changing the proportion of components and the degree o~ substi.tution o~ the dia.minopropyl-carboxymethyl-levan used.

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Table 4. Tolerogenicity of high mol. Pen-DAP-CM-Levans on IgE response of CBA mice*

Anti~Pen immune response~
Tolerogen 2 weeks aft:er boost 3 weeks after boost ~CAt PCA HCT~ (mm ) . _ Pen-DAP(l)-CM-NLE ~1/10 ~1/5 '1 Pen-DAP(2)-CM-NLE ~1/10 ~1/5 '1 Nil 1/63 (1.73)1/23 (2.2) 12.6 (1.59) . . .

Immunization acoording to Fig. 1 *Priming: days 0, 28 and 59.
Tolerogen: day 78 Infection (N.b): day 79 Boost: day 85 tGeometric average of 5 mice. Standard error in paren-thesis.
~Area of the blue spot (mm2).

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'~ ''

Claims (39)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A process for preparing a medicament-substituted hapten conjugate having a molecular weight greater than 20,000 and comprising a hapten selected from the group consisting of levans and dextrans, in which a number of the hydroxy groups are substituted by at least one chain of formula wherein X is selected from the group consisting of alkylene of 1 to 8 carbon atoms, hydroxy-substituted alkylene of 1 to 8 carbon atoms, and alkyleneamino of 1 to 7 carbon atoms, and Y is a medicament, or a derivative thereof, which causes an allergic reaction on administration and which contains at least one carboxy group or is modified to contain a carboxy group, said carboxy group in Y, and the amino group in said chain forming an amide linkage between said hapten and said medicament, or medicament derivative, which process comprises reacting said medica-ment, or derivative thereof, with a hapten selected from the group consisting of levans and dextrans in which a number of the hydroxy groups are substituted by a group of the formula (V):
-CH2-CO-NH-X-NH2 (V) wherein X is as defined above.
2. A process according to claim 1, wherein Y is a group of formula (IV):

(IV) or a pharmaceutically acceptable salt or ester thereof, wherein R is a hydrogen atom or an acyl side-chain con-ventionally linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins.
3. A process according to claim 2, wherein R is a phenylacetyl group.
4. A process according to claim 1 wherein Y is a group of formula or a pharmaceutically acceptable salt thereof.
5. A process according to claim 1, wherein X is a propylene group.
6. A process according to claim 2, wherein X is a propylene group.
7. A process according to claim 3, wherein X is a propylene group.
8. A process according to claim 4, wherein X is a propylene group.
9. A process according to claim 1, wherein the molecular weight is at least in the order of 106.
10. A process according to claim 1, wherein the hapten is a levan.
11. A process according to claim 2, wherein the hapten is a levan.
12. A process according to claim 3, wherein the hapten is a levan.
13. A process according to claim 4, wherein the hapten is a levan.
14. A process according to claim 10, wherein the levan is substituted by at least 90 Y groups per 1,000 fructosyl residues.
15. A process according to claim 11, wherein the levan is substituted by at least 90 Y groups per 1,000 fructosyl residues.
16. A process according to claim 12, wherein the levan is substituted by at least 90 Y groups per 1,000 fructosyl residues.
17. A process according to claim 13, wherein the levan is substituted by at least 90 Y groups per 1,000 fructosyl residues.
18. A process according to claim 1, wherein the hapten is a dextran.
19. A process according to claim 1, wherein Y is present in an amount effective for inducing tolerance to the medicament in humans.
20. A medicament-substituted hapten conjugate having a molecular weight greater than 20,000 and com-prising a hapten selected from the group consisting of levans and dextrans, in which a number of the hydroxy groups are substituted by at least one chain of formula wherein X is selected from the group consisting of alkylene of 1 to 8 carbon atoms, hydroxy substituted alkylene of 1 to 8 carbon atoms, and alkyleneamino of 1 to 7 carbon atoms, and Y is a medicament, or a derivative thereof, which causes an allergic reaction on administration and which contains at least one carboxy group or is modified to contain a carboxy group, said carboxy group in Y, and the amino group in said chain forming an amide linkage between said hapten and said medicament, or medicament derivative, whenever prepared by the process of claim 1, or by an obvious chemical equivalent.
21. A medicament-substituted hapten conjugated as defined in claim 1, wherein Y is a group of formula (IV) (IV) or a pharmaceutically acceptable salt or ester thereof, wherein R is a hydrogen atom or an acyl side-chain con-ventionally linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 2, or by an obvious chemical equivalent.
22. A medicament-substituted hapten conjugate, as defined in claim 1, wherein Y is a group of formula (IV) (IV) wherein R is a phenylacetyl group linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 3, or by an obvious chemical equivalent.
23. A medicament-substituted hap-ten conjugate, as defined in claim 1, wherein Y is a group of formula:

or a pharmaceutically acceptable salt thereof, whenever prepared by the process of claim 4, or by an obvious chemical equivalent.
24. A medicament-substituted hapten conjugate, as defined in claim 1, wherein X is a propylene group, whenever prepared by the process of claim 5, or by an obvious chemical equivalent.
25. A medicament-substituted hapten conjugate, as defined in claim 1, wherein X is a propylene group and Y is a group of formula(IV) (IV) or a pharmaceutically acceptable salt or ester thereof, wherein R is a hydrogen atom or an acyl side-chain conventionally linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 6, or by an obvious chemical equivalent.
26. A medicament-substituted hapten conjugate, as defined in claim 1, wherein X is a propylene group and Y is a group of formula:

(IV) wherein R is a phenylacetyl group linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 7, or by an obvious chemical equivalent.
27. A medicament-substituted hapten conjugate, as defined in claim 1, wherein X is a propylene group and Y is a group of formula or a pharmaceutically acceptable salt thereof, whenever prepared by the process of claim 8, or by an obvious chemical equivalent.
28. A medicament-substituted hapten conjugate, as defined in claim 1, having a molecular weight at least in the order of 106, whenever prepared by the process of claim 9, or by an obvious chemical equivalent.
29. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan, whenever prepared by the process of claim 10, or by an obvious chemical equivalent.
30. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan and Y is a group of formula (IV) (IV) or a pharmaceutically acceptable salt or ester thereof, wherein R is a hydrogen atom or an acyl side-chain con-ventionally linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 11, or by an obvious chemical equivalent.
31. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan, and Y is a group of formula (IV);

(IV) in which R is a phenylacetyl group linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 12, or by an obvious chemical equivalent.
32. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan and Y is a group of formula or a pharmaceutically acceptable salt thereof, whenever prepared by the process of claim 13, or by an obvious chemical equivalent.
33. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan sub-stituted by at least 90 Y groups per 1,000 fructosyl residues, whenever prepared by the process of claim 14, or by an obvious chemical equivalent.
34. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan substituted by at least 90 Y groups per 1,000 fructosyl residues and Y is a group of formula (IV) (IV) or a pharmaceutically acceptable salt or ester thereof, wherein R is a hydrogen atom or an acyl side-chain con-ventionally linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 15, or by an obvious chemical equivalent.
35. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan sub-stituted by at least 90 Y groups per 1,000 fructosyl residues and Y is a group of formula (IV) (IV) wherein R is a phenylacetyl group linked to the amino group attached to the 6-position in naturally occurring or semi-synthetic penicillins, whenever prepared by the process of claim 16, or by an obvious chemical equivalent.
36. A medicament-substituted hapten conjugate, as defined in claim 1, wherein said hapten is a levan sub-stituted by at least 90 Y groups per 1,000 fructosyl residues and Y is a group of formula or a pharmaceutically acceptable salt thereof, whenever prepared by the process of claim 17, or by an obvious chemical equivalent.
37. A medicament-substituted hapten conjugate, wherein Y is present in an amount effective for inducing tolerance to the medicament in humans, whenever prepared by the process of claim 19, or by an obvious chemical equivalent.
38. A process according to claim 1, wherein said hapten substituted by said group of formula (V) is prepared by reacting a hapten selected from the group consisting of levans and dextrans, in which a number of the hydroxy groups are substituted by a group of formula or a reactive N-acylating derivative thereof, with a com-pound of formula:

wherein X is selected from the group consisting of alkylene of 1 to 8 carbon atoms, hydroxy substituted alkylene of 1 to 8 carbon atoms, and alkyleneamino of 1 to 7 carbon atoms.
39. A medicament-substituted hapten conjugate, as defined in claim 1, whenever prepared by the process of claim 38, or by an obvious chemical equivalent.
CA309,347A 1977-08-16 1978-08-15 Hapten polysaccharide conjugates Expired CA1114812A (en)

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SE445013B (en) * 1979-06-21 1986-05-26 Landstingens Inkopscentral Means for preventing or treating infections by humans and animals
US4457918A (en) * 1982-05-12 1984-07-03 The General Hospital Corporation Glycosides of vitamins A, E and K
US5480642A (en) * 1982-12-20 1996-01-02 The Board Of Regents Of The University Of Nebraska Synthetic immunoreglators, and methods of use and preparation
US4719182A (en) * 1985-03-18 1988-01-12 Eastman Kodak Company Fluorescent labels and labeled species and their use in analytical elements and determinations
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US2768097A (en) * 1954-03-08 1956-10-23 Ohio Commw Eng Co Shaped articles comprising regenerated cellulose
US2880105A (en) * 1955-09-02 1959-03-31 Ohio Commw Eng Co Dextran containing sizing composition
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US3063906A (en) * 1960-09-13 1962-11-13 Central Pharmacal Company Benzyl dextran-amphetamine and method of making same
US3130126A (en) * 1962-09-14 1964-04-21 Central Pharmacal Company Oral pharmaceutical products prepared with carboxymethyl benzyl dextran having a low carboxymethyl d. s. of 0.15
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