Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

Подождите немного. Документ загружается.

confer bacterial resistance by inactivating penicillins and cephalosporins (see Chapter

9), are useful in the sterility testing of certain antibiotics (see section 4.5) and, prior to

culture, in inactivating various /3-lactams in blood or urine samples in patients undergoing

therapy with these drugs. One other important therapeutic application is in the rescue

of patients presenting symptoms of a severe allergic reaction following administration

of a /3-lactamase-sensitive penicillin. In such cases, a highly purified penicillinase

obtained from Bacillus cereus is administered either intramuscularly or intravenously

and in combination with other supportive measures such as adrenaline or antihistamines.

Applications of microorganisms in the partial synthesis

of pharmaceuticals

Whole microbial cells as well as microbially derived enzymes have played a significant

role in the production of novel antibiotics. The potential of microorganisms as chemical

catalysts, however, was first fully realized in the synthesis of industrially important

steroids. These reactions have assumed increasing importance following the discovery

that certain steroids such as hydrocortisone have anti-inflammatory activity, whilst

derivatives of the steroidal sex hormones are useful as oral contraceptive agents. More

recently, chiral inversion of non-steroidal anti-inflammatory drugs (NS AIDs) has been

demonstrated.

Production of antibiotics

In the antibiotics industry, the hydrolysis of benzylpenicillin to give 6-aminopenicillanic

acid by the enzyme penicillin acylase is an important stage in the synthesis of many

clinically useful penicillins (see Chapters 5 and 7). The combination of genetic

engineering techniques to produce hybrid microorganisms with significantly higher

acylase levels, together with their entrapment in gel matrices (which appears to improve

the stability of the hybrids), has resulted in considerable increases in 6-aminopenicillanic

acid yields.

A second example is provided by the production by fermentation of cephalosporin

C, which is used solely for the subsequent preparation of semisynthetic cephalosporins

(Chapters 5 and 7).

Furthermore, antibiotics produced by fermentation of various moulds or, especially,

Streptomyces spp. can be employed by medicinal chemists as starting blocks in the

production of what might be more effective antimicrobial compounds.

Steroid biotransformations

Since steroid hormones can only be obtained in small quantities directly from mammals,

attempts were made to synthesize them from plant sterols which can be obtained

cheaply and economically in large quantities. However, all adrenocortical steroids are

characterized by the presence of an oxygen at position 11 in the steroid nucleus. Thus,

although it is easy to hydroxylate a steroidal compound it is extremely difficult to

obtain site-specific hydroxylation, so that many of the routes used for synthesizing the

desired steroid are lengthy, complex and consequently expensive. This problem was

Additional pharmaceutical applications of microorganisms All

Fig. 25.4 Conversion of progesterone to 11 a-hydroxyprogesterone by Rhizopus nigricans.

overcome when it was realized that many microorganisms are capable of performing

limited oxidations with both stereo- and regio-specificity. Thus, by simply adding a

steroid to growing cultures of the appropriate microorganism, specific site-directed

chemical changes can be introduced into the molecule. In 1952, the first commercially

employed process involving the conversion of progesterone to 11 a-hydroxyprogesterone

by the fungus Rhizopus nigricans was introduced (Fig. 25.4). This reaction is an

important stage in the manufacture of cortisone and hydrocortisone from more readily

available steroids. Table 25.4 gives several other examples of microbially directed

oxidations employed in the manufacture of steroidal drugs.

More recent advances involving the employment of microorganisms in bio-

transformation reactions utilize immobilized cells (both living and dead). Immobilization

of microbial cells, usually by entrapment in a polymer gel matrix, has several important

advantages. Whole microbial cells contain complex multistep enzyme systems and

there is therefore no longer a need to extract enzymes or enzyme systems which may

be inactivated during purification procedures. It also increases the stability of membrane-

associated enzymes which are unstable in the solubilized state, as well as permitting

the conversion of water-insoluble compounds like steroids in two-phase water-organic

solvent systems.

Chiral inversion

Several clinically used drugs, e.g. salbutamol (a /^-adrenoceptor agonist), propanol (a

j3-adrenoceptor antagonist) and the 2-arylpropionic acids (NSAIDs) are employed in

Table 25.4 Examples of biological transformations of steroids

Starting material Product

Progesterone 11 a-hydroxyprogesterone

Compound S* Hydrocortisone

11a-hydroxyprogesterone A-11a-hydroxyprogesterone

Hydrocortisone Prednisolone

Cortisone Prednisone

* Derived from diosgenin by chemical transformation.

Type of reaction

Hydroxylation

Dehydrogenation

the racemic form. In the last series, e.g. ibuprofen, activity resides almost exclusively

in the S(+) isomers; chiral inversion, in the undirectional manner R(-) —> S(+),-occurs

in vivo over a 3-hour period. The S(+) form is a more effective inhibitor of prostaglandin

synthesis, and enzymes from some fungal enzymes convert a racemic mixture into the

S(+) isomer in vitro. It has thus been suggested that the enantiomerically pure S(+)

form could be administered clinically to give a reduced dosage and possible less toxicity.

Use of microorganisms and their products in assays

Microorganisms have found widespread uses in the performance of bioassays for:

1 determining the concentration of certain compounds (e.g. amino acids, vitamins,

some antibiotics) in complex chemical mixtures or in body fluids;

2 diagnosing certain diseases;

3 testing chemicals for potential mutagenicity or carcinogenicity;

4 monitoring purposes involving the use of immobilized enzymes;

5 sterility testing of antibiotics.

Antibiotic bioassays

Antibiotics may be assayed by a variety of methods (see Chapter 8, pages 166-188, in

Pharmaceutical Microbiology, 5th edition 1992). Only microbiological and radio-

enzymatic assays will be considered briefly here: see Figs 25.5 and 25.6 and sections

4.1.1 and 4.1.2.

Fig. 25.5 Graphical

representation of a two-by-two

assay response. X is the

horizontal distance between the

two lines. The antilog of X gives

the relative potency of the

standard and test.

Fig. 25.6 Relationship between concentration of

aminoglycoside antibiotic and the transfer of radioactivity

from adenosine triphosphate to phosphocellulose.

t.1.1

Microbiological assays

In microbiological assays the response of a growing population of microorganisms to

the antimicrobial agent is measured. The usual methods involve agar diffusion assays,

in which the drug diffuses into agar seeded with a susceptible microbial population and

produces a zone of growth inhibition.

In the commonest form of microbiological assay used today, samples to be assayed

are applied in some form of reservoir (porcelain cup, paper disc or well) to a thin layer

of agar seeded with indicator organism. The drug diffuses into the medium and after

incubation a zone of growth inhibition forms, in this case as a circle around the reservoir.

All other factors being constant, the diameter of the zone of inhibition is, within limits,

related to the concentration of antibiotic in the reservoir.

During incubation the antibiotic diffuses from the reservoir, and that part of the

microbial population away from the influence of the antibiotic increases by cell division.

The edge of a zone is formed when the minimum concentration of antibiotic which

will inhibit the growth of the organism on the plate (critical concentration) reaches, for

the first time, a population density too great for it to inhibit. The position of the zone

edge is thus determined by the initial population density, growth rate of the organism

and the rate of diffusion of the antibiotic.

In situations where the likely concentration range of the tests will lie within a

relatively narrow range (e.g. in determining potency of pharmaceutical preparations)

and high precision is sought, then a Latin square design with tests and calibrators

at two or three levels of concentration may be used. For example an 8 x 8 Latin

square can be used to assay three samples and one calibrator, or two samples and

two calibrators at two concentrations each (over a two- or fourfold range), with a

coefficient of variation of around 3%. Using this technique, parallel dose-response

lines should be obtained for the calibrators and the tests at the two dilutions (Fig. 25.5).

Using such a method, potency can be computed or determined from carefully prepared

nomograms.

480 Chapter 25

Conventional plate assays require several hours' incubation and consequently the

possibility of using rapid microbiological assay methods has been studied. Two such

methods are:

1 Urease assay. When Proteus mirabilis grows in a urea-containing medium it

hydrolyses the urea to ammonia and consequently raises the pH of the medium. This

production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein

synthesis; Chapter 8). In practice, it is difficult to obtain reliable results by this

method.

2 Luciferase assay. In this technique, firefly luciferase is used to measure small

amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced

by the inhibitory action of aminoglycoside antibiotics. This method may find more

application in the future as more active and reliable luciferase preparations become

available.

4.1.2 Radioenzymatic (transferase) assays

These depend on the fact that bacterial resistance to aminoglycosides (Chapter 9), such

as gentamicin, tobramycin, amikacin, netilmicin, streptomycin, spectinomycin, etc., and

chloramphenicol is frequently associated with the presence of specific enzymes (often

coded for by transmissible plasmids) which either acetylate, adenylylate or phosporylate

the antibiotics, thereby rendering them inactive (Chapter 9). Aminoglycosides may

be susceptible to attack by aminoglycoside acetyltransferases (AAC), aminoglycoside

adenylyltransferases (AAD), or aminoglycoside phosphotransferases (APH). Chloram-

phenicol is attacked by chloramphenicol acetyltransferases (CAT). Acetyltransferases

attack susceptible amino groups and require acetyl coenzyme A, while AAD or APH

enzymes attack susceptible hydroxyl groups and require ATP (or another nucleotide

triphosphate).

Several AAC and AAD enzymes have been used for assays. The enzyme and the

appropriate radiolabeled cofactor ([1-

C] acetyl coenzyme A, or [2-

H] ATP) are used

to radiolabel the drug being assayed. The radiolabeled drug is separated from the

reaction mixture after the reaction has been allowed to go to completion; the amount of

radioactivity extracted is directly proportional to the amount of drug present.

Aminoglycosides are usually separated by binding them to phosphocellulose paper,

whereas chloramphenicol is usually extracted using an organic solvent. An example of

a standard curve (for gentamicin) is provided in Fig. 25.6.

These types of assay are rapid, taking approximately 2 hours, show good precision

and are much more specific than microbiological assays.

4.2 Vitamin and amino acid bioassays

The principle of microbiological bioassays for growth factors such as vitamins and

amino acids is quite simple. Unlike antibiotic assays (see section 4.1) which are based

on studies of growth inhibition, these assays are based on growth exhibition. All that is

required is a culture medium which is nutritionally adequate for the test microorganism

in all essential growth factors except the one being assayed. If a range of limiting

concentrations of the test substance is added, the growth of the test microorganism will

Additional pharmaceutical applications of microorganisms 481

Fig. 25.7 Standard curve in a

vitamin assay.

Table 25.5 Some examples of

microorganisms used as

bioassays for vitamins and amino

acids

be proportional to the amount added. A calibration curve of concentration of substance

being assayed against some parameter of microbial growth, e.g. cell dry weight, optical

density or acid production, can be plotted. An example of a standard curve is presented

in Fig. 25.7 and from this the concentration of growth factor in the unknown solution

can be determined. One example of this is the assay for pyridoxine (vitamin B

) which

can be assayed using a pyridoxine-requiring mutant of the mould Neurospora. Lactic

acid bacteria have extensive growth requirements and are often used in bioassays. It

is possible to assay a variety of different growth factors with a single test organism

simply by preparing a basal media with different growth-limiting nutrients. Table 25.5

summarizes some of the vitamin and amino acid bioassays currently available. In

practice, only vitamins are assayed by bioassay procedures, because most amino acids

are currently determined chemically.

4.3

Phenylketonuria testing

Phenylketonuria (PKU) is an inborn error of metabolism by which the body is unable

to convert surplus phenylalanine (PA) to tyrosine for use in the biosynthesis of, for

482 Chapter 25

Assay microorganism

Lactobacillus casei

L. arabinosus

L leichmannii

L. casei

Saccharomyces uvarum

L arabinosus

Acetobacter suboxydans

L. casei

Neurospora crassa or

S. carlsbergiensis

L. casei

L viridans

Vitamin or amino acid

Biotin

Calcium pantothenate

Cyanocobalamin

Folic acid

Inositol

Nicotinic acid

Pantothenol

Pyridoxal

Pyridoxine

Riboflavine

Thiamine

Fig. 25.8 (a) Normal metabolism, in which phenylalanine is converted by phenylalanine 4-mono-

oxygenase to tyrosine, (b) Phenylketonuria, in which there is a transamination reaction between

phenylalanine and a-ketoglutaric acid. Phenylalanine 4-mono-oxygenase is absent in about 1 in

every 10000 human beings because of a recessive mutant gene.

example, thyroxine, adrenaline and noradrenaline. This results from a deficiency in the

liver enzyme phenylalanine 4-mono-oxygenase (phenylalanine hydroxylase). A secondary

metabolic pathway comes into play in which there is a transamination reaction between

PA and a-ketoglutaric acid to produce phenylpyruvic acid (PPVA), a ketone and glutamic

acid. Overall, PKU may be defined as a genetic defect in PA metabolism such

that there are elevated levels of both PA and PPVA in blood and excessive excretion of

PPVA (Fig. 25.8).

Control of PKU can be achieved simply by resorting to a low PA-containing diet.

Failure to diagnose PKU, however, will result in mental deficiency, and early diagnosis

is essential. In 1968, the UK Medical Research Council Working Party on PKU

recommended the adoption of the Guthrie test as a convenient method for screening

newborn infants. This assay employs Bacillus subtilis as the test organism. In minimal

culture media, growth of this bacterium is inhibited by /3-2-thienylalanine (Fig. 25.9a)

and its competitive reversal in the presence of PA (Fig. 25.9b) or PPVA. The use of

filter-paper discs impregnated with blood or urine permits the detection of elevated

levels of PA and PPVA. The test can be quantitated by the measurement of the diameter

of the growth zone around the filter-paper disc and comparing it with a calibration

curve constructed from known concentrations of PA or PPVA (Fig. 25.9c).

If positive, the Guthrie test provides presumptive evidence for the presence of PKU.

It should be confirmed by other, chemical, means.

Additional pharmaceutical applications of microorganisms 483

Fig. 25.9 (a) /3-Thienylalanine, (b) phenylalanine, (c) standard curve in Guthrie test.

4.4

Carcinogen and mutagen testing

A carcinogen is a substance which causes living tissues to become carcinomatous (to

produce a malignant epithelial tumour). A mutagen is a chemical (or physical) agent

which induces mutation in a human (or other) cell.

Mutagenicity tests are used to screen a wide variety of chemicals for their ability to

cause a mutation in the DNA of a cell. Such mutations can occur at:

1 gene level (a 'point' mutation);

2 individual chromosome level;

3 chromosome set level, i.e. a change in the number of chromosomes (aneuploidy).

Some compounds are only mutagenic or carcinogenic after metabolism (often in

the liver). This aspect must, therefore, be considered in designing a suitable test method

(see section 4.4.2).

4.4.1 Mutations at the gene level

Forward mutation refers to mutation of the natural ('wild-type') organism to a more

stringent organism. By contrast, reverse (backward) mutation is the return of a mutant

strain to the wild-type form, i.e. it is a heritable change in a previously mutated gene

that restores the original function of that gene.

There are two types of reverse mutation:

1 frame-shift: in these mutants, the gene is altered by the addition or deletion of one

or more basis so that the triplex reading frame for RNA is modified;

2 base-pair: in these mutants, a single base is altered so that the triplex reading frame

is again modified.

These principles of reverse mutation are utilized in one important method, the Ames

test (section 4.4.2), which is used to detect compounds that act as mutagens or

carcinogens (most carcinogens are mutagens).

4.4.2 The Ames test

The Ames test is used to screen a wide variety of chemicals for potential carcinogenicity

484 Chapter 25

or as potential cancer chemotherapeutic agents. The test enables a large number of

compounds to be screened rapidly by examining their ability to induce mutagenesis in

several specially constructed bacterial mutants derived from Salmonella typhimurium.

The test strains contain mutations in the histidine operon so that they cannot synthesize

the amino acid histidine. Two additional mutations increase further the sensitivity of

the system. The first is a defect in their lipopolysaccharide structure (Chapter 1) such

that they are in fact deep rough mutants possessing only 2-keto-3-deoxyoctonate (KDO)

linked to lipid A. This mutation increases the permeability of the mutants to large

hydrophobic molecules. The second mutation concerns a DNA excision repair system

which prevents the organism repairing its damaged DNA following exposure to a

mutagen.

The assay method involves treatment of a large population of these mutant tester

strains with the test compound. Histidine-requiring mutants are used to detect mutagens

capable of causing base-pair substitutions (in some strains) or frame-shift mutations

(other strains). This can be carried out by incorporating both the test strain and test

compound in molten agar (at 45°C), which is then poured onto a minimal glucose agar

plate. Alternatively, the mutagens can be applied to the surface of the top agar as a

liquid or as a few crystals. The medium used for the top agar contains a trace of histidine

which permits all the bacteria on the plate to undergo several divisions, since for many

mutagens some growth is a necessary prerequisite for mutagenesis to occur. After

incubation for 2 days at 37°C the number of revertant colonies can be counted and

compared with control plates from which the test compound has been omitted. Each

revertant colony is assumed to be derived from a cell which has mutated back to the

wild type and thus can now synthesize its own histidine: see Fig. 25.10 for a summary.

A further refinement to the Ames test permits screening of agents which require

metabolic activation before their mutagenicity or carcinogenicity is apparent. This is

achieved by incorporating into the top agar layer, along with the bacteria, homogenates

of rat (or human) liver whose activating enzyme systems have been induced by exposure

to polychlorinated biphenyl mixtures. This test is sometimes referred to as the

Salmonella/microsome assay since the fraction of liver homogenate used, called the

S9 fraction, contains predominantly liver microsomes.

It is important to realize that this test is flexible and is still undergoing modification

and development. Almost all the known human carcinogens have been tested and shown

Fig. 25.10 Summary of the

Ames test.

Additional pharmaceutical applications of microorganisms 485

to be positive. These include agents such as /3-naphthylamine, cigarette smoke

condensates, aflatoxin B and vinylchloride, as well as drugs used in cancer treatment

such as adriamycin, daunomycin and mitomycin C. Whilst the test is not perfect for the

prediction of mammalian carcinogenicity or mutagenicity and for making definitive

conclusions about potential toxicity or lack of toxicity in humans, it nevertheless

represents a significant advance providing useful information rapidly and cheaply. The

Ames test forms an important part of a battery of tests, the others of which are non-

microbial in nature, for detecting mutagenicity or carcinogenicity.

4.5 Use of microbial enzymes in sterility testing

Sterile pharmaceutical preparations must be tested for the presence of fungal and

bacterial contamination before use (see Chapters 18 and 23). If the preparation contains

an antibiotic, it must be removed or inactivated. Membrane filtration is the usual

recommended method. However, this technique has certain disadvantages. Accidental

contamination is a problem, as is the retention of the antibiotic on the filter and its

subsequent liberation into the nutrient medium.

Enzymic inactivation of the antibiotic (see also Chapter 9) prior to testing would

provide an elegant solution to this problem. Currently, the only pharmacopoeial method

permitted is that of using an appropriate /^-lactamase to inactivate penicillins and

cephalosporins. Other antibiotics which are susceptible to inactivating enzymes are

chloramphenicol (by chloramphenicol acetyltransferase) and the aminoglycosides, e.g.

gentamicin, which can be inactivated by phosphorylation, acetylation or adenylylation.

A method for acetylating and consequently inactivating aminoglycosides prior to testing

and using 3 -N- acetyl transferase (an enzyme with wide substrate specificity) in

combination with acetyl coenzyme A has been described, but this method has yet to be

adopted.

4.6 Immobilized enzyme technology

The therapeutic uses of microbially derived enzymes have already been examined

(section 2.4). However, enzymes also form the basis of many diagnostic tests used in

clinical medicine. For example, glucose oxidase, an enzyme used in blood glucose

analysis, is obtained commercially from Aspergillus niger. Future development and

improvement of such diagnostic tests is likely to involve the immobilization of enzymes

in enzyme electrodes. Several types of glucose oxidase electrodes have been developed,

although none is yet in clinical use. One basic system employs glucose oxidase layered

over a platinum electrode. As the reaction proceeds and oxygen is consumed, i.e. glucose

+ oxygen —> gluconic acid + hydrogen peroxide, the reduction in oxygen levels is

detected by the underlying electrode. However, problems of enzyme inactivation in

vivo, competition between glucose and oxygen in body fluids and calibration have

prevented the adoption of this system as an implantable glucose monitor in diabetic

patients. However, there are currently a number of major research efforts in this area

and it is likely that biosensors employing immobilized enzymes which are potentially

useful for monitoring many substances of clinical importance will become readily

available in the not-too-distant future.

486 Chapter 25