SHIKHA KUMARIM.TECH-I

BIOCHEMICAL ENGINEERING

Production of Antifungal Antibiotics

Introduction

Antifungal antibiotics are used to treat fungal infections, which are most commonly found on the skin, hair and nails.

They come as creams, sprays, solutions, shampoos, tablets designed to go into the vagina (pessaries), medicines to take by mouth, and injections.

The length of treatment depends on what type of fungal infection you have, how severe it is and if you have any other health problems - for example, problems with your immune system.

Site of Action of Antifungal Drugs

GriseofulvinInhibits mitoticspindle formation

Classification of Antifungal Drugs

1- Antifungal Antibiotics :GriseofulvinPolyene macrolide : Amphotericin- B &

Nystatin2- Synthetic :Azoles :A) Imidazoles : Ketoconazole , MiconazoleB) Triazoles : Fluconazole , Itraconazole

THE COST OF RESEARCH AND DEVELOPMENT

Candidate compounds have to pass tests for: ToxicityAllergic effectsMutagenicityCarcinogenicity

The estimated cost of a new drug is between $100 million to $500 million.

Development can take as long as 5-10 years.

Manufacturers Of Antifungal Antibiotics

Versicor and Biosearch Italia, US based company

PURE SOURCE, FLORIDAAvni Chlorochem Pvt. Ltd., VadodaraAlichem Pvt. Ltd., NilangaRemedica Products, Europe

ORIGIN, CHEMICAL NATURE, AND ACTIVITY OF ANTIFUNGAL ANTIBIOTICS

Antibiotic Producing organism

Chemical nature

Active upon

Clavicin Aspergillus clavatus

Unsaturated ketone

Bacteria and fungi

Gliotoxin Trichoderma Contains sulfur andnitrogen

Bacteria and fungi

Trichothecin Trichothecium roseum

Unsaturated ketone

Fungi

Viridin Triclhoderma viride

Contains carbon,hydrogen, oxygen

Fungi

Eumycin Bacillus subtilis

Alcohol -soluble

Bacteria and fungi

Pyocyanin Pseudomonas aeruginosa

a-ketophenazine

Bacteria and fungi

Griseofulvin

Griseofulvin is an antifungal antibiotic first isolated from a Penicillium species in 1939.

It was discovered by Harold Rastrick and his team. It is a secondary metabolite produced by the fungus

Penicillium griseofulvum. The compound is insoluble in water, and slightly soluble in

ethanol, methanol, acetone, benzene, CHCl3, ethyl acetate, and acetic acid.

Griseofulvin binds microtubule proteins, inhibit cell wall synthesis ,inhibits nuclear division.

Induces apoptosis in human tumor cell lines.

Production Of Griseofulvin

• Fermentation has been widely used for the production of a wide variety of substances that are highly beneficial to individuals and industry.

• Over the years, fermentation techniques have gained immense importance due to their economic and environmental advantages.

• Ancient techniques have been further modified and refined to maximize productivity.

• This has also involved the development of new machinery and processes.

• Two broad fermentation techniques have emerged as a result of this rapid development: Submerged Fermentation and Solid State Fermentation .

Preparation of media

Medium Czapek Dox Medium

Chemicals Glucose 5% NaNO3 0.2% KH2PO4 0.1% Mg2SO4. 7H2O 0.05%

Steps involved in the manufacturing process

Fermentation

Pretreatment Of fermentation broth

Filtration

Extraction

Decolorization

Isolation and Separation

Precipitation and Purification

Schematic Representation

Submerged-State Fermentation

The pH of Czapek-Dox medium adjusted between 6.0-7.2. Medium dispensed in the fermenter .Fresh sample of mycelial suspension of fungus Penicillium

griseofulvum obtained.Solution autoclaved for 200 minutes at 120°C at 15lbs

pressure and fermented for 14 days at 24°C.Broth heated above 60°C for 20-30minutesPeriod of heating may be short, 5-10 minutes at 80°C

having been found to provide a satisfactory increase in filtration rate.

Pre treatment of fermentation broth

The broth is heated above 60°C for 20-30minutes.

After heating, sufficient coagulation of material occurs to produce a valuable improvement in separation characteristics of the broth.

Filtration

Drum covered with diatomaceous earth matter and allowed to rotate under vacuum with half immersed in the slurry tank.

Small amount of coagulation agent added to broth and pumped into the slurry tank.

As drum rotates in the slurry tank under vacuum thin layer of coagulated particles adhere to drum.

The layer thickens to form cake. As the cake portion in the drum comes to the upper region which is

not immersed in the liquid it is washed with water and dewatered immediately by blowing air over it.

Then before the dried portion is again immersed into the liquid it is cut off from drum by knife.

Extraction

Griseofulvin is extracted in the cold acetone when it is used as an extraction agent.

The extractions with the cold acetone may be carried out with the efficiencies between 75-96% or even upto 99.5%.

The quantity of the solvent used in the extraction at large scale production should be kept minimum.

The volume of acetone should be 3-5 times of the mycelial left.

Decolorization

The color of the extract can be improved by the addition of calcium hydroxide usually 2.5-50 g/liter preferably 5-30 g/liter.

The pH of the extract should be above 10. It can be neutralize by the removal of lime or by using

mineral acid.

Isolation and Separation

The impurities or waxy substances are removed by washing the extract with a solvent in which extract is immiscible and also griseofulvin is insoluble.

Hydrocarbon solvents, generally aliphatic hydrocarbons such as hexane or petroleum containing a high portion of hexane are in general suitable for this step.

Precipitation and Purification

Griseofulvin can be precipitated from the solvent extract in various ways. One of the method is using the liquid solvent in which griseofulvin is substantially insoluble. Griseofulvin non-solvent is preferably water.

The alkaline water is more effective for the removal of colored impurities present in the crystals of the griseofulvin.

Water is made alkaline with ammonia or an alkali metal carbonate or alkali metal hydroxide. The suitable pH is about 8.5.

The purity of the precipitate is generally improved by washing with a solvent for the small quantities of impurities remaining. The suitable washing media are dry or wet acetone, a lower alkanol for example methanol or butanol. Marked purification is obtained with the use of methanol for this step.

•Submerged fermentation utilizes free flowing liquid substrates, such as molasses and broths. •The bioactive compounds are secreted into the fermentation broth.•The substrates are utilized quite rapidly ; hence need to be constantly replaced/supplemented with nutrients. •This fermentation technique is best suited for microorganisms such as bacteria that require high moisture content. •An additional advantage of this technique is that purification of products is easier. •Submerged Fermentation is primarily used in the extraction of secondary metabolites that need to be used in liquid form.

Submerged Fermentation (SmF)

Solid State Fermentation Solid state Fermentation utilizes solid substrates like bran,

bagasse, and paper-pulp. The main advantage of using these substrates is that nutrient-

rich waste materials can be easily recycled as substrates. In this fermentation technique, the substrates are utilized

very slowly and steadily, so the same substrate can be used for long fermentation periods.

Hence, this technique supports controlled release of nutrients.

SSF is best suited for fermentation techniques involving fungi and microorganisms that require less moisture content.

However, it cannot be used in fermentation processes involving organisms that require high water activity, such as bacteria. (Babu and Satyanarayana, 1996).

Uses

Anti-inflammatory propertiesInhibits keratolytic actionIt is used in the treatment of

Ringworm of the Scalp Ringworm of the Body Ringworm of beard Fungal Disease of the Nails Ringworm of Groin Area

Diseases Being Treated

Side-effects

The most common side-effects are• Nausea• Vomiting• Diarrhoea• Flatulence, cracking at the side of the mouth• Soreness • Blackening of the tongue and thirst• Headache• Peripheral neuritis• Mental confusion• Fatigue• Blurred vision• Increases alcohol intoxication

References

Hazen et al., U.S. Pat. No. 2,797,183, supraDutcher et al., Antibiotics Annual, l953l954,

pages 191-194, Medical Encyclopedia, Inc., New York, NY.

U.S. Pat. No. 2,786,781 to Vandeputte et al.http://patient.info/health/antifungal-medicine

s

http://www.life-worldwide.org/fungal-diseases/griseofulvin

References

US patents US Patent 3069329A Published on Dec 18, 1962 published by

Dorey Michael John, Ivor L S Mitchell, David W Rule, Walker Cecile US Patent 3616247 Published on Nov 4, 1968 published by

Harold George Hemming, Malcolm Lehan, David Giles US Patent 2986496 Published on May 30, 1961 published by

Alan Rhodes, Bracknell And Derek L. Fletcher US Patent 3152150 Published on Oct 6, 1964 published by

Wilson Alan, Arthur P Best, Arthur R Lockwood, Alan H Raper, Gordon C Sayer, Richards John Willis