DK164107B - Conjugates of vinblastine or derivatives thereof, processes for preparing them, and pharmaceutical preparations which comprise them - Google Patents

Abstract

Previously unknown conjugates of vinblastine, or derivatives thereof, have the general formula <IMAGE> where A denotes an "arm" such as <IMAGE> or <IMAGE> where n is an integer from 1-5 and R is a hydrogen atom or an acyl group, R1 is an optionally modified protein radical, R2 is a methoxy group, an amino group or an α-amino acid ester which is bonded using a bond of the amide type and where the ester group contains 1-6 carbon atoms, and where R3 is a hydrogen atom or an hydroxyl group and is in all cases in the two possible configurations. These conjugates have a powerful effect when treating leukaemias, lymphosarcomas and other malignant tumours, including the so-called "solid" tumours, and when treating Hodgkin's disease.

Description

DK 164107 B

The present invention relates to novel vinblastine conjugates or derivatives thereof with certain proteins. The conjugates of the invention are useful as antifungal agents and peculiar to having the general formula I shown in claim 5 1, wherein A, R, 2 3 R and R have the same meanings stated. The invention also relates to processes for preparing these conjugates and pharmaceutical compositions containing them.

Vinblastine and some of its derivatives, in particular vincristine and vindesin, have already been previously linked to proteins, for example albumin or various immunoglobulins. As a result, coupling products or compounds called "conjugates" appear. Specifically, reference may be made to the following literature references: J.D. Teale, 15 Jacqueline M. Clough and V. Marks, Br.J.Clin.Pharmac. 4, 169-172, 1977; C.H.J. Ford, C.E. Newman, J.R. Johnson, C.S. Woodhouse, T.A. Reeder, G.F. Rowland, R.G. Simmonds,

Br. J. Cancer 47, 35-42, 1983; MJ Embleton, G.F. Rowland, R.G. Simmonds, E. Jacobs, C.H. Marsden, R.W. Baldwin, Br.

J. Cancer 47, 43-49, 1983; J.R. Johnson, C.H.J. Ford, C.E.

Newman, C.S. Woodhouse, G.F. Rowland, R.G. Simmond's, Br.J. Cancer 44, 472-475, 1981; Eli Lilly European Patent Application Publication No. 56,322 of July 21, 1982 (corresponding to DK Patent Application No. 59/82) and R.A. Conrad, G.J. Cullinan, 25 K. Gerzon, G.A. Poore, J.Med.Chem. 22, 391, 1979.

European Patent Application No. 84 870057.1 (published as No. 124,502 and corresponding to DK Patent Application 2107/84) contains a description of coupling bis-indole derivatives to a protein, fragment 30 of protein or an amine by means of an arm such as -CCX 4 - or -CO (CH 2) n ~ CO, where n ranges from 1-5 optionally substituted with an alkyl or amino group.

Coupling of these bis-indole derivatives has been carried out not only to develop new immunological reagents, but in particular to produce anti-tumor agents which are more active, more selective and less toxic than the known ones.

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2 In the latter respect, numerous conjugates of proteins with other antifungal agents are currently being investigated.

This is particularly true of antibody conjugates and a fragment of ricin or albumin and methotrexate conjugates (French Patent Application No. 2,437,213, CM Industries; and BCFChu, SB Howell, J. of Pharmacology and Exp. Therapeutics, 219 (2), 389-393 (1981).

It should also be mentioned that the use of 1: 1 complexes of bis-indole alkaloids with tubulin has been described in Belgian Patent Specification No. 854,053. In some cases, lower toxicity and a more significant chemotherapeutic activity may be achieved than that of the corresponding free alkaloids.

The 15 conjugates known from the aforementioned DK 2107/84 (EP 124,502) can be reproduced by the general formula I, such that R and R have the meanings given in the preceding claim 1, R may have the value specified in claim 1, but may also be a substituted amino group, but wherein the linking group A differs by being a straight chain group such as -COCH 2 'or -CO (CH 2) n -CO- where n is a number 1 -5. In contrast, group A of the conjugates of the present invention has a side chain -NH-R where R is hydrogen or an acyl group.

Comparative experiments have been made between, on the one hand, two compounds representing the conjugates known from DK 2107/84 (one compound of Example 9, where A is a hemisuccinate group) and the compound vinblastine, on the other hand the conjugate of Example 3. The experiments were carried out as described at the end of Example 3 and aimed to determine the effect of the respective compounds against leukemia in female mice.

The results are shown in the following table:

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3

Dose of wine- Dose al- Survivor

Forb caalkaloid bumin, MF Number of% ILS mice day 60 no mg Ag mg / kg mice 5 ----------; - 30 293 8.5 7> 614 4/7. 45 370 10 7> 669 4/7 60 383 13 5> 650 5/5 60 454 11 8> 606 5/8 60 644 7,8 7 [> 532 6/7 10 2__75__356__2ty> __ 10__57__0 70 536 12.1 7 109 0 3 80 690 12.1 7 120 0 100__844__09__3 __-_ 25____0 3 10 77 0 4 4 10 0 0 15 11 --------- ----------- l —-. ..-........ ...

In the table, MF represents the molar ratio of vinca alkaloid to galactosylated human serum albumin (whereby doses of albumin correspond to the amount of group R) and ILS is the average survival time of treated mice, expressed as% of survival time of untreated mice.

The compounds tested are: 1: Conjugate of the invention (Example 3), namely 4-O-desacetyl-4-O-L-N-acetyl hemiaspartate-vinblastine-galactosylated human albumin, i.e. a compound of formula I wherein A-R 2 is a group 30

- C - CH - (CH2) - C - galactosylated human albumin O NHR O

Wherein n = 1, R is an acetyl group, R is -OCH 2 and R is hydroxyl.

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2: Conjugate of Example 9 of DK 2107/84, namely vindesin-4-O-hemisuccinate-galactosylated human albumin, ie a compound of formula I wherein A-R1 is a group of 5 - C - CH 2 - CH ~ - C - galactosylated human albumin, il λ 2 il 0 O is -NH 2 and R 2 is hydroxyl.

3: Conjugate according to claim 3 of DK 2107/84, namely desacetylvinblastin-0-4-hemisuccinate-galactosylated human albumin, i.e. a compound of formula I wherein A-R is a group - C - CH - - CHO - C - galactosylated human albumin,. Wherein R is -OCH 2 and R is hydroxyl.

4: Vinblastine corresponding to the general formula I

1 2 3 wherein A-R is a group - C - CH 2, R is -OCH ^ and R is

Hydroxyl. ISLAND

It can be seen from the table that several mice were alive more than 60 days after injection of the conjugate according to the inventor, while none of the treated compounds were. Furthermore, it is apparent from the 25% ILS column that the conjugate of the invention is highly effective against the tested leukemia strain, while the tested compounds of DK 2107/84 are substantially less effective; at the high dose, the effect of compound # 3 is even negative.

The derivative of vinblastine may be a derivative of 4-0-diacetylvinblastine, vindesin, 4-0-desacetylvinblastine coupled at C-3 with an amino acid, 4-0-desacetyl-deoxy-4'-: vinblastine or 4-0-desacetyl -desoxy-4'-vinblastine coupled at C-3 with an amino acid.

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5

The therapeutic effect of the compounds of the invention is higher than the therapeutic activity of the compounds described so far and their toxicity is significantly lower.

The derivatives of the present invention are first obtained by condensing an anhydride, for example, as-paraggic anhydride or glutamic anhydride or a higher homolog, optionally substituted on the amine group, to the hydroxyl group at 04 in 4-O-desacetylvinblastine or 10 desacetyldesoxyvinblastine or any of the derivatives of 4-0-desacetyldesoxyvinblastin-3-carboxamide or 4-0-desacetyldesoxyvinblastin-4-carboxamide type.

The hemiaspartate or hemiglutamate or homologous derivatives thus obtained are then condensed with the desired serum albumin in a solvent in which these compounds are soluble. For this purpose, a mixture of water and dioxane can be used which is kept at the appropriate pH by means of a buffer, for example a phosphate buffer. The condensation is confirmed by chromatography or electrophoresis. In the latter case, radioactive (e.g. tritiated) vinblastine may be used to facilitate characterization.

From a chemical point of view, condensation with the albumin can be explained by the formation of covalent bonds resulting from the reaction of the amine groups in the protein lysine with the activated ester group derived from the optionally substituted hemiaspartate or hemiglutamate.

For example, bovine serum albumin contains 56 amine residues of lyin. The number of vinblastine molecules per molecule elbow min varies as a function of working conditions, but is generally between 1 and 34.

The activation can be performed in conventional manner by treatment with an alkyl chloroformate, preferably ethyl or isopropyl chloroformate, in the presence of an amine base such as N-methylpiperidine or N-methylmorpholine.

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6

The condensation can be carried out in situ on the reaction mixture containing the activated anhydride. However, in most cases, the activated anhydride can also be isolated.

The obtained conjugate is isolated by conventional methods. Accordingly, the albumin conjugate is precipitated from the reaction mixture by the addition of acetone and then centrifuged, rinsed, lyophilized and purified by gel filtration. If desired, the derivative 10 thus obtained can be subjected to a conventional succinylation reaction, which allows to avoid the aggregation problems that characterize certain conjugates and non-conjugated material.

The albumin used may be galactosylated so that conjugates are obtained which, when used in therapy, are preferably concentrated in certain tissues, for example in the liver. The galactic tosylation is carried out, for example, using the method described by G. Wilson in Journal of Biochemistry 253 (7) 2070-2072, 1978.

In vitro experiments with the conjugates of the invention to demonstrate their anti-tumor effect show that formation of the conjugate by a bond at C-4 may be more advantageous than if the bond is at C-3.

It is also possible to condense a compound of the type -AR, wherein A and R have the above meanings, in a suitable solvent, to the hydroxyl group in C-4 of 4-O-desacetylvinblastine or 4-O-desacetyldes. -oxyvinblastine or one of the derivatives of 4-O-desacetyl-vinblastine-3-carboxamide or 4-O-desacetyldesoxyvinoblastin-3-carboxamide. The activation can advantageously be effected by treatment with an alkyl chloroformate such as ethyl or isobutyl chloroformate, in the presence of an amine base such as N-methylpiperidine, N-methylmorpholine or triethylamine. The following examples describe some conjugates of the invention wherein bis-indole derivatives are coupled at position C-4 to human serum albumin by a compound group A of the substituted hemiaspartate or hemi-glutamate type.

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7

Indeed, the compounds of the invention have particularly valuable anti-tumor properties which can be used in human therapy.

In particular, these vinblastine derivatives can be used to treat leukemias, gliomas, lymphosarcomas and other malignant tumors, including so-called "solid" tumors.

Thus, in human therapy, they are used to treat Hodgkin's disease and against other tumors that may be affected by 10 treatment with vinblastine, vineristin or vindin.

For use in the therapy, the compounds of the invention are administered, optionally in lyophilized form, parenterally, in solution or in a pharmaceutically acceptable solvent.

Suitable solvents are physiological NaCl solution or other salt solutions buffered, eg with phosphate.

The active substance is generally administered at a dose which can range from 50 mg to several grams. The compounds of the invention may also be used in combination 20 with other antifungal agents.

Example 1 a) α and β-Isomer of 4-O-desacetyl-4-O-LN-acetylhemias-2β-partate-vinblastine 250 mg of N-acetyl-L-aspartic anhydride are added to a solution of 700 mg (0.91 mmol) 4-O-desacetylvinblastine in 20 ml of anhydrous dichloromethane.

The solution is stirred for 20 hours at room temperature.

After distillation under vacuum of the solvent, the residue obtained is purified by chromatography on silica (elution with ether / methanol / 25% NH 4 OH 60: 49.75: 0.25).

After trituration in a mixture of dichloromethane and petroleum ether, 650 mg of 4-O-desacetyl-4-O-L-N-25 acetyl hemiaspartate vinblastine is obtained in the form of a white powder and in a yield of 77%.

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8 HPLC analysis of the product shows the presence of the two isomers α and M ratio 85:15.

IR spectrum (KBr): 3420, 2960, 2880, 1737, 1660, 1613, 1501, 1360, 1430 cm cm 1.

Mass Spectrum: DCI (isobutane): 925 (M +), 939 (M + + 14), 857, 769, 693, 635.

b) α- and β-Isomer of 4-O-desacetyl-4-O-LN-acetylhemiasate partate methyl ester vinblastine A solution of 500 mg (0.540 mmol) of the oc and β-isomer of 4- O-desacetyl * 4-O-LN-acetylhemiaspartate-vinblin in 10 ml of absolute methanol saturated with dry HCl is stirred for 20 hours at room temperature.

After distillation of the solvent under vacuum 15, the residue is recovered in a mixture of 15 ml of distilled water and 15 ml of dichloromethane. The mixture is made alkaline by the addition of NH 2 OH. The aqueous phase is extracted with 3 x 20 ml of dichloromethane. The organic extracts are combined, washed successively with 40 ml of water and 40 ml of an aqueous solution saturated with NaCl, then dried over magnesium sulfate and evaporated under reduced pressure. The residue is purified by chromatography on silica (elution with CH 2 Cl 2 / CH 3 OH 95: 5). Thereby, 410 mg of α and β isomers of 4-O-des-acetyl-4-O-L-N-acetylhemiasiaspartate methyl ester vinblastine 25 are obtained in a yield of 81%.

Fractions resulting from chromatography and inf. containing the individual isomers is analyzed.

Mass Spectrum (DCI, isobutane): 939 (M +), 940 (M + + 1), 953 (M + + 14).

IR spectrum (KBr): 2950, 2880, 1740, 1675, 1615, 1503 cm -1.

NMR spectrum (CDCl3, 360 MHz), δ: 9.65 (OH), 8.02 (NH), 7.52 (H-9 '), 7.16-7.05 (H-10'), H-11 ·, H-12 ′), 6.61 (H-9), 6.52 (NH), 6.07 (H-12), 5.75 (H-14), 5.52 (H -17), 5.17 (H-15), 4.75 (-CH-NH), 3.95 (H-17A '), 3.80 (-OMe),

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3.77 (-OMe), 3.70 (-OMe), 3.60 (-OMe), 2.80 (H-21A ', H-21B'), 2.67 (NMe), 2.62 (H-21), 2.00 (MeCO), 0.92-0.75 (2 Me).

Rf: 0.51 (CH 2 Cl 2 / CH 3 OH 90:10 silica).

5

Isorner_in_Small_ quantity:

Mass spectrum (DCI-isobutane): 939 (M +), 940 (M + + 1).

IR Spectrum (KBr): 1740, 1675, 1615 cm -1.

NMR Spectrum (CDCX3, 360 MHz), 5: 9.25 (OH), 8.02 (NH), 7.50 (H-91), 7.16-7.05 (Η-10 ', Η -11 ', H-12'), 6.61 (H-9), 6.52 (NH), 6.08 (H-12), 5.82 (H-14), 5.47 (H- 17), 5.28 (H-15), 4.75 (CH-NHAc), 3.93 (Η-17A '), 3.77 (2 x OMe), 3.60 (OMe), 2.70 (NMe), 2.02 (MeCo-), 0.95-0.77 (2 Me).

Rf: 0.39 (CH2 C19 / CH, OH 90:10 silica).

15 J

Example 2 α- and γ-Isomer of 4-O-desacetyl-4-O-LN-carbobenzyloxyhemiglutamate-vinblastine A solution of 300 mg (0.39 mmol) of 4-O-desacetyl-vinblastine and 144 mg (0.519 mmol) N-carbobenzyloxy-L-glucamic anhydride in 5 ml of dichloromethane is stirred for 20 hours at room temperature. The solvent is evaporated under vacuum. The residue obtained is purified by chromatography on silica (elution: ether / methanol / 25% NH 2 OH 50: 49.5: 0.5).

In this way, 230 mg of 4-O-desacetyl-4-O-L-N-carbobenzyloxyhemiglutamate vinblastine is obtained in the form of a mixture of the α and γ isomer.

HPLC analysis of the product shows the presence of the 30 α and γ isomer in the 60:40 ratio.

IR spectrum (KBr): 3450, 2960, 2880, 1730, 1612, 1593, 1501, 1459, 1432, 1228 cm -1.

Mass Spectrum (DCI-isobutane): 1032 (M + + 1), 984, 928, 723.

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Example 3 10

Coupling of 4-O-desacetyl-4-O-LN-acetylhemiaspartate-wine blastin with galactosylated albumin of human origin ml of dioxane. The solution is then poured into an ice bath. Add 24.4 μΐ triethylamine in 0.5 ml dioxane and 22.6 μΐ isobutyl chloroformate in 0.5 ml dioxane. The mixture is stirred for 1 hour.

Further, a solution of 200 mg of lactosylated human albumin in 37 ml of H2 O is prepared. The pH is adjusted to 8.5 using 0.1N NaOH.

The solution is cooled to 4 ° C. After 1 hour, the activated ester is added to the solution of galactosylated human albumin. The solution is stirred for 14 hours at 4 ° C while maintaining the pH of 8.5 by the addition of 1N NaOH. Then, the solution is purified by filtration on "Sephadex" gel G 25 equilibrated using a solution of NaCl 9: 1000, pH 7.5. The fractions containing the conjugate are combined, concentrated by filtration and sterilized. The protein content is determined by the Lowry method and the content of alkaloids is assessed by radioactivity determination.

The conjugate obtained contains 13 moles of vinblastine per ml. moles of galactosylated human albumin. HPLC assay 25 of monomers, dimers and polymers of the conjugate composition shows 82% monomers and 18% dimers.

b) Dissolve 80.6 mg of 4-O-desacetyl-4-O-L-N-acetyl hemiaspartate vinblastine in 2 ml of dioxane. The solution is then poured-3Q into an ice bath. Add 24.4 μΐ triethylamine in 0.5 ml dioxane and 22.6 μΐ isobutyl chloroformate in 0.5 ml dioxane.

In addition, a solution of 200 mg galactosylated human albumin in 37 ml of 0.1M phosphate buffer pH 8.2 is prepared. The pH is adjusted to 8.5 using 1N NaOH.

55 The solution is cooled to 4 ° C. After 1 hour, the activated ester is added to the solution of galactolyzed human albumin. The solution is stirred for 14 hours at 4 ° C while

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11 pH is maintained at 8.5 by addition of 1N NaOH. The solution is purified by filtration on Sephadex® gel G 25 equilibrated with a solution of NaCl 9: 1000, pH 7.5. The fractions containing the conjugate are combined, concentrated by ultrafiltration and sterilized. The protein content is measured by the Lowry method and the content of alkaloids is assessed by radioactivity determination. The conjugate obtained inside The conjugate obtained contains 9.3 moles of vinblastine per ml. moles of galactosylated human albumin. HPLC assay 10 of monomers, dimers and polymers in the conjugate composition shows 91.5% monomers, 7% dimers and 1.5% polymers.

The sensitivity of the conjugate to lysosomal enzymes was examined by incubating for 48 hours at 37 ° C in the presence of 5 mM cysteine, 40 mM acetate buffer and lysosomial enzymes. Samples are taken and the undegraded proteins are precipitated by the addition of a volume of trichloroacetic acid (TCA? 40% TCA). After incubation at 4 ° C for 1 hour, the samples are centrifuged and the radioactivity of the above liquid is assessed by counting the scintillations in a sample of the liquid.

The soluble radioactivity is a measure of digestion of the conjugate. Practical experiments have shown that 75% of the conjugate has been digested after 24 hours. No further development was observed up to 48 hours.

The chemotherapeutic activity of this conjugate 25 has been determined on P 388 leukemia with female mice of the strain g BDF.J: 10 tumor cells are intraperitoneally injected on day 0. The conjugate is administered intraperitoneally on day 1. The test results show that the conjugate shows important effect in this experimental model. leads to an increase of 30 in the life of more than 650% if administered in an amount of 60 mg / kg and the number of surviving mice is 5/5 after 60 days.

35

Example 4

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12 α- and β-Isomer of ethyl N- (4-O-desacetyl-4-O-LN-acetyl-hemiaspartate-vinblastinoyl-23) -L-tryptophanate-2 By proceeding in the manner described in Example 1 ethyl N- (desacetyl-4-O-vinblastinoyl-23) -L-tryptophanate to ethyl N- (4-O-desacetyl-4-O-LN-acetyl-hemi-aspartate-vinblastonoyl-23) -tryptophanate (mixture of isomers α and / 3).

The obtained residue is purified by chromatography on silica (elution with ether / methanol / 25% NH 2 OH 50: 49.75: 0.25).

200 mg of the product is obtained from 314 mg of the starting product.

+ 1

Mass Spectrum (DCL, acetone): 1126 (M), 1066, 984, 970, 951, 911.

IR spectrum (KBr): 3400, 2960, 2940, 1665, 1618 cm -1. Example 5 α and β isomers of ethyl N- (4-O-desacetyl-4-O-LN-acetyl-hemiaspartate) -vinblastonoyl-23) -L-isoleucinat_

By proceeding as described in Example 4, ethyl N- (desacetyl-4-O-vinblastinoyl-23) -L-isoleucinate was converted to ethyl N- (4-O-desacetyl-4-O-LN-acetyl hemiaspartate). wine-blastinoyl-23) -L-isoleucinate.

Mass Spectrum (DCI-acetone): 1051 (M +), 1036, 1009, 977, 897, 838, 755, 709, 652.

IR spectrum (KBr): 3410, 2963, 2929, 2880, 1734, 1676, 1612, 1500, 1460, 1430, 1372, 1333, 1293 cm -1.

Example 6 cc and γ-Isomer of 4-O-desacetyl-4-O-L-N-acetylhemiglutamate vinblastine

Proceeding as described in Example 2, 4-O-desacetylvinblastine was treated with N-acetyl-L-glutamic anhydride to give 271 mg of 4-O-desacetyl-4-O-LN-acetyl-hemiglutamate. vinblastine (mixture of isomers α

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13 and r) from 380 mg of 4-O-desacetylvinblastine.

Mass spectrum (DCI-acetone): 940 (M + 1), 871, 707. IR spectrum (KBr): 3470, 2960, 2880, 1740, 1665, 1617 cm -1.

5 10 15 20 25 30 35

Claims (9)

A vinblastine conjugate or a derivative thereof, characterized in that it has the general formula OT3U h / A / \ R ch3ooc J 10 / i CH, 0 0-A-R Conjugate according to claim 1, characterized in that the α-amino acid ester from which R is derived is ethyl tryptophanate or ethyl isoleucinate. Conjugate according to claim 1, characterized in that R is a methoxy group or amino group. 3 CH, Γ0Η LcO 23 \ -RZ where A is a linking group such as NH-R NH-R II -COCH- (CHo) CO- or -CO- (CHo) CHCO-2U 2. n 2 n where n is an integer of 1- 5, R is a hydrogen atom or an acyl group such as acetyl, trifluoroacetyl or carbobenzyloxy, R 1 is an optionally galactosylated residue of bovine or human serum albumin, R by means of an amide-type bond and wherein the ester group contains 1-6 carbon atoms and R is a hydrogen atom or a hydroxyl group, at least in the two possible configurations. A process for preparing conjugates as claimed in any one of claims 1-3, characterized in that an anhydride of an acid of the formula NH-R HOOC-CH- (CH2) nCOOH 5 wherein n and R has the meanings given in claim 1, condensed to the hydroxyl group at C-4 in the vinblastine derivative, and the derivative thus obtained is condensed with the optionally galactosylated residue of bovine or human serum albumin. Process according to claim 2, characterized in that the solvent in the first condensation consists essentially of dichloromethane and that the solvent in the second condensing step is a mixture of water and dioxane. Process for preparing a conjugate according to any one of claims 1-3, characterized in that a group of the type NH-R NH-R I 1 I 1 Wherein R is the residue of a bovine or human serum albumin and n and R have the meanings specified in claim 1, are condensed to -CO-CH- (CHo) -COR1 or -CO- (CH-) -CHCOR 2 n 2 n 35 the hydroxyl group at C-4 in the vinblastine derivative. Process according to claim 6, characterized in that an activation of the starting product is carried out by treatment with an alkyl chloroformate such as ethyl chloroformate, in the presence of an amine base such as N-methylpiperidine, N-methylmorpholine or triethylamine. Process according to any one of claims 4 to 7, characterized in that a further centrifugation, a rinse, a freeze drying and / or a purification by gel filtration is carried out and, if desired, a conventional succinylation reaction. DK 164107 B 16 A pharmaceutical composition for the treatment of leukemias, lymphosarcomas or other malignant tumors, including so-called "solid" tumors and for the treatment of Hodgkin's disease, characterized in that it contains a conjugate as claimed in any one of claims 1-3. in combination with a diluent, solvent, excipient or pharmaceutically acceptable carrier wherein the active compound is in lyophilized form or in solution in a buffered solvent.