CA1337431C - Sustained release compositions - Google Patents

Abstract

A novel sustained release composition is provided herein. The composition includes: (I) a biologically-active substance; and (II) a specific monomeric compound which is obtainable by reacting: (if required, in the presence of a Bronsted acid or a Lewis acid) (a) at least one acidic, neutral or amphoteric organic compound which contains one or more active hydrogen atom-containing groups and which may be otherwise substituted or unsubstituted, or which may be saturated or may be unsaturated, with a least one olefinic bond of (b) at least one olefinically unsaturated cyclic ether of the formula:

Description

This invention relates to sustained release compositions comprising materials having biological activity and a monomeric compound suitable for use as diluent , and more particularly, but not exclusively, to such compounds which are desirably liquids suitable for use as injectable diluents.
A defect of oral dosage forms of medication is patient failure; that is, the failure of the patient properly to adhere to the complete oral administration schedule prescribed. This failure is exacerbated for extended prescription and for prophylactic, rather than therapeutic, treatment. To overcome this defect, attempts have been made to administer materials having biological activity, for example, drugs, as a subcutaneous implant, for example, in the abdominal wall, or as an injectable preparation. The latter has the advantage that it can be administered by paramedical personnel.
It is also desirable that injectable preparations should be long acting. Diluents for such preparations should be neutral and stable; in particular, they should not react with the materials having biological activity to form toxic products.
Also, they should not be too viscous so as readily to pass through a hypodermic needle. Moreover, they should be inert and non-irritating to the tissue at the injection site; they should be essentially free from antigenic properties and be rapidly z5 absorbed from the injection site soon after the medication period is terminated leaving no residue.

B

Mineral oils have been used as a diluent in intramuscular injection, for example, of steroid hormones, but they can, on occasion, give rise to painful local reactions including anaphylactic irritation; moreover, the toxicological testing required for mineral oil preparations is both extensive and expensive. Silicone fluids have been used as diluents but it has been found that compositions containing such silicone fluids lose their efficacy com-paratively rapidly, for example, in three weeks.
The present invention seeks to provide a diluentcomposition which overcomes, or reduces, at least some of the above-mentioned disadvantages.
According, therefore, to a first aspect of this invention, a sustained release composition is provided which comprises: (I) a biologically-active substance; and (II) a monomeric compound which is obtainable by reacting, if required in the presence of a Bronsted acid or a Lewis acid, (a) at least one acidic, neutral or amphoteric organic compound which contains one, two or a plurality of active hydrogen atom-containing groups and which may be otherwise substituted or unsubstituted, or which may be saturated or may be unsaturated, with at least one of the olefinic bonds of (b) at least one olefinically-unsaturated cyclic ether of the formula:

R2 /~ n3 ~ O ~ x ~5 2a l 3 3 7 4 3 1 in which: Rl, R2, and R4, which may be the same or different, each represent a hydrogen atom or a substituted or unsubstituted hydrocarbyl or hydrocarbyloxy group; R3 represents a substituted or an unsubstituted methylene, ethylene or 1,3-propylene group; R5 represents a monovalent group reactive with the organic compound (a), or a hydrogen atom, or a Cl to C4 alkyl grou~; and X represents:

O
(CH2)~t C~c d in which: Y represents an oxygen atom or an -NR6-group, wherein R6 represents any of the values which R~ may assume;
a is O or 1; b is O or 1; and c is 1 or 2; d is O or 1;
with the proviso that at least one of b or d is 1.
In one variant thereof, (a) is unsubstituted, and in another variant thereof, (a) is saturated.
According to other variants: (a) may be an organic compound containing 1, 2 or a plurality of carboxy, hydroxy, amido or mercapto groups; or (a) may be a mono-carboxy or hydroxy aliphatic hydrocarbyl with less than 18 carbon atoms; or (a) may be a di-carboxy or hydroxy ali-phatic hydrocarbylene with less than 18 carbon atoms, or (a) may be octadecanol; or (a) may be a di-carboxy or hydroxy aliphatic hydrocarbylene with less than 18 carbon atoms; or (a) may be a C2 to C4 alkanol or alkandiol, e.g., octadecanol.

C

2b 1 33743 1 In some such variants, at least one of Rl, R2 or R4 may represent a hydrogen atom. In other such variants, R3 may represent a mono- or poly-substituted or unsubstituted ethylene group. In still other such variants, X may represent -COO- or CH20CO-.
According to other variants thereof, (b) may have the formula:

nl ~ / coo~5 or nl ~ / cll2ocoR5 in which: R " R2, R4 and R5 are as defined above; or (b) may have the formula:

--r COOC~I 2 in which: R'l, R'2, and R'4, which may be the same or different, represent any of the values which Rl, R2 and R4, respectively, may assume; or (b) may be acrolein tetramer;
or (b) may have the formula:

~ ' \ , " î ~a coocll2 ~,j .

t 33743 1 in which: R8 and R'8, which may be the same or different, each may represent a Cl to C6 alkyl group or a C2 to C6 hydroxyalkyl group; or both R8 and R'8 may be an octadecyl group.
In still another variant thereof, (II) may be a liquid at a temperature of from 35O to 40OC and at standard pressure.
In still another variant, (I) may be intimately admixed with the compound (II). In a still further variant, the composition may be a suspension, dispersion or solution.
According to all aspects of the present invention, the active substance (I) comprises a biologically-active sub-stance. Such substance may comprise a natural or syntheticsteroid hormone; or it may be one which is effective against a tropical disease; or it may be pyrimethamine; or it may be a natural or synthetic prostaglandin. In the sustained-release composition of aspects of this invention, at least part of organic compound (a) comprises a mono-, di- or polycarboxy, hydroxy or mercapto-substituted, biologically-active compound (I).
By another aspect of this invention, a novel compound is provided, which is of the formula:

~-~J~ /~ J-I~

C

2d l 33 7 4 3 1 in which: R8 and R'8, which may be the same or different, each represent a Cl to C6 alkyl group, or a C2 to C6 hydroxy-alkyl group; or in which both R8 and R'8 may be an octadecyl group.
By "unsaturated cyclic (thio)ether", as used herein, is meant a compound comprising at least one >C=C-0 or >C=C-S- group, wherein the, or each, oxygen (or sulphur) atom is a hetero atom and the, or each, double bond conjugated therewith is contained in the hetero ring.
Apart from the active hydrogen atom-containing functional group, the organic compound (a) may be substituted, for example, by one or more halogen, e.g., chlorine or bromine, atoms. Desirably, the substituent should not be basic and it is generally convenient to utilise unsubstituted organic compounds (a).
The organic compound (a) may be aliphatic, alicyclic or aromatic and may have olefinic unsaturation. It is generally convenient to utilise aliphatic, preferably saturated aliphatic, organic compounds (a); for example, mono-, di- or polycarboxy, hydroxy, amido or mercapto group-containing organic compounds, and preferably mono- or dicarboxy or hydroxy group-containing organic compounds.
The organic compound (a) may be monomeric. Where the derivative of an aspect of this invention is formable from a monomeric organic compound (a), that organic compound is preferably a mono- (or di-) carboxy or hydroxy C~ (or C2) to ClO aliphatic hydrocarbyl(ene), preferably a C2 to C4 alkan (di) ol.

1 33~431 The organic compound (a) may be polymeric. Where the derivative of this invention is formable from a polymeric organic compound (a), that organic compound is preferably a mono-, di- or poly- hydroxy homo- or co- poly (C3 to C5 alkylene oxide).
05 A mixture of a plurality of monomeric organic compounds (a) or of a plurality of polymeric organic compounds (a) or of at least one monomeric with at least one polymeric organlc compound (a) may be used.
Examples of mono-carboxy or hydroxy hydrocarbyls(a) include monocarboxyllc acids, phenols and alcohols, desirably those with less than 18, preferably less than ln, carbon atoms; for example, formic, acetic, propionic, butyric and valeric acids, benzoic and phenylacetic acids; phenol and cresols; methanol, ethanol, propanol, butanol and n-octanol. Alcohols are most suitable, especially the Cl to C6, preferably C2 to C4, alcohols.
Mixtures of monocarhoxylic acids, phenols and alcohols ~ay be used. Preferably the monocarboxylic acids, phenols and alcohols are linear.
Examples of di-carboxy or hydroxy hydrocarbylenes (a) include dicarboxylic acids, dihydric phenols, hydroxyacids and glycols, desirahly those with less than 18, preferably less than 10, carbon atoms; 1,2-cyclohexanedicarboxylic, 1,3-cyclohexanedicarboxylic and 1,4-cyclohexanedicarboxylic acids, phthalic, isophthalic and terephthalic acids, 4,4'-dihydroxypheny1-2,2-propane, resorcinol, quinol and orsinol, lactic, 2-hydroxyisobutyric, 10-hydroxydecanoic, 12-hydroxyoctadecenoic, 12-hydroxy-cis-9-octadecenoic, 2-hydroxycyclohexane carboxylic, 2-hydroxy-2-phenyl (D) propionic, diphenylhydroxyacetic, 2-hydroxybenzoic, 3-hydroxybenzoic and 4-hydroxybenzoic acids, glycol, propanediols and butanediols.
Glycols are most suitable, especially the C2 to C6, preferably C2 to C4 glycols. Mixtures of dicarboxylic acids, dihydric phenols, hydroxy acids and glycols may be used. Preferably the dicarboxylic acids, dihydric phenols, hydroxy acids and glycols are linear.
Examples of organic compounds (a) substituted by more than two carboxy or hydroxy groups include polycarboxylic acids, polyhydric _ 4 _ t 33743 1 phenols, hydroxy acids and polyhydric alcohols, deslrably those with less thsn 18, preferably less than 10, carbon atoms; for example aliphatic polyols such as glycerol, erythritol, pentaery-thritol, sorbitol, dulcitol, inositol, 2-ethyl-2-hytroxy-05 methylpropane-1,3-diol and 1,2,6-hexanetriol; aromatic polyols such as 1,2,3-trihydroxybenzene, 1,2,4-trihydroxybenzene, 1,3,5-trihydroxybenzene; araliphatic polyols; hydroxy aliphatic, alicyclic and aromatic carboxylic acids, including Krebs cycle acids, such as citric acid, malic acid, tartaric acid, 2-hydroxy-3-methyl (D) succinic acid, ascorbic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,5-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid. Mixtures of polycarboxylic acids, polyhydric phenols, hydroxy acids and polyhydric alcohols may he used. Preferably the polycarhoxylic acids, polyhydric phenols, hydroxy acids and polyhydric alcohols are linear.
Mixtures of mono-, di- and poly-substituted organic compounds (a) may be used to obtain the desired rheological and release properties in the diluent composition.
Desirably the mono-, di- or poly-unsaturated cyclic (thio)ether (b) has the formula:

R2~-rR
~, ~ ~\~X Rs in which:
Rl, R2 and R4, which may be the same or different, each represent a hydrogen atom or a substituted or unsubstituted hydrocarbyl or hydrocarbyloxy group;
R3 represents a suhstituted or unsubstituted methylene, ethylene or l,3-propylene group;
R5 represents a monovalent group reactive with the organic compound (a) or a hydrogen atom or a Cl to C4 alkyl group;

Q represents an oxygen or a sulphur ato~; and X represents:

- (C t~ ) O l ( C ) in which:
Y represents an oxygen atom or an -NR6- group wherein R6 5 represents any of the values which Rl may assume;
a is 0 or 1;
b is 0 or 1;
c is 1 or 2;
d is 0 or 1; 0 with the proviso that at least one of b or d is 1.
X may, as shown, represent any hydrolysable carboxylic acid ester, carbonate ester or oxalate ester function, or an amide analogue. Preferably, however, X represents -C00- or -C~20C0-.
Particularly preferred cyclic unsaturated ethers have the 5 formula:

R ~ Cc)o~ C - R ~< C 1~20C0~5 in which:
Rl, R2 and R4, which may be the same or different, and R5 are as herein defined. R5 may suitably represent any group provided that it is reactive with the organic compound (a). Preferred examples are vinyl (thio)ether and epoxy groups.

6 ~ 337431 It is particularly preferred that R5 represents a group derived from a cyclic unsaturated vinyl (thio)ether, especially of the formula:

R~
~ ~3~
f~4 Q

in which:
05 Rl', R2', R3' and R4', which may be the same or different, represent any of the values which Rl, R2, R3 and R4 may assume;
M represents the group -ZX'-;
~' represents an oxygen or sulphur stom;
X' represents any of the values which X may assume; and Z represents a single bond or a carbon-linked mono-, oligo-or homo- or co- poly(substituted or unsubstituted hydrocarbylene or hydrocarbyleneoxy) residue, such as a substituted or unsubstituted arylene, alkylene or alkylene oxide residue.
Especially preferred cyclic unsaturated ethers are the reaction products formed by subjecting one or a mixture of dihydropyran aldehydes to disproportionation by the Tischenko reaction; they have the for~ula:

~< Cooc ~ R' in which:
Rl', R2' and R4', which may be the same or different, represent any of the values which Rl, R2 and R4 respectively may assume. A
preferred such compound is acrolein tetramer (in which the Ri all represent hydrogen atoms).

_ 7 _ 133743~
Cyclic unsaturated ethers wherein X comprises a -C00- or -CH20C0- group may conveniently be prepared from the tetramer of the corresponding unsaturated aldehyde produced by the Tlschenko reaction; namely:

~,2.~CQ

05 Thus, cyclic ethers wherein X comprises a -C00- group may be prepared by reaction of the tetramer with an alcohol R50H using a transesterification catalyst and reaction conditions:

R~ ~'3 ~3 R~ ~ R~ R;, ~3 + QsOH ~ ~ ¢ ~ ~ + R ¢~R~
o ~c~oco^~O ~Rt O ~oDRs I o CH~o~

Cyclic ethers wherein X comprises a -CH20C0- group ~ay be prepared by reaction of the tetra~er with a lower alkyl carboxylic acid ester R5C00 R7 in which R7 represents a lower alkyl group using a transesterification catalyst and reaction conditions:

R R3 ~3 R~ R R3 ~ ~3 ~ + ~5~~7 R~ r, ,<~ oco~R I ~O ~ CooR 7 The respective by-products may also be transesterified with R5C00 R7 or R50H to give, correspondingly:

R L ~ ,, R3 ~o ~CI~oco~5 ~>~Coo~5 The corresponding amides may be prepared analogously.
Cyclic ethers where X comprises a -C00- group may also he 05 obtained by mild oxidation of the dimer of the correspondin~
unsaturated aldehyde, followed by esterification of the salt, for example the silver salt.
Meta-carbonates and oxalates may be obtained, respectively, by esterification:

- ~< R } ~ ~ oG~

(Chlorofor~ic esters, such as ethyl chloroformate, may also be used~.

R~ ~ ~ L4 ~ ~ t(~O~7~ ~~ R ~

It is, however, to be stressed that acrolein tetramer is readily prepared fro~ acrolein which is a commercially available material; can readily be purified; and has heen found to he satisfactory in the practice of this invention.

- 9 - t337431 Particularly preferred derivatives of the invention are those wherein the derivative has the formula:

/

coOcll2 - in which:
R8 and R'8, which may be the sa~e or different, each represent 05 a Cl to C6 alkyl group, a C2 to C6 hydroxyalkyl group or a ho~D-or co- poly(propylene or butylene oxide).
The mono-, di- or poly-unsaturated cyclic (thio)ether (b) ~ay also desirably have the formula:
1~_ R3 in which:
Rl, R2, R3, R4, R5 and Q are as hereinabove defined; and X"
represents:

- (~ H~ ( C )~(Y~C

in which:
Y represents an oxygen atom or an -NR6- group wherein R6 represents any of the values which Rl may assume;
a is O or l;
b is O or l;

- -lo- 1337431 cisOor 1;
with the proviso that b + c is 1.
The mono-, di- or poly-unsaturated cyclic(thio)ether (b) may also comprise the aldol condensation product of at least one dimer of the corresponding unsaturated aldehyde:

R, R3 - R2 ~ ~CHO

namely:

Rl~ ~CHOH ~ ~ R'l ; or OHC
", CH ~ R' CHOH

,.

RIR~Q/~R"4 CH
A

The mono-, di- or poly-unsaturated cyclic (thio)ether (b) may further comprise the Diels-Alder product of reacting at least one unsaturated aldehyde with a polyvinyl ether of a polyhydroxy compound:

R~ R3 R2 ~ ~ O R9 n wherein:
R9 represents an n-valent hydrocarbon or poly(oxyhydrocarbon) residue of an n-hydroxy compound.
the polymerisable cyclic (thio)ether (i) may further comprise R~ R3 " R2 ~ ~\~ - Rlo n wherein:
Rlo represents an n-valent non-hydrolysable bridging residue.
These last four depicted compounds give rise to compositions of the invention which are less readily hydrolysed. Their use gives a first method for providing a controlled release composition according to the invention with a desired rate of hydrolysis.
The degree of hydrolytic stability provided can also be accurately tailored by anumber of other different mechanisms. Thus, homologues of acrolein tetramer, for example methacrolein tetramer or the mixed acrolein/methacrolein tetramer, exert steric hindrance, which increases as the size of R4 increases, to the hydrolysable ester or amide function X and thereby increases the stability of the derivative. Mono-, di and X

poly-carboxy and phenolic hydroxy suhstltuted hydrocarbons form derivatives which are prone to hydrolysis and which yield catalytic acidic species thereon; derivatives wherein X and X', which may be the same or different, each represent a -COO- or -C~20C~- group 05 and Z represents a substitued or unsubstituted hydrocarbylene group also facilitate hydrolysis, Examples include:

Cl I~OCO~CH ~ COOCH2--~ o/l ~- C`t;Z'?Cc>(c~l2)~ooc~

I~L CH~OCO~COOC~-__,o,t Furthermore, derivatives which retain one or more carboxy or hydroxy ~roups comprise a relatively high level of water which itself facilitates hydrolysis.
The ~roups R1, R2 and R4 may each represent a substituted or unsubstituted hydrocarbyl or hydrocarbyloxy ~roup: examples include unsubstituted or halo-substituted C1 to C4 alkyl, such as methyl or ethyl; unsubstituted C6 to C10 aryl or arakyl such as phenyl or benzyl; and oxy analogues. In the case of R4, increase in the size of the group increases the steric hindrance to any hydrolysable ester or amide function and thereby lncreases the stability of the hydrogel. It is preferred, however, from the standpoint of ease of preparation and availability, that at least one, and preferably all, of Rl, R2 and R4 represents a hydrogen 1 33743 ~
_ 13 -atom. The group R3 may represent a mono- or poly-substituted ethylene group, preferably an unsubstituted ethylene group; that is, a dihydro(thia~pyran derivative.
Preferred such compounds (i) are ethers; that is, those 05 compounds of the above fo mula wherein Q represents an oxygen atom, especlally dihydropyrans.
A proportion of polyethylene glycol of the formula R11-(OC~2CH2~ -0~ wherein Rll represents a hydrogen atom or an alkyl group and n is a numher such that the number avera~e molecular weight, M , is less than 1000 may also be incorporated to facilitate hydrolysis.
The derivatives formed by reacting acrolein tetramer and C
to C12 monohydric alcohols are oils of relatlvely hi~h boiling point and low vapour pressure at ambient temperature. They have a relatively low viscosity (typically 100-300 cP at 20 to 25 C) which allows their administration by in~ection and, on cooling below -2n C, form colourless glasses. Their densities at 2~ C
are close to that of water with a progressive decrease as the alkyl chain length is increased. They are insoluble in water and body fluids.
~hile it is preferred, in accordance with this invention, that the derivative is a liquid at a temperature from 35 to 40 C and at standard pressure, solids are of interest as implant materials and in co-precipitation with active substances.
~oth solid and liquid derivatives may be extended with pharmaceutially- acceptable oils, such as olive oil and also with other derivatives of the invention of lower molecular weight in order to increase their plasticity or decrease their viscosity, and to alter the release properties of a sustained release composi-tion formed therefrom.
In accordance wlth a preferred feature of this invention, there is provided a derivative which is a monomeric adduct and wherein the molar ratio of (a) to (b) is no greater than n:l wherein n is the numher of ring double bonds in (b). There is also provided a derivative which is a thermoplastic polymer and wherein the molar ratio of (a) to (b) is no less than (n-l):l 14 l 337431 wherein n is the number of ring double bonds in (b) and n > 1. Preferably, the derivative is formed by the reaction of (a) with (b) in the presence of a Bronsted acid or a Lewis acid.
The composition provided by broad aspects of this invention, comprises a derivative as herein described and a biologically-active substance. It is preferred that the biologically-active substance be intimately admixed with lo the derivative as a suspension, dispersion or solution.
The biologically-active substance may be presented as a controlled release composition, in accordance with the teachings of Canadian Patent No. 1,242,144, suspended in a derivative of aspects of this invention.
Such compositions may, for example, on injection, provide a timed release (typically 1 to 7 days) of biologi-cally-active substance from a depot. It is preferred, however, that such compositions provide a sustai~ed release of biologically-active substance for 1 to 3 months or even longer. These differing rates of release may be brought about by tailoring the derivative as above-described or by forming a pro-drug as is hereinafter described.
The present invention, in its various aspects, is of broad applicability in the formulation of biologically-active substances releasable at a sustained rate. Examplesof classes of biologically-active substances which may be incorporated in the sustained-release compositions of aspects of the present invention include flavourings, pharmaceuticals, bacteriostatis, viruscides, pesticides, C

_ 15 1 337431 e.g., insecticides, nematicides, molluscicides, and larvi-cides, herbicides, fungicides, algaecides, topical or dermatological agents, antifoulants for marine growth prevention, proteins, for example, enzymes, peptides, microbiological and plant hydroculture salts and nutrients and preservatives, veterinary trace metal formulations, and other growth promoting factors used in animal husbandry, for example, antianaemia preparations, and anabolic steroids. Of particular interest are compositions of the present invention comprising, as biologically-active sub-stance, at least one pharmaceutical.
The compositions of aspects of this invention thus find wide application in medical and surgical, including veterinary, contexts and in horticulture and agriculture as well as outside these areas.
Specific classes of drug which may be utilised in a sustained-release composition of aspects of the invention include: abortiffacients, e.g., prostaglandins; hypnotics;
sedatives; tranquilizers; antipyretics; anti-inflammatory agents; preparations for the treatment of allergies, for example, anti-histamines; anti-tussives; anti-convulsants;
muscle relaxants; anti-tumour agents, for example, those for the treatment of malignant neoplasia; local anaesthe-tics; anti-Parkinson agents; topical or dermatological agents; diuretics, for example, those containing potassium, e.g., potassium iodide; preparations for the treatment of mental illness, for example, preparations containing lithium for use in the treatment of manic depression or 16 l 33743 1 containing prostaglandins for the treatment of schizo-phrenia; anti-spasmodics; anti-ulcer agents; preparations containing various substances for the treatment of infec-tion by pathogens including anti-fungal agents, for example, metronidazole, anti-parasitic agents and other anti-microbials; anti-malarial; cardiovascular agents;
preparations containing hormones, for example, androgenic, estrogenic and progestational hormones, notably steroids, e.g., oestradiol; sympathomimetic agents; hypoglycaemic agents; contraceptives; nutritional agents; preparations containing enzymes of various types of activity, for example, chymotrypsin; preparations containing analgesics, for example, acetylsalicylic acid; and agents with many other types of action including nematocides and other agents of veterinary application. Mixtures of active sub-stances may be incorporated into the sustained-release compositions of aspects of this invention.
The sustained-release compositions of aspects of this invention may be used as a contraceptive composition suit-ably containing, as active substance, at least one natural or synthetic steroid sex hormone, for example, an oestrogen or progestogen. Suitably progestogens include the natural progesterone and its synthetic analogues, including 11-dehydroprogesterone, delalutin, 21-fluoro-17-acetoxy-6-a-methylprogesterone, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, ethisterone, dimethister-one, A-norprogesterone, 19-norprogesterone, 21-norproges-terone, normethandrone, norethynodrel, norethindrone and ~' -_ 17 l 337431 its acetate, DL- and D-norgestrel, norgestrienone, ethyno-diol diacetate, lynstrenol, ethynylestradiol, retropro-gesterone, dydrogesterone, norvinodrel, quingestranol acetate, norethisterone and its acetate and oenanthate, anagesterone acetate, medrogestone, clomagestone, allyl estrenol and cingestol; but preferably progesterone.
Suitably, oestrogens include the natural ~-oestradiol and its synthetic analogues, principally ethinyloestradiol or mestranol; but preferably ~-oestradiol.
The sustained-release compositions of aspects of this invention are also useful in the treatment of diabetes and pernlclous anaemia where, for example, the controlled release of insulin and cobalamin, respectively, may be utilised.
Moreover, the sustained-release compositions of aspects of this invention are particularly suited to treat-ment, both prophylactic and therapeutic, of tropical diseases, for example, malaria, leprosy, schistosomiasis and clonorchiasis. Examples of drugs which can be used as biologically-active substance in sustained-release composi-tions of aspects of this invention for the treatment of these and other tropical diseases include quinine, sulphon-amides, rifamcin, clofazimine, thiambutosine, chlorphenyl derivatives, chlorguamide, cycloguanil, pyrimethamine, sulphadiazine, trimethoprim, quinoline derivatives, e.g., pamaquine, chloroquine, chloroquine, pentaquine, primaquine and amodiquine, pararosaniline, sulphametizole, quinacrine, dapsone, sodium sulphoxone, sulphetrone, sodium hydnocar-pate and sodium chaulmoograte. Drugs of particular effec-tiveness are cycloguanil, pyrimethamine and sulphadiazine.
Antibiotics, e.g., tetracycline (both as free base and hydrochloride or a mixture thereof), have also been found to be efficacious in the treatment of tropical diseases in compositions according to aspects of this invention.
The sustained-release compositions of aspects of this--invention are also very well suited to veterinary appli-cations. Examples include liquid depot preparations of antibiotics for general antibacterial activity and also in the treatment of anaplasmosis in cattle; preparations for provision of a wide spectrum of activity against both ecto-parasites, for example, termites and endoparasites including arthropods, arrested larvae stages of nematodes, lungworms and general strongyles, e.g., avermectins;
preparations for provision of activity against tremotode, cestode and roundworm infections, e.g., amoscanate and praziquantel; preparations for provision of activity against theileria in cattle, e.g., biologically-active naphthoquinones, e.g., menoctone; preparations for provi-sion of activity against babesiosis in cattle, horses and dogs, e.g., berenil, amidocarb and diampron; and prepara-tions for provision of activity against liver fluke in sheep and cattle and against Haemonchus species, e.g., closantel.

In accordance with a particularly preferred feature of aspects of this invention, a pro-drug composition is pro-vided wherein at least part of the organic compound (a) comprises a mono-, di- or polycarboxy, hydroxy or mercapto-substituted biologically-active compound.
Examples of such biologically-active compounds include hydroxylated steroids, e.g., norethisterone or laevonor-gestrel; prostaglandins, e.g., PGEI, PGFIa, PGE2, PGF2a, PGE3, PGF3a, 15-methyl PGE2, 16,16-dimethyl-PGE2, 16-phenoxy-17,18,19,20-tetramer-PGE2, 16,16-dimethyl-trans-~2-PGE, and 16-(3-trifluoromethylphenoxy)-17,18,19,20-tetranor-PGF2a;
and acetylsalicylic acid. Certain of the previously-men-tioned substituted hydrocarbons are themselves biologi-cally-active, for example, alkyl phenols.
Where all of the organic compound (a) is a biologi-cally-active compound, the pro-drug composition may be a solid. Where some, preferably at least an equimolar amount, of the organic compound (a) is a mono-, di- or polycarboxy, hydroxy or mercapto-substituted hydrocarbon as described above, for example, a trihydric, dihydric or monohydric alcohol, and, preferably, where the above-described molar ratios apply, the pro-drug composition may be a hydrophobic or hydrophilic liquid. In this manner, not only may a solid biologically-active compound be con-verted to a liquid formulation, but it amy also have its lyphilicity tailored to the pharmaceutical needs.

~ 33743~
A solid pro-drug in accordance with aspects of the invention may be dissolved or dispersed in a derivative of aspects of the invention or in a mixture of such deriva-tives with each other and/or one or more conventional injectable diluents, for example, olive oil. A liquid pro-drug in accordance with aspects of the invention may be master-batched and then diluted with a derivative of an aspect of the invention or with a mixture of such deriva-tives with each other and/or one or more conventional injectable diluents, for example, olive oil. Liquid pro-drugs also act as surface active agents for their conjugate biologically-active compound in the conjugate derivative.
In this manner, substantial quantities of insoluble or poorly soluble biologically-active compound may be homo-geneously dispersed in derivatives according to aspects of this invention. Other biologically-active compounds, and even other pro-drugs, may be incorporated in such composi-tions.
Where the biologically-active compound includes an amino or amido group, especially an aliphatic amino or amido group, and where the derivative includes a carboxylic ester group, that ester group could be replaced by an amido group through which the biologically-active compound is combined in a pro-drug. This may obtain even when all the substituted hydrocarbon is not a biologically-active compound, or when at least a part of the substituted hydro-carbon is the same or a different biologically-active com-pound.

.C

20a In accordance with a further aspect of this invention, - a sustained release composition is provided for use in prophylactic, therapeutic or contraceptive methods of treatment of an animal or human patient.
This invention also provides a process for the preparation of a derivative, especially a liquid diluent derivative, which process comprises reacting at least one monomeric or polymeric, substituted or unsubstituted, saturated or unsaturated, mono-, di- or poly- and, neutral or amphoteric active hydrogen atom - containing organic compound with at least one mono-, di- or poly- unsaturated (thio)ether in the presence of, if required, a Bronsted acid or a Lewis acid.
The process of this aspect of this invention may be effected at a temperature from -70 to 120C, but advan-tageously the process is effected at an elevated tempera-ture, e.g., from 50 to 110C, suitably from 60 to 100C, and especially from 70 to 90C. Ambient or autogenous pressure may be used.
Suitable Bronsted acids and Lewis acids include strong mineral acids which are not redox reagents under the reac-tion conditions, e.g., hydrochloric acid, sulphuric acid, tosylic acid, brosylic acid and the adduct of methanol and boron trifluoride. Suitable Lewis acids include boron trifluoride and its etherate, boron trichloride, boron tribromide, aluminum tetrachloride, aluminum tribromide, gallium trichloride, germanium tetrachloride, tin tetrabro-mide, zinc chloride and ferric chloride; ferric chloride is C

20b 1 33743 1 preferred. From 0.01% to 2%, particularly from 0.04% to 1%, based on the total weight of the reaction mixture may suitably be used.
Where the, or each, organic compound (a), for example a mono-, di- or poly-carboxy, hydroxy or mercapto hydro-carbon, is insoluble, as is often the case with polycar-boxylic acids, in the cyclic unsaturated (thio)ether, or mixture thereof, it is necessary to use a solvent, rather than mass, reaction theatre.
Where a plurality of organic compounds (a), for example, mono-, di- or polycarboxy, hydroxy or mercapto hydrocarbon, is used, stoichiometry and addition sequence is important. For example, to prepare the ethyl propyl, or propyl butyl mixed acetal of acrolein tetramer, it is pre-ferred to react the acrolein tetramer with an equimolar amount of one of the alcohols (for example, the ethanol or propanol, respectively); and then to react that product with an equimolar amount of the other alcohol.
Likewise, three moles of acrolein tetramer react with one mole of glycerol to give:

Coo~ , ~OC~2 C~ C~O~ Cit ~ ~_ C oOC H2 J\ i--O C 1~2 C

20c 1 33743 1 which can then be reacted with three moles of an alcohol to provide an oil of aspects of the invention.
Where the biologically-active substance includes a free amino group, and is soluble in the reaction mixture, this can prevent the reaction. Where, therefore, such biologically-active substance is incorporated in the com-position of an aspect of this invention before the process is completed, it has either (i) to be rendered insoluble or (ii) to be converted to a soluble quaternary ammonium salt, either of which may be effected in situ.
It may be desired to incorporate the active substance before the process is completed (iii) where it is intended that the active substance, as the organic compound (a) is incorporated into the derivative as a pro-drug as aforemen-tioned, or (iv) where the viscosity of the final composi-tion would be too high to allow satisfactory dispersion of the active substance therein. In the latter case, a quan-tity of base sufficient to neutralize the remaining acid or acid catalyst is added; the active substance admixed there-with; and the process continued with a fresh quantity of catalyst.
The following examples illustrate the invention.
Acrolein tetramer (ex Canadian Industrial Ltd.) was carefully triple distilled under reduced pressure with a dry nitrogen bleed. That middle fraction which distilled between 78 and 80C was collected and sealed under an inert atmosphere into small glass ampoules ready for use in the Examples.

C

All liquid reactants which could be distilled without risk of decomposition were redlstilled, collectet and sealed under an lnert atmosphere prior to use. Glycols were dried by being main-tained, at ambient temperature, for 4 hours under reduced pressure 05 with a dry nitrogen bleed and sealed under an inert atmosphere.
Anhydrous ferric chloride (ex Aldrich) was sublimed in a cold finger condenser to remove any trace water.

7.86g of acrolein tetramer where added to 2.24g of dried methanol (ex Aldrich) containing 0.1Z w/w anhydrous ferric chloride. The mixture was then maintained ln a sealed vessel at 70C for 1.5 hours.
A clear oil resulted which was qualitatively more viscous than the starting acrolein tetramer. The oil gave no charac-teristic >C = C< IR absorption at 1645cm indicating that complete reaction of the acrolein tetramer double bonds had occurred.

38.85g of acrolein tetramer were added to 11.10g of dried methanol containing 0.1~ w/w anhydrous ferric chloride. The mixture was then maintained in a sealed vessel at 75 C for 2.5 20 hours.
A clear, orange oil resulted which gave no characteristic >C = C< IR absorption at 1645 cm 1.

140.99g of acrolein tetramer were added to 58.02g of dried ethanol containing 0.1~ w/w anhydrous ferrlc chloride. The mixture 25 was than maintained in a sealed vessel at 80C for 2.5 hours.
A clear, pale brown oil of density 1.075gcm 3 and viscosity of 130cP at 21C resulted. The oil gave no characteristic >C = C<
IR absorption at 1645cm 1.47g of acrolein tetramer were added to 3.00g of dried 30 ethanol containing O.lZ w/w anhydrous ferric chloride. The mixture was then maintained in a sealed vessel at 80C for 7 hours.

A clear, pale brown oil resulted which gave no characteristic >C ~ C< IR absorption at 1645cm 1, EXAMPL~ 5 30.50g of acrolein tetramer were added to 14.60g of dried ethanol containing O.IX w/w anhydrous ferric chloride. The oS mixture was then maintained in a sealed vessel at 75C for 2.5 hours.
A clear, pale brown oil resulted which gave no characteristic >C - C< IR absorption at 1645cm 1.

0.415g of anhydrous ferric chloride was dissolved, at room temperature, in 41.50g dried ethanol to form a 0.1~ w/w solution.
lOOg of acrolein tetramer were then gradually added, with stirrlng, to the ethanolic solution. The clear yellow liquid so formed was first heated for 0.5 hours at 50 to 60 C and then heated for 3 hours at 80 C. A clear viscous yellow oil resulted which gave no characteristic >C ~ C< IR absorption at 1645cm 1, This oil was next purified by first neutralising the acidic catalyst by passage through a basic alumina column and thereafter distilling under vacuum in a Kugalruhr distillation unit to give a clear viscous colourless oil (bp 170 to 175 C at O.lm~Hg). The product is believed to have the formula:

H3C CH20 O COOCH2 ~ 0CH2CH3 with which the analysis is consistent:-calculated (~) found (~) gravimetric: C 60.73 60.76 H 8.91 9.00 IR: 1075. 1061 and 1031cm 1 bands of tetrahydropyran No bands in the hydroxyl absorption region No bands in the olefinic unsaturation absorption region -- ~ 33743~

H-NMR~ 1.3 -c~3 1.5 - 1.9 -CH2 - (ring) 3.3. - 4.2 -CH2 - (ester and ethyl) 4.4 - 4.6 ) -CH<
4.8 - 5.0 45.39g of acrolein tetramer were added to 30.00g of dried n-butanol containing O.lZ w/w anhydrous ferric chloride. The mixture was then maintained, with continuous stirring, at 80 C for 70 minutes.
05 A clear, pale brown oil of density 1.020gcm 3 and viscosity of 125cP at 21 C resulted. The oil gave no characteristic >C = C<
IR absorption at 1645cm 1.

43.39g of acrolein tetramer were added to 30.00g of dried butan-2-ol containing O.lX w/w anhydrous ferric chloride. The 10 mixture, with continuous stirring, was then maintained in a sealed vessel at 80 C for 4 hours.
The resulting oil gave no characteristic >C - C< IR
absorption at 1645cm 23.10g of acrolein tetramer were added to 26.90g of dried lS n-octanol containing 0.1% w/w anhydrous ferric chloride. The mixture was then maintained in a sealed vessel at 80 C for 2.5 hours.
A clear, pale brown oil of density 0.96gcm 3 ant viscosity of 170cP at 22 C resulted. The oil gave no characteristic >C ~ C<
20 IR absorption at 1645cm 1.

1.72g of acrolein tetramer were added to 2.00g of dried n-octanol containing 0.1~ w/w p-toluenesulphonic acid. The mixture was then maintained in a sealed vessel st 80 & for 6 hours.
A clear, pale straw-coloured oil resulted which gave no characteristic >C ~ C< IR absorption at 1645cm 1.

16.455g of acrolein tetramer were added to 27.334g of melted n-dodecanol containing 0.1~ w/w anhydrous ferric chloride. The mixture was then maintalned in a sealed vessel at 80C for 4 hours.
A c]ear, pale brown oil of density 0.944gcm 3 and viscosity 05 of 180cP at 22C resulted (mp -7C). The oil gave no charac-teristic >C = C< IR absorption at 1645cm 1.

4.587g of acrolein tetramer were added to 11.002g melted n-octadecanol containing 0.1 w/w anhydrous ferric chloride. The mixture was then maintained with continuous stirring in a sealed vessel at 100C for 0.5 hours.
The solid product (mp 39C) gave no characteristic >C = C< IR
absorption at 1645cm 1.

1.252g of acrolein tetramer were added to 1.11lg of dried cyclohexanol containing 0.1~ w/w anhydrous ferric chloride. The mixture was then maintained at 80C for 1 hour after which almost complete conversion had occurred; however, after a further 4 hours at 80C some IR absorption at 1645cm 1 remained.

15.3204g of 1,4-butanediol (ex Aldrich) were mixed with 0.0534g of anhydrous ferric chloride and left in an oven for 2 to 4 hours at 80C, with occasional stirring, to form a 0.1~ w/w solution.
38.1225g of acrolein tetramer were then added dropwise, with warming and stirring, to the solution at 80C and the reaction mixture became homogeneous within a short time. The resulting clear brown, viscous liquid was then maintained at 80C for 3 to 4 hours until no characteristic >C = C< IR absorption at 1645cm 1 occurred.

Proceeding in essentially the same manner described ln Example 14, 1,4-butanediol and acrolein tetramer were reacted together in the non-stoichiometric ratios shown in Table 1.

~ 1 337431 mole ratio 1,4-butane-diol: wt wt anhydrous acrolein acrolein wt 1.4-butane- ferric Example tetramer tetamer (g) diol(g) chloride(g) - 15 1: 1.25 38.1225 19.108 0.0572 16 1: 1.50 35.8800 21.6288 0.0575 17 1:1.20 29.1525 23.4312 0.0526 Procee~ing in essentially- the same manner as described in Example 14, 1,4-butanediol, ethanol and acrolein tetramer were reacted together in the stoichiometric ratios shown in Table 2.

mole ratio Weight acrolein Weight anhydrous tetramer: 1,4- acroleinWeight Weight ferric butanediol: tetramer l ,4-butanediol ethanol chloride ~xample ethanol (g) (g) (g) (g) 18 3:2:2 40.365 10.814 5.528 0.0579 19 2:1:2 40.365 8.111 8.294 0.0568 3:1:4 40.365 5.407 11.057 0.0568 38.250g of polypropylene glycol 425 (ex Aldrich) were heated to 80C and mixed with 0.0584g of anhydrous ferric chloride to form a 0.1% w/w solution. 20.183g of acrolein tetramer were then gradually added dropwise, with warming and stirring, over a period of 0.5 hours. The resulting light yellow, viscous liquid was then maintained at 80C until no characteristic > C = C ~ lR absorption at 1645cm~l occurred.

EXAMPLES 22 to 24 Proceeding in essentially the same manner as described in Example 21, polypropylene glycol 425 and acrolein tetramer were heated together in the non-stoichiometric ratios shown in Table 3-mole ratio Weight Weight I Weightacrolein acrolein polypropylene anhydrous tetramer: tetramer glycol 425 ferric chloride polypropylene (g) (g) (g) Example glycol 425 22 1:1.25 17.940 42.500 0.0604 23 1:1.50 15.968 44.625 0.0603 24 1:2.00 11.225 42.500 0.0537 Proceeding in essentially the same manner as described in 05 Example 21, pluronic 72 (M 2750) and acrolein tetramer were heated together in stoichiometric ratio.
The number average molecular weights (Mn) and the glass transition temperatures (Tg) of the polymers of Examples 14 to 25 are shown in Table 4.

Example Mna Tg& b _able 4 (Cont'd) Example Mna Tg& b a Determined by vapour phase osmometry b Determined by DuPont 990 differential scanning calorimeter In the following experiments several derivative oils of this invention, prepared as aforesaid, were evaluated and compared for 05 suitability as matrixes for active substances in the preparation of injectable depot antimalarial systems.
The following key correlates the derivative notation with the preparative Example:
S1 Example 2 S9 24 S17 25 S2 Example 5 S10 14 S3 Example 9 S11 15 S4 Example 11 S12 16 S5 Example 7 S13 17 S6 Example 8 S14 18 S7 Example 22 S15 19 S8 Example 23 S16 20 Depot preparations were made up by adding, by hand mixing, powdered active substance to the respective oil at 30Y wtw.

Twenty-eight groups of mice, 5 mice per group, were selected at random from pooled Tuck TFW male white mice of approximate body weight 20g. The mice were provided with food and water ad libitum.
Care was taken that the diet contained adequate levels of p-amino-benzoic acid and that the mouse stock was free from lnfection with ~perythrozoon coccoides.

On day 0 the groups of mice were processed as follows:-Groups (cages) no. 2, 9, 16 & 23 - all mice recieved 50mg Sl subcutaneously (sc) " 1, 8, 15 & 22 - all mice received 50mg Sl +
05 pyrlmethamine (sc) " 4, 11, 18 & 25 - all mice received 50mg S2 sc " 3, 10, 17 & 24 - all mice received 50mg S2 +
pyrimethamine ( 6C ) " 6, 13, 20 & 27 - all mice received 50mg S3 sc " 5, 12, 19 & 26 - all mice received 50mg S3 +
pyrimethamine (sc) " 7, 14, 21 & 28 - all mice were employed as untreated controls Subcutaneous injections were admlnistered in the mid dorsal, suprascapular position. Cages were exmained daily and all deaths recorded. Mice were examined for evidence of local irritation at the site of injection.
On day 7, groups (cages) 1 to 7 inclusive were infected with Plasmodium berghei (N straln). 0.2ml of an inoculum containing 1 x 10 infected mouse erythrocytes was injected into the tail vein of each recipient mouse. The presence or absence of patent malarial infection was determined by blood film examination three days post infection and at intervals thereafter. Standard procedures were employed in the examination of Glemsa stained blood films and in the determination of psrasitaemia levels. On days 21, 32, 45 further groups of mice were infected as shown in Table 5 below. The method of infection and the techniques for assessment of parasitaemia were as described previously. Some groups in which no parasitaemia developed after the initial inoculation with P. berghel were subsequently reinfected on the days illustrated in Table 5.

Table 5 The time (in days~ after injection of derivative oil on which mice were inoculated with P. berghei ~N strain) Time (Day) Groups (caRes inoculated with P. berghei) 7 1, 2, 3, 4, 5, 6, 7 21 8. 9, 10, 11, 12, 13, 14 32 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27,28 48 24, 25 49 15, 17 63 22, 24 An apparent anomaly in Table 5 is the reinfection of groups 24 and 25 only 3 days after the initial infection with P. berghei.
05 These animals were reinfected since on day 3 post-infection no malaria parasite was observed in the blood of any mouse in group 24, which had received S2 ~ pyrimethamine, or group 25, which had rece~ved S2 alone. Group 28, the untreated control group infected on day 45, had a mean parasitaemia of 7.9X on that day and it was assumed that experimental error was responsible for the failure to infect group 25 and/or 24. Group 17, 18 and 21 may be co~pared with 24, 25 and 28. It should be noted that 3 days after initial infection, Group 18 possessed a mean parasitaemia of 11.67, and in the untreated controls, Group 21, the parasitaemia on that day was 12X, The result~ obtained in this experlment are pre~ented in Table 6. At 7 days after in~ection of the oils ~ pyrimethamine all three groups of mice, namely Groups 1, 3 and 5, were protected from P. berBhei challenge, whereas those groups which received oil alone, Groups 2, 4 and 6, and Group 7 succumbed to infection.
By 21 days post in~ection animals which had received S3 ~ pyri-methamine were not protected against malaria challenge but both Sl ~ pyrimethamine and S2 I pyrimethamine were still effective.
-Table 6 The susceptibility to lnfectlon with P. berghei of ~lce challenged at varying lntervals subsequent to 8C ln~ection with pyrimethamine (pyr) in the oll of thls lnventlon, or oll alone.
Dosages employed are glven ln the text.
Interval between Parasitaemia (~) on drugging and (days post infection) G _ p Dosed with infection (days) 3 7 10 17 1 S1 + pyr 7 0 0 2 S1 7 9.9 80*
3 S2 + pyr 7 0 0 4 S2 7 11.2 80*
S3 + pyr 7 0 0 6 S3 7 ~0.2 80*
7 - 7 8 ns 8 Sl + pyr 21 0 0 9 Sl 21 16.2 80*
S2 + pyr 21 0 0 11 S2 21 24 80*
12 S3 + pyr 21 19 74*
13 S3 21 18.8 80*
14 - 22.2 80*

Sl + pyr 32 0 4 25 16 S1 32 4.8 57 17 S2 + pyr 32 0 0 0 18 S2 32 11.6 56 19 S3 + pyr 32 11.2 32 S3 32 12.2 72 21 - 12.0 38 22 S1 + pyr 45 2.2 20.3 40 23 S1 45 10.3 55 ns 24 S2 + pyr 45, 48 0 0 0 S2 45, 48 0/8.8 16.4 80 Table 6 (cont'd.) Interval between Parasitaemla (~) on drugglng and (days post lnfection) Group Dosed withlnfection (days) 3 7 10 17 26 S3 + pyr 45 11.7 65 80 27 S3 45 10.5 81 80 28 - 45 7.9 82 80 S1 + pyr49 (reinfection) 6.5 49 17 S2 + pyr49 (reinfection) 0 0 Control - - 6.8 5.0 22 S1 + pyr63 (reinfection) 6.7 40 24 S2 + pyr63 (reinfection~ 0.4 0 Control - - 30 50 ns - no survivors * - estimated prasitaemia. 1 only survivor with marked ana- ia and very high parasitaemia.
At 45 days only that group of animals which had been injected with pyrimethamine in S2 (Group 24) were uniformly protected against challenge. In Group 22, 2 out of 4 mice remained negative by blood film ~r in~tion at 45 days and of the two surviving mice 05 from Group 15 which were reinfected on day 49, only 1 of the 2 developed a parasitaemia. Four mice from Group 24 (S2 + pyrl-methamine) were reinfected on day 63. Two of these mice had patent parasitaemias on day 3 post-reinfection but were oegative on day 4 and remianed negative through the period of examination 10 (11 days).
Mice from Groups 23, 25 and 27 were sacrificed and necropsied 54 days after the injection of 50mg of S1, S2 and S3, respectively.
No evidence of adverse tissue reaction to the oils was evident at the site of injection. The relative amounts of lnjected oil 15 remaining in individual mice demonstrated that little, if any, degradation or dispersion of the oil S3 had occurred in 54 days.
No signs of the in~ected oil were observed in 2 of 4 mice which received Sl and small discrete globules of an opaque brown 'fluid' were observed ln the remaining two mice. No obvious tlssue reaction was observed at the site of the residual injected material. Residues of the injected material were found in each of the 5 mice which had 05 received S2. The fluid appeared well tolerated with no apparent host reaction.
It should be noted that there is no direct correlation with the duration of effect of pyrimethamine/oil mixtures and the life of the oil itself. S3 appears, from the preliminary observations made above, to persist in vivo for a significantly greater period than S1 or S2 but the period of antimalarial protection afforded by the S3 oil + pyrimethamine was very much shorter than either of the others. S1 appears to persist for, or possess a life expectancy of, approximately 8 weeks and, whilst it was effective with pyrimethamine at three weeks, an intermediate level only was observed at six to seven weeks.
S2 appears to show considerable promise. It appears to be stahle for a surprisingly long time in vivo and remained at the site of injection as discrete globules. It also, with pyrimetha-mine, conferred protection on mice for a minimum period of 9 weeks.

This experiment was designed to extend the observationspresented above in Experiment 1. All basic experimental procedures, including the preparation of 30~ w/w dispersions of pyrimethamine base in S1, S2 and S3, were performed as described ahove. The primary objective was to obtain confirmation of the results obtained above whilst extending the period of observation. Since no adverse host reactions were observed to the oils (without pyrimethamine) and since they also lack any antimalarial activity the experimental design was simplif~ed by including only S2 as an oil without anti-malarial compound. Twenty-seven groups of mice, with 5 mice in each, were again selected at random from pooled male white mice (Tuck TFW) of 20gm body weight. Individual groups received 50mg injections sc of S1 + pyrlmethamine, S2 + pyrimethamine, S3 + pyri-methamine or S2 above. The distribution of groups is shown below:-Inoculum Groups S1 + pyrimethamine 1, 6, 11, 16, 20, 24 S2 + pyrimethamine 2, 7, 12, 17, 21, 25 S3 ~ pyrimethamine 3, 8, 13 S2 - 4, 9, 14, 18, 22, 26 Control, untreated 5, 10, 15, 19, 23, 27 At intervals of 14 days one group of mice from each of the different classes of inocula were infected by the intravenous in~ection of 1 x 106 mouse erythrocytes parasitised with P. berghei (N strain~. Details are given in Table 7.
Table 7 05 The time (in days) after injection of derivative oil and pyrimethamine mixtures at which mice were infected with Plasmodium berghei (N strain~

Time (days) Groups (cages~ of mice infected with P. berghei 14 1, 2, 3, 4, 5 28 6, 7, 8, 9, 10 42 11, 12, 13, 14, 15 56 16, 17, 18, 19 20, 21, 22, 23 84 Z4, 25, 26, 27 The examination of mice for P. berghei infection was per-formed as described above in Experiment 1. The results are 10 presented in Table 8. These results demonstrate that 50mg of the oil S2, contsining a 30~ w/w dispersion of pyrimethamine base, when in~ected sc in mice afforded antimalarial protection for not less than 12 weeks.
S1 + pyrimethamine protected mice for between 6 and 8 weeks 15 but high mortality rates were obtained in some groups which received this mixture. S3 + pyrimethamine protected mice for less than 2 weeks.

The experiment is considered adequately to confirm the efficacy of S2 as a matrix for pyrimethamine in the preparation of injectahle sustained-release antimalarial systems.
Table 8 The susceptibility to infection with P. berghei of mice 05 challenged at varying times subsequent to injection sc with pyri-methamine (pyr) in derivative oil or with oil alone.
The dosages employed are given in the text.
Parasitaemia on (days post infection) Group Dosed withDay of challenge 314 7/8 10 1 Sl + pyr 14 0 0 0 2 S2 + pyr 14 0 0 0 3 S3 + pyr 14 50a 7la 6 Sl + pyr 28 0 0 0 7 S2 + pyr 28 0 0 0 8 S3 + pyr 28 b b b 11 Sl + pyr 42 0 0 0 12 S2 + pyr 42 0 0 0 13 S3 + pyr 42 b b b 14 S2 42 3.7 32 - 42 2.3 23 63 16 Sl + pyr 56 0.2 23 62 17 S2 + pyr 56 0 0 0 Sl + pyr 70 b b b 21 S2 + pyr 70 0 0 0 1 33743t Table 8 (cont'd.) Parasltaemia on (days post lnfection) Group Dosed withDay of challenge 3/4 7/8 10 24 S1 + pyr 84 4.5 33 S2 + pyr 84 0 0.02 0 26 S2 84 29.6 70 80 a - only one mouse surviving in this group b - all mice dead prior to challenge c - one mouse of 5 patent (parasitaemia 0.1~ day 8) This experiment was designed to extend, in relation both to other derivatives and other active substances, the observations presented above. All basic experi~ental procedures were as described above except that the P. berghei challenge was presented at 7, 14, 05 28, 42, 56 and 84 days after administration of active substance.
The results are presented in Table 9.
Table 9 DerivativeMinimum duration of protection (days) wlth:
oilpyrimethamine (free base) Sulphadiazine Sulphadoxine S1 42 14 < 2~ ~ 14 S2 84 70 C 84 < 28 S3 < 7 56 < 70 < 14 S4 < 28a < 14 < 28 S5 28 < 56a 42 < 28 S6 28 < 56a 42 < 28 a - Acute toxicity was associated with administration of this mixture.
These results demonstrate that no significantly prolonged duration of the effect of sulphadoxlne was obtained with any of the oils tested. Sulphadiazine gave extended protection when 10 blended with each of the oils, with the exception of S4.

Pyrimethamine gave extended protection with all oils except S3 and S4. The greatest duration of protection was obtained with S2. The injection of pyrimethamine blended in oils S3, S4, S5 and S6 was associated with manifestations of severe acute 05 toxicity which was sometimes fatal. This aspect is discussed further below.
Toxicity and tissue irritability of the derivative oils An extensive toxicological investigation of the oils, either alone or in combination with antimalarials, has not been undertaken.
The observations reported here are based on the behaviour and condition of mice immediately following injection of test substances, and on the development of any obvious gross pathology subsequent to drugging. All animals in experimental groups were examined at intervals for evidence of ulceration or other tissue reactions at the site of iniection and mice were killed at intervals and examined ~5 for residual oil deposits and evidence of adverse tissue reactions to such deposits.
Not one of the oils studied, when administered without drug, induced any apparent acute or chronic reaction in mice. Immediately following injection, even at lOOmg/mouse, the animals were normal in their behaviour and no adverse reaction was noted. The oils were extremely well tolerated by the tissues, no evidence of adverse reaction to any oil being observed.
Animals which received the oils with either sulphadiazine or sulphadoxine likewise developed no adverse response to the mixtures, either immediately after injection or on long-term observation.
No adverse response was observed to 50mg blends of pyrimethamine with Sl or S2 but when lOOmg of either blend was employed 1/5 mice died by 24 hours although no symptoms of acute toxicity were observed by 6 hours after drugging. A severe toxicosis developed within a short interval of the administration of 50mg of 30æ pyri-methamine with each of the other oils (S3, S4, S5 and S6). This toxicosis was first clearly evident 4 hours after drugging, the mice appearing distressed and hyperactive with erratic movement.
Later mice collapsed, their limbs jerking. Bleeding from the mouth resulted from the tongue being protruded and bitten by the use. One mouse from each of the groups dosed with S3, S4 and S5 + pyrimethamine was dead at 24 hours but all surviving mice were normal and showed no persistent drug effect. 100mg dosages 05 of S3, S4, S5 and S6 + pyrimethamine proved highly to~ic to mice and all mice which received these blends were dead within 24 hours (2 mice frnm each of the groups with S4, S5 and S6 + pyrimethamine were euthanised 6 hours after drugging since they were in great distress). The behavioural signs of drug toxicity were even more pronounced in these groups than in those which received 50mg doses of the blends.
The effects described above were not observed in mice which received pyrimethamine base in an aqueous suspension at doses of either 15mg or 30mg/mouse (750 and 1500mg/kg~. One of 5 mice which received pyrimethamine base at 1500mg/kg was dead at 24 hours. It is presumed that the base is released from some oils at a greater rate than from depots of powdered drug in water.
This effect may reflect the degree of solubility of pyrimethamine base in the oils, a conclusion supported by studies with pyri-methamine isethionate. When administered sc at dosages equivalentto 750mg/kg and 1500mg/kg base, the isethionate induced distress in animals within minutes and all mice died within 1 hour of dosing.
Mice which received 50mg of pyrimethmine base in S3, S4, S5, and S6, and survived to 24 hours appeared normal and showed no further evidence of reaction to the oil + drug.
None of the oils tested, alone or with pyrimethamine base, sulphadiazine or sulphadoxine gave rise to visible signs of chronlc tissue irritation. There was no hair loss, swelling, or signs of inflammatory reaction in the skin at the site of in~ection and the mice were normal to handle, giving no indicatlon of tenderness at that site. Evidence that the oils were well tolerated was also obtained from autopsy. At 54 days after administratlon of oils mice were killed, the skin from the dorsal surface of the thorax removed and the connective tissue examined.

1 ~3743 1 Of the oils tested the S1 preparation was the least durable in vivo. At 8 weeks no visible residues were found ln some mice and in the remainder 6mall, dark brown deposits at the lnjection site represented the residue. The duration of protection sfforded 05 mice by pyrimethamine S1 mixtures correlates with the life of the matrix, no mouse with thls preparation being protected to 8 weeks, Somewhat greater quantities, albeit small amounts, of S2 were visible in mice at 54 days. The oil appeared hyaline and light brown and there was no marked fibrosis or vascularisation of the oil depot site.
With each of the S3, S4, S5 and S6 preparations greater residual masses were obtained at 54 days than with the S2 preparation.
The oils appeared unchanged and had elicited no appreciable host tissue reaction. S3 + pyrimethamine protected mice for less than 7 days and this, together with the acute toxicosis observed after injection of the oil/drug blend is consistent with an extremely rapid release of drug from the oil in vivo. The intermediate periods of antimalarial protection provided by pyrimethamine in S4, S5 and S6 indicate that the release of pyrimethamine in these oils is slower than from S3 but there is presumably still an extremely rapid drug loss after doslng.
Combined pyrimethamine and sulphadiazine preparations The encouraging results obtained with both pyrimethamine and sulphadiazine in oil S2 indicated that these systems could be exploited for studies on the release of drug mixture from a single oil depot. As a preliminary step 10~ pyrimethamine-base-S2 mixtures were evaluated and it was shown that 50mg of 10~ pyrimethamine in S2 injected sc gave at least three months protection in mice.

This experiment was designed to ascertain whether the sustained release composition of this invention could be utilised for the release of a mixture of active susbtances from a single depot. Preparations of 30~ sulphadiazine + 10~ pyrimethamine base in S2 (a new batch prepared as in Example 6~ were blended and injected sc in 50mg aliquots into mice in the supra6capulsr position. Additional groups of mice received S2 alone or no drug.
Three groups of mice with S2 + drugs and 1 group with S2 slone were prepared for challenge on each of days 28, 42, 56 and 70. At each 05 time interval one group each of the S2 + drug mice were challenged by the inoculation lv of 1 x 10 mouse erythroctyes lnfected with the drug sensitive N strain of P. berghei, the pyrimethamine resistant PYR strain and the sulphonamide resistant ORA strain, respectively.
The group of mice with S2 alone was challenged with N straln parasites. Additional groups of control mice without oil were dosed with pyrimethamine (60mg/kg~ and orisulf (lOOOmg/kg) immediately pos t-infection.
The results obtained in thls experiment are summarised in Table 10 below.
The results of this experiment showed that 4 weeks sfter the injection of S2 contalning a mixture of pyrimethamine and sulpha-diazine, all mice challenged were protected against drug sensitive, pyrimethamine and sulphonamide resistant parasites. At 6 weeks one mouse in each of the groups challenged with N, PYR and ORA strains respectively, developed a parasitaemia but at 8 weeks, 4 of 5, 4 of 5 and 2 of 4 mice challenged with the N, PYR and ORA strains respectively were susceptible to challenge. A residual drug effect at 8 weeks was however demonstrated by the slowness with which parasitaemia developed in cage 8 mice where, following N strain challenge a parasitaemia (0.5X) was observed in only a single mouse on day 3. The day 3 N strain parasitaemias in control cages 4, 5 and 11 were 15.3, 25.8 and 9.0 respectively, with 15fl5 mice patent.
Table 10 The duration of protection against drug sensitive (N strain~
30 pyrlmYthamlne-res~Rtant (PYR strain) and sulphonamide-resistant (ORA strain) P. berghei afforded mice by 50mg subcutaneous depots of derivative oil S2 containing 30X sulphadiazine snd lOX
pyrimethamine.

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Ancillary tests were then initiated to determine the duration of effect of sulphadiazine (30X) and pyrimethamine (10~) in S2.
The results of these tests and comparison with the results obtained with the combination of the drugs ls shown in Table 11 below.
Table ll 05 The duration of effect of pyrimethamine, sulphadiazine and a combination of the two drugs in S2 when administered to mice sc as 50mg or lOOmg of oil/drug blend. Mice were challenged with N strain P. berghei and examined for parasitaemia for 14 - 17 days after challenge Number of mice Dosage of developing oiltdrug blendparasitaemia after Drug and ~O composition (mg) challenge on day:

pyrimethamin~ (107.) + sulphadiazine (30~) 50 0/5 0/5 1/4 5/5 pyrimethamine (107)50 0/5 0/5 4/5 sulphadiazine (30~)50 0/5 0/5 4/5 sulphadiazine (30X)100 0/4 Ot4 4/5 As noted ahove, the rate of development of parasitaemia in mice with the drug combination in S2 demonstrated that a residual drug effect was present at day 56 but Table 11 shows that there was a very clear difference ln the degree of protection in mice with pyrimethamine and sulphadiazine and in those with the drug combination. 50mg and lOOmg of S2 containing 307 sulphadiazine fully protected mice against N strain challenge at days 14 and 28.
A failure of protection occurred by day 42 however, with 4/5 mice on both regimens patent by day 3 post infection. lOX pyrimethamlne in 50mg S2 likewise protected mice against N strain challenge on days 14 and 28 but by day 42 4 of 5 mice were patent on day 3 of infection. If earlier results obtained with the first batch of S2 are excluded from this study then the results of this experiment ~ 337431 with the drug combination are encouraging since it appears that pyrimethamine + sulphadiazine together exert a greater and more prolonged antimalarial effect than either drug singly.

This experiment (which is incomplete) was designed to ascertaln 05 the suitability of the derivatives of this invention in the adminis-tration of antibiotics. All basic experimental procedures were as described above except that the mice received 100mg of the 30 mixtures of active substance (tetracycline as free base, or tetracycline as hydrochloride).
The results are summarised in Table 12.
Table 12 Duration of protection against P. berghei infection afforded mice by 30~ mixtures of tetracycline free base or hydrochloride in derivative oils. Each use received 100mg of mixture subcutaneously.

Oil TetracyclineDuration of protection (days) S1 free base 7 < 17 HCl < 7 S3 free base < 7 HCl < 7 S2 free base 7 < 17 HCl < 7 S4 free base < 17 HCl < 7 S5 free base < 7 HCl < 7 S6 free base 7 < 17 HCl < 7 A conspicuous feature of these results was the greater duration of protection afforded mice with tetracycline free base compared with the hydrochloride, in oils Sl, S2, S3 and S6. The greatest duration of effect was obtained with oil S2 and tetra-cycline base.

~ 33743 1 With all preparations tissue necrosis, with ulceration of the dermal tissues, occurred at the injection site. These lesions were more pronounced in animals which received the hydrochloride.
EXPF.RIMENT 6 This experiment (which is incomplete) was designed to ascertain O5 the sultability of the polymeric derivatives of this invention in formulating sustained release compositions. All basic experimental procedures were as described above except that the mice received lOOmg of the 30~ mixture of active substance (pyrimethamine).
The results are summarised in Table 13.
Table 13 Duration of protection against P. berghei Acute toxicity challenge Preparation ,observations (days) pyrimethamine + S7 16/25 mice dead by 3 days > < 35 pyrimethamine + S8 16/25 mice dead by 3 days > < 35 pyrimethamine + S9 16/25 mice dead by 3 days > < 35 pyrimethamine + S10 1/10 mlce dead by 6 days > 21 pyrimethamine + S11 0/10 mice dead by 12 days > 21 pyrimetha~ine + S12 3tlO mice dead by 3 days > 21 pyrimethamine + S13 5tlO mice dead by ~ 1 day > 21 pyrimethamine + S14 0/10 mice dead by 12 days > 21 pyrimethamine + S15 0/10 mice dead by 12 days > 21 pyrimethamine + S16 0/10 mice dead by 12 days > 21 pyrimethamine + S17 4/10 mice dead by 3 days +N 21 Sll, S14, S15 and S16 all appear to be of promise.
As to the derivatives per se, each of the polymers tested was well tolerated by the mice. In no instance was evidence found of acute or chronic toxicity in mice which received 100mg oil by sc _ 44 _ 1 337431 injection. No acute tlssue reaction was observed with any oil and autopsy examination at 7 days revealed no evidence of significant inflammatory reaction at the site of the oil depot.
Each of the oils S7, S8 and S9 (the only ones on which longer 05 term observations have been made~ persisted, as pure compounds, for at least 56 days; at 56 days the oil depot was encapsulated in a thin-walled fibrous 'sacl. The effect of mixing pyrimethamine with the oils apparently reduces their persistence since nelther residue of oil in drug was evident after 41 days.

Claims (23)

1. A sustained-release composition which comprises:
(I) a biologically-active substance; and (II) a monomeric compound which is obtainable by reacting, if required, in the presence of a Bronsted acid or a Lewis acid:
(a) at least one acidic, neutral or amphoteric organic compound which contains one, two or a plurality of active hydrogen atom-containing groups and which may be otherwise substituted or unsubstituted, or may be saturated or may be unsaturated, with at least one of the olefinic bonds of (b) at least one olefinically unsaturated cyclic ether of the formula:

in which:
R1, R2 and R4, which may be the same or different, each represent a hydrogen atom or a substituted or unsubstituted hydrocarbyl or hydrocarbyloxy group;
R3 represents a substituted or unsubstituted methylene, ethylene or 1,3-propylene group;

R5 represents a monovalent group reactive with the organic compound (a) or a hydrogen atom or a C1 to C4 alkyl group; and X represents:

in which:
Y represents an oxygen atom or an -NR6-group, wherein R6 represents any of the values which R1 may assume;
a is 0 or 1;
b is 0 or 1;
c is 1 or 2;
and d is 0 or 1;
with the proviso that at least one of b or d is 1.

2. The sustained-release composition according to claim 1, wherein (a) is unsubstituted.

3. The sustained-release composition according to claim 1, wherein (a) is saturated.

4. The sustained-release composition according to claims 1, 2 or 3, wherein (a) is an organic compound con-taining 1, 2 or a plurality of carboxy, hydroxy, amido or mercapto groups.

5. The sustained-release composition according to claims 1, 2 or 3, wherein (a) is mono-carboxy or hydroxy aliphatic hydrocarbyl with less than 18 carbon atoms; or is a di-carboxy or is an hydroxy aliphatic hydrocarbylene with less than 18 carbon atoms; or is octadecanol.

6. The sustained-release composition according to claims 1, 2 or 3, wherein (a) is a C2 to C4 alkanol or alkandiol.

7. The sustained-release composition according to claims 1, 2 or 3, wherein at least one of R1, R2 or R4 represents a hydrogen atom.

8. The sustained-release composition according to claims 1, 2 or 3, wherein R3 represents a mono- or poly-substituted or unsubstituted ethylene group.

9. The sustained-release composition according to claims 1, 2 or 3, wherein X represents -COO- or CH2OCO-.

10. The sustained-release composition according to claims 1, 2 or 3, wherein (b) has the formula:

or in which:
R1, R2, R4 and R5 are as defined in claim 1.

11. The sustained-release composition according to claims 1, 2 or 3, wherein (b) has the formula:

or in which:
R1, R2, R4 and R5 are as defined in claim 1 and further wherein (b) has the formula:

in which:
R'1, R'2 and R'4, which may be the same or different, represent any of the values which R1, R2 and R4, respec-tively, may assume.

12. The sustained-release composition according to claims 1, 2 or 3, wherein (b) is acrolein tetramer.

13. The sustained-release composition according to claims 1, 2 or 3, wherein (b) has the formula:

in which:

R8 and R'8, which may be the same or different, each represent a C1 to C6 alkyl group or a C2 to C6 hydroxyalkyl group; or both R8 and R8' are an octadecyl group.

14. The sustained-release composition according to claims 1, 2 or 3, wherein (II) is a liquid at a temperature from 35° to 40°C and at standard pressure.

15. The sustained-release composition according to claims 1, 2 or 3, wherein said active substance (I) is intimately admixed with the compound (II).

16. The sustained-release composition according to claims 1, 2 or 3, which is a suspension, dispersion or solution.

17. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active substance is provided as a controlled-release composition.

18. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active sub-stance (I) comprises a natural or synthetic steroid hormone.

19. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active sub-stance (I) comprises a substance which is effective against a tropical disease.

20. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active sub-stance (I) comprises pyrimethamine.

21. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active sub-stance (I) comprises a substance which comprises a natural or synthetic prostaglandin.

22. The sustained-release composition according to claims 1, 2 or 3, wherein said biologically-active sub-stance (I) comprises a substance which is effective against a tropical disease, and wherein at least part of organic compound (a) comprises a mono-, di- or polycarboxy, hydroxy or mercapto substituted biologically-active compound (I).

23. A compound of the formula:

in which:
R8 and R'8, which may be the same or different, each represent a C1 to C6 alkyl group, or a C2 to C6 hydroxyalkyl group; or both R8 and R'8 are an octadecyl group.