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LAB MANUAL OF THE

CHEMOTHERAPY

(For Students of Pharmacology of Veterinary, Medical and Pharmacy Disciplines)

By

DR. GOVIND PANDEY

BVSc & AH, MVSc & AH, PhD Hon. (Pharmacol.), DSc (std.), LLB, LLM, MBA,

MA (Soc.), MA (Hin.), MA (Eng.), MA (Pol.), Acharya (Jyotish), PGDPA & LSG,

PGDCA, AvR, MDEH, SR, DNHE, AIT, PGPHT, FSLSc, FASAW, FISCA

Professor / Principal Scientist & Sectional Head

Department of Pharmacology & Toxicology, College of Veterinary Science &

Animal Husbandry, Rewa (The Nanaji Deshmukh Veterinary Science University,

Jabalpur), MP, India

2013

International E - Publication

www.isca.me , www.isca.co.in

International E - Publication 427, Palhar Nagar, RAPTC, VIP-Road, Indore-452005 (MP) INDIA

Phone: 91-731-2616100, Mobile: 91-8057083382, E-mail: [email protected]

Website: www.isca.me , www.isca.co.in

© Copyright Reserved

2013 All rights reserved. No part of this publication may be reproduced, stored, in

a retrieval system or transmitted, in any form or by any means, electronic,

mechanical, photocopying, reordering or otherwise, without the prior

permission of the publisher.

ISBN: 978-93-83520-01-5

International E – Publication


International Science Congress Association




iv

PREFACE

“Chemotherapy” in its most general sense, is the treatment of disease by chemicals,

especially by killing microorganisms or cancerous cells. In popular usage, it refers to

‘antineoplastic/anticancer drugs’ used to treat cancer; or the combination of these drugs

is used into a cytotoxic standardized treatment regimen. In its non-cancerous use, the

chemotherapy refers to ‘antibiotics or antibacterial chemotherapy’. An older and broader

usage of the term “chemotherapy” encompassed any chemical treatment of disease (e.g.,

treatment of infections by antimicrobial agents). However, this usage has become archaic.

The word “chemotherapy” without a modifier usually refers to ‘cancer treatment’, but its

historical meaning is broader.

Keeping the above definition of chemotherapy in view, this “Lab Manual of the

Chemotherapy” has been putforth to enable the students to develop a strong knowledge

of chemotherapy drugs involved in the treatment of various diseases or infections. As

described in this manual, such chemotherapy drugs may be antibacterial (viz.,

sulphonamides, nitrofurans and antibiotics), antifungal, anthelmintic, antiprotozoan or

antineoplastic (cytotoxic) drugs, etc. The students will also learn how the safe and

rational use of these drugs is potential against the disease. Thus, the aim of the manual is

also to enable the students to have a great understanding of the chemotherapy drugs,

particularly in respect to the treatment of clinical diseases. Overall, the manual will fulfil

the practical objective of the students as well as the concerned teachers. The experiments

described here may be fruitful and resourceful materials for not only to the Pharmacology

of Veterinary Science but also for the Pharmacology of Medical or Pharmacy Sciences.

Mainly the experiments included in the manual are: chemotherapy in general, various

chemotherapy drugs, some chemotherapeutic preparations, spectrophotometry, testing of

bacterial sensitivity, assay of antibiotics, estimation of sulphonamides, estimation of

oxytetracycline, estimation of tetracycline and doxycycline, estimation of

chloramphenicol, estimation of pefloxacin/ofloxacin, and estimation of cefotaxime.

In this context, it is my immense pleasure by putting the “Foreword” of Dr. Y.P.

Sahni, Director of Research Services, Nanaji Deshmukh Veterinary Science University

(NDVSU), Jabalpur, MP, India. My sincere thanks and deep regards are devoted to him. I

am extremely thankful to Dr. Nitesh Kumar, Dr. Arpita Shrivastav, Dr. Swatantra Kumar

Singh and Dr. Anjana Panicker (all teachers of Veterinary Pharmacology and Toxicology

in the NDVSU, Jabalpur). Last but not the least, I gratefully acknowledge to the

‘Laboratory Manual: Veterinary Chemotherapy’ (compiled by Dr. Arpita Shrivastav) and

other authors/publishers/books/websites, from where the matters and photographs have

been chosen and incorporated in the manual; I especially acknowledge to all those.

26th

July, 2013 DR. GOVIND PANDEY




v

CONTENTS

S. No. Description Page

- Foreword iii

- Preface iv

- Contents v

1 Chemotherapy in General

Objective

Meaning and Definition of Chemotherapy

History of Chemotherapy

Action of Chemotherapy

Chemotherapy Treatment Schemes in Cancer

Cytotoxic Antibiotics

Uses of Chemotherapy

Dosage of Chemotherapy

Resistance by Chemotherapeutic Drugs

Limitations of Chemotherapy

Side Effects or Toxicity of Chemotherapy

Occupational Precautions

1

1

1

2

3

3

4

4

5

7

8

9

12

2 Various Chemotherapy Drugs : Antibacterials

(Sulphonamides and Nitrofurans)

Objective

Sulphonamides (Antibacterial Drugs)

Nitrofurans (Antibacterial Drugs)

13

13

13

14

3 Various Chemotherapy Drugs : Antibacterials (Antibiotics)

and Antifungal Drugs Objective

Antibacterial Antibiotics

Antifungal Drugs

15

15

15

19

4 Various Chemotherapy Drugs : Anthelmintics and

Ectoparasiticides Objective

Anthelmintics

Ectoparasiticides and Ectoparasiticides

20

20

20

22

5 Various Chemotherapy Drugs : Antiprotozoans

Objective

Antiprotozoans

23

23

23

6 Various Chemotherapy Drugs : Antineoplastic (Cytotoxic)

Drugs Objective

Antineoplastic/Anticancer Drugs

25

25

25

7 Some Chemotherapeutic Preparations : Solutions

Objective

Potassium Permanganate Solution

Lugol’s Iodine Solution

Tincture Iodide (Tincture of Iodine)

Trypan Blue Solution

Gentian Violet Solution

26

26

26

27

27

28

28




vi

8 Some Chemotherapeutic Preparations : Ointments

Objective

Zinc Oxide Ointment

Whit’s Fluid Ointment (Ointment of Salicylic Acid with

Benzoic Acid)

Boric Acid Ointment

29

29

29

29

30

9 Spectrophotometry

Objective

Principle

Calculation of Concentration of Unknown Substance

General Procedure of Spectrophotometry

31

31

31

33

34

10 Testing of Bacterial Sensitivity

Objective

Methods of Bacterial Sensitivity Testing

Determination of Minimum Inhibitory and Bactericidal

Concentrations

36

36

36

37

11 Assay of Antibiotics

Objective

Biological Assay of Antibiotics

Physicochemical Assay of Antibiotics

38

38

38

38

12 Estimation of Sulphonamides

Objective

Principle

Requirement

Method

39

39

39

39

40

13 Estimation of Oxytetracycline

Objective

Principle

Requirement

Method

42

42

42

42

43

14 Estimation of Tetracycline and Doxycycline

Objective

Principle

Requirement

Method

44

44

44

44

45

15 Estimation of Chloramphenicol

Objective

Principle

Requirement

Method

46

46

46

46

47

16 Estimation of Pefloxacin/Ofloxacin

Objective

Requirement

Method

48

48

48

48

17 Estimation of Cefotaxime

Objective

Requirement

Method

50

50

50

50

- About Author : Dr. Govind Pandey 52




1

1

CHEMOTHERAPY IN GENERAL

OBJECTIVE

To know about the chemotherapy and its various aspects regarding treatment of

diseases in humans and animals.

MEANING AND DEFINITION OF CHEMOTHERAPY

“Chemotherapy” is the ‘use of medications (chemicals) to treat disease’.

Chemotherapy may include the use of antibiotics or other medications to treat any illness

or infection. On the other hand, chemotherapy in its most general sense, is the treatment

of disease by chemicals, especially by killing microorganisms or cancerous cells. In

popular usage, it refers to ‘antineoplastic drugs’ used to treat cancer; or the combination

of these drugs is used into a cytotoxic standardized treatment regimen. In its non-

cancerous use, the chemotherapy refers to ‘antibiotics or antibacterial chemotherapy’.

In relation to treatment of cancer, the chemotherapy (also abbreviated as ‘chemo’) is

the ‘treatment of cancer with one or more chemotherapeutic agents (or cytotoxic

antineoplastic drugs) as part of a standardized regimen’. In other words, ‘chemotherapy

typically refers to the destruction of cancer cells’. It may be given with a curative intent,

or it may aim to prolong the life or to palliate the symptoms.

An older and broader usage of the term “chemotherapy” encompassed any chemical

treatment of disease (e.g., treatment of infections by antimicrobial agents). However, this

usage has become archaic. The word “chemotherapy” without a modifier usually refers to

‘cancer treatment’, but its historical meaning is broader.

However, in respect to this course book, the chemotherapy broadly includes the

following categories of drugs:

1. Antibacterial drugs.

2. Antitubercular drugs.

3. Antileprotic drugs.

4. Antiviral drugs.




2

5. Antifungal drugs.

6. Antiprotozoal drugs.

7. Antihelminthic drugs (Anthelmintics).

8. Antineoplstic/anticancer/cytotoxic drugs.

HISTORY OF CHEMOTHERAPY

The first modern chemotherapeutic agent was ‘arsphenamine’ (an arsenic

compound) discovered by Paul Ehrlich in the year 1909, and used to treat the syphilis.

This was later followed by the ‘sulphonamides’ (sulpha drugs, discovered by Domagk)

and ‘penicillin’ (discovered by Alexander Fleming, 1928-29).

The use of minerals and plant-based medicines is believed to date back to prehistoric

medicine. Therefore, in the 1940s, several patients with advanced lymphomas (cancers of

certain white blood cells) were given the drug by vein, rather than by breathing the

irritating gas. Their improvement, although temporary, was remarkable. That experience

led researchers to look for other substances that might have similar effects against the

cancer. As a result, many other drugs have been developed to treat the cancer, and drug

development since then has exploded into a multibillion-dollar industry, although the

principles and limitations of chemotherapy discovered by the early researchers still apply.

After a military operation in World War II, some sailors were accidentally exposed

to mustard gas. They were later found to have very low white blood cell (WBC) counts.

The WBCs usually grow very quickly and cancer cells also divide and grow very quickly.

The doctors wondered whether the effect of mustard gas- slowing down the rapid growth

of WBCs- may have same effect on cancer cell growth. There are more than 100 different

types of chemotherapeutic drugs (Fig. 1) today, which can treat the most cancers.

Fig. 1: Some Chemotherapy Drugs (Source: Google website, gratefully acknowledged)




3

ACTION OF CHEMOTHERAPY

Chemotherapy mostly acts by killing the cells which divide rapidly, one of the main

properties of cancer cells. It means that it also harms cells which divide rapidly under the

normal circumstances: cells in the bone marrow, digestive tract and hair follicles. The

newer anticancer drugs act directly against abnormal proteins in the cancer cells; this is

termed as ‘targeted therapy’.

Chemotherapy drugs attack cells in the process of growth and division. Individual

drugs may work through many different mechanisms, such as damaging a cell’s genetic

material (DNA) or preventing the cell from dividing. However, the chemotherapeutic

drugs can not distinguish between malignant cancer cells and normal cells. All rapidly

dividing cells are potentially sensitive to chemotherapy.

Cytotoxic drugs may lead to complete remissions for some disseminated cancers

(e.g., lymphoma), may be effective in decreasing tumour size and may prolong the life in

many other types of metastatic cancers like osteosarcoma. The choice of specific

therapies depends on tumour type (what it is), histologic grade of tumour (how aggressive

it is), stage of disease (where it is) and patient’s (but mainly the pet owner’s) tolerance

for the side effects of various treatments. Most of the chemotherapy protocols designed

for veterinary patients have a < 5% incidence of severe, life-threatening complications.

Most veterinary chemotherapy patients enjoy a good quality of life while on therapy.

CHEMOTHERAPY TREATMENT SCHEMES IN CANCER

There are a number of strategies in the administration of chemotherapeutic drugs

used today for the treatment of cancer/tumour. The ‘combined modality chemotherapy’ is

the use of drugs with other cancer treatments like radiation therapy or surgery. The most

cancers are now treated in this way. The combination chemotherapy is a similar practice

which involves treating a patient with a number of different drugs simultaneously. The

drugs differ in their mechanism and side effects. The biggest advantage is minimizing the

chances of resistance developing to any one agent.

In ‘neoadjuvant chemotherapy’ (pre-operative treatment), the initial chemotherapy is

designed to shrink the primary tumour, thereby rendering the local therapy (surgery or

radiotherapy) less destructive or more effective. The ‘adjuvant chemotherapy’ (post-

operative treatment) can be used when there is little evidence of cancer present, but there

is risk of recurrence. This can help reduce chances of developing resistance if the tumour




4

does develop. It is also useful in killing any cancerous cells which have spread to other

parts of the body. This is often effective as the newly growing tumours are fast-dividing,

and therefore, very susceptible. The ‘palliative chemotherapy’ is given without curative

intent, but simply to decrease the tumour load and increase life expectancy. For these

regimens, a better toxicity profile is generally expected.

All chemotherapy regimens require that the patient be capable of undergoing the

treatment. Performance status is often used as a measure to determine whether a patient

can receive chemotherapy, or whether dose reduction is required. Because only a fraction

of the cells in a tumour dies with each treatment (fractional kill), the repeated doses must

be administered to continue to reduce the size of tumour. The current chemotherapy

regimens apply the drug treatment in cycles, with the frequency and duration of

treatments limited by toxicity to the patient.

CYTOTOXIC ANTIBIOTICS

The ‘cytotoxic antibiotics’ are a varied group of drugs which have various

mechanisms of action. This class of drugs includes anthracyclines and other drugs,

including actinomycin, bleomycin, plicamycin and mitomycin. Doxorubicin and

daunorubicin were the first two anthracyclines (obtained from the bacterium

Streptomyces peucetius). The derivatives of these compounds are epirubicin and

idarubicin. Other clinically used drugs in the anthracyline group are pirarubicin,

aclarubicin and mitoxantrone. The mechanisms of anthracyclines include the DNA

intercalation (molecules insert between the two strands of DNA), generation of highly

reactive ‘oxygen free radicals’ (which damage the intercellular molecules) and

topoisomerase inhibition. Actinomycin is a complex molecule which intercalates DNA

and prevents RNA synthesis. Bleomycin (a glycopeptide isolated from Streptomyces

verticillus) also intercalates DNA, but produces free radicals that damage the DNA. This

occurs when bleomycin binds to a metal ion, becomes chemically reduced and reacts with

the oxygen. Mitomycin is a cytotoxic antibiotic with the ability to alkylate the DNA.

USES OF CHEMOTHERAPY

Chemotherapy was historically used for non-oncological (non-cancerous) references,

such as the use of antibiotics (antibacterial chemotherapy). But, it is often used in

conjunction with other cancer treatments, e.g., radiation therapy or surgery. Certain




5

chemotherapeutic agents also have a role in the treatment of other conditions, including

ankylosing spondylitis, multiple sclerosis, Crohn’s disease, dermatomyositis, psoriasis,

psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, polymyositis,

scleroderma and suppression of transplant rejections.

Some newer anticancer drugs (e.g., various monoclonal antibodies) are not

indiscriminately cytotoxic, but rather target proteins that are abnormally expressed in

cancer cells and that are essential for their growth. Such treatments are often referred to

as ‘targeted therapy’ (as distinct from ‘classic chemotherapy’), and are often used

alongside traditional chemotherapeutic agents in antineoplastic treatment regimens. As

said, the chemotherapy is often used alongside other treatments. The doctors and patients

should take physical exercise seriously, because of the benefits for many cancer patients.

Patients who regularly exercise after the chemotherapy treatment, have a much lower risk

of cancer recurrence, compared to the people who are physically inactive.

Chemotherapy is likely to be recommended for cancer that has already spread to

other areas of the body (metastatic disease), for tumours which occur at more than one

site (multicentric disease), or for tumours that can not be removed surgically (non-

resectable disease). In some cases, chemotherapy can be used to try to shrink the large

tumours prior to surgery or to help eradicate certain types of microscopic cancer cells that

can not or have not been completely removed surgically. For cancers that are at high-risk

for metastasis early in the course of disease, chemotherapy can be used after surgery or

radiation therapy to help slow down the growth of cancer cells in other parts of the body.

In animals also, chemotherapy is used to treat the systemic cancers, the cancers that

have already spread or metastasized, or the cancers that are likely to spread or

metastasize. Sometimes, the chemotherapy can used to treat the cancers that can not be

treated with other methods, such as surgery or radiation therapy.

DOSAGE OF CHEMOTHERAPY

The dosage of chemotherapy can be difficult. If the dose is too low, it will be

ineffective against the tumour; whereas, at excessive doses, the toxicity (side effect) will

be intolerable to the patient. This has led to the formation of detailed ‘dosing schemes’ in

most hospitals, which give guidance on the correct dose and adjustment in case of

toxicity. In immunotherapy (treatment of autoimmune disorders), the chemotherapeutic

agents are used in smaller dosage than in the treatment of malignant diseases.




6

The standard method of calculating dosage is based on the calculated ‘body surface

area’ (BSA), a measure which correlates with blood volume. The BSA is usually

calculated with a mathematical formula or a nomogram, using a patient’s weight and

height, rather than by direct measurement. This method was originally used in the 1960s

to calculate a uniform dose to patients in clinical trials. Recently, the validity of this

method in calculating uniform doses has been questioned. The reason for this is that the

formula only takes into account of the individual’s weight or height. Other factors, like

drug clearance, have major effects on the actual dose to an individual on chemotherapy,

which can lead to sub-optimal dosing. Also, calculating the BSA for obese patients could

provide a dose that is too high (overdosing), and therefore, most chemotherapy drugs are

dose-capped (an upper limit of dose). Carboplatin is one of the only drugs that are not

dosed depending on the BSA method. Another method, ‘area under the curve’ (AUC), is

employed to individualize the dosing. In this method, the drug levels in the blood plasma

are measured overtime. The area under this curve is used in conjunction with the renal

function of the individual receiving the chemotherapy to obtain the optimal dose.

Most chemotherapy is delivered intravenously, although a number of agents can be

administered orally (e.g., melphalan, busulfan, capecitabine). There are many intravenous

(iv) methods of drug delivery, known as ‘vascular access devices’. These include the

winged infusion device, peripheral cannula, midline catheter, peripherally inserted central

catheter (PICC), central venous catheter and implantable port. The devices have different

applications regarding duration of chemotherapy treatment, method of delivery and types

of the chemotherapeutic drugs.

Depending on the patient, the cancer, the stage of cancer, the type of chemotherapy,

and dosage, the iv chemotherapy may be given on either an inpatient or an outpatient

basis. For continuous, frequent or prolonged iv chemotherapy administration, different

systems may be surgically inserted into the vasculature to maintain access. Commonly

used systems are the Hickman line, the Port-a-Cath, and the PICC line. These have a

lower infection risk, are much less prone to phlebitis or extravasation, and eliminate the

need for repeated insertion of peripheral cannulae. Isolated limb perfusion (often used in

melanoma), or isolated infusion of chemotherapy into the liver or lung have been used to

treat some tumours. The main purpose of these approaches is to deliver a very high dose

of chemotherapy to tumour sites without causing overwhelming systemic damage. These

approaches can help control the solitary or limited metastases, but they are by definition




7

not systemic, and therefore, do not treat the distributed metastases or micrometastases. If

the cancer has central nervous system (CNS) involvement, or with meningeal disease, the

intrathecal chemotherapy may be administered.

In animals, chemotherapy can be given intravenously (the most common method),

intramuscularly, subcutaneously or orally for tumour/cancer. The type of cancer, extent

of disease and general health of animal help the oncologists to formulate a treatment

protocol (type of drugs, dose and schedule used) appropriate for the pet. Some drugs are

oral medications (pills) which can be given at home. Others are brief injections that

require an outpatient appointment. In some instances, slow infusions or repeated

treatments throughout the day may require an animal to spend the day in the hospital. The

treatments are typically repeated weekly for every third week. Blood tests may be needed

to monitor the effects of chemotherapy during the weeks between the treatments.

Duration of chemotherapy depends on the type of cancer and extent of disease. Some

animals need to receive the chemotherapy for the rest of their lives. In others, the

treatments may be spread out or discontinued after a period of weeks to months provided

that the cancer is in remission, i.e., there is no detectable evidence of cancer in the body.

The chemotherapy can be resumed when the cancer relapses. We usually recommend that

every patient should receive at least two cycles of chemotherapy, and then be evaluated

for response before we decide to continue the treatment, change the drugs or discontinue

the chemotherapy. It is imperative that you, as a pet owner, are committed to treatment

and that you bring your pet to the veterinary hospital when scheduled for therapy.

RESISTANCE BY CHEMOTHERAPEUTIC DRUGS

Development of resistance by the chemotherapeutic drugs is a major cause of

treatment failure. There are a few possible causes of resistance in cancer, one of which is

the presence of small pumps on the surface of cancer cells that actively move

chemotherapy from inside the cell to the outside. The cancer cells produce high amounts

of these pumps, known as p-glycoprotein, in order to protect themselves from

chemotherapeutics. Another mechanism of resistance is gene amplification, a process in

which multiple copies of a gene are produced by the cancer cells. This overcomes the

effects of drugs that reduce the expression of genes involved in replication. With more

copies of the gene, the drug can not prevent all expression of the gene, and therefore, the

cell can restore its proliferative ability. The cancer cells can also cause defects in the




8

cellular pathways of apoptosis (programmed cell death). As most chemotherapy drugs

kill cancer cells in this manner, the defective apoptosis allows survival of these cells,

making them resistant. Many chemotherapy drugs also cause DNA damage, which can be

repaired by enzymes in the cell that carry out DNA repair. Upregulation of these genes

can overcome the DNA damage and prevent the induction of apoptosis. Mutations in

genes that produce drug target proteins like tubulin, can occur which prevent the drugs

from binding to the protein, leading to resistance to these types of drugs.

Some chemotherapy drugs are used in diseases other than the cancer, such as in

autoimmune disorders. They are often used at lower doses, which means that the side

effects are minimized. Methotrexate is used in the treatment of rheumatoid arthritis,

psoriasis, ankylosing spondylitis and multiple sclerosis. Antiinflammatory response seen

in the rheumatoid arthritis is thought to be due to increases in adenosine, which causes

immunosuppression; effects on immunoregulatory cyclooxygenase-2 enzyme pathways;

reduction in proinflammatory cytokines; and antiproliferative properties. Although

methotrexate is used to treat both multiple sclerosis and ankylosing spondylitis, its

efficacy in these diseases is uncertain. Cyclophosphamide is used to treat lupus nephritis.

LIMITATIONS OF CHEMOTHERAPY

Chemotherapy does not always work, and even when it is useful, it may not

completely destroy the cancer. Patients frequently fail to understand its limitations. The

blood brain barrier (BBB) poses a difficult obstacle to pass to deliver chemotherapy to

the brain. This is because the brain has an extensive system in place to protect it from

harmful chemicals. The drug transporters can pump out drugs from the brain and brain’s

blood vessel cells into the cerebrospinal fluid (CSF) and blood circulation. These

transporters pump out most chemotherapy drugs, which reduce their efficacy for

treatment of brain tumours. Only small lipophilic alkylating agents like temozolomide are

able to cross this BBB.

The blood vessels in tumours are very different from those seen in normal tissues. As

a tumour grows, the tumour cells furthest away from the blood vessels become low in

oxygen (hypoxic). To counteract this, they then signal for new blood vessels to grow. The

newly formed tumour vasculature is poorly formed and does not deliver an adequate

blood supply to all areas of the tumour. This leads to issues with drug delivery because

many drugs will be delivered to the tumour by the circulatory system.




9

SIDE EFFECTS OR TOXICITY OF CHEMOTHERAPY

Toxicity to normal, rapidly growing or self-renewing tissues in the body is the reason

for most of the side effects seen with chemotherapy. Fortunately, these normal tissues

continue to grow and repair themselves, so the injury caused by chemotherapy is rarely

permanent. The most common side effects of chemotherapy include: myelosuppression

(decreased production of blood cells), mucositis (inflammation of the lining of digestive

tract) and alopecia (loss of hair).

Chemotherapeutic techniques have a range of side effects which depend on the type

of medications used. The most common medications affect mainly the fast-dividing cells

of the body, such as blood cells, and the cells lining the mouth, stomach and intestines.

Chemotherapy related toxicities can occur acutely after administration, within hours or

days, or chronically, from weeks to years. Less common side effects include red skin

(erythema), dry skin, damaged fingernails, dry mouth (xerostomia), water retention and

sexual impotence. Some medications can trigger allergic or pseudoallergic reactions.

The specific chemotherapeutic agents are associated with organ-specific toxicities,

including cardiovascular disease (e.g., doxorubicin), interstitial lung disease (e.g.,

bleomycin) and occasionally secondary neoplasm (e.g., therapy for Hodgkin’s disease).

Compared to people who receive chemotherapy, the pet animals have fewer and less

severe side effects because we use lower doses in them, and do not combine as many

drugs as in humans. Normal tissues which typically are most sensitive to chemotherapy,

are the intestinal lining, bone marrow (which makes red and white blood cells) and hair

follicles. The toxic effects to gastrointestinal tract (GIT) are responsible for decreased

appetite, vomiting and diarrhoea. These effects may be mild, moderate or severe. In most

cases, these signs are mild and usually resolve on their own or with oral medication given

at home. Although infrequent, some dogs (and cats) may develop severe diarrhoea

requiring hospitalization and fluid therapy. In many cases, the GIT side effects from

chemotherapy are not seen on the day of treatment; they often occur 3 to 5 days later.

Suppression of the bone marrow by chemotherapeutic drugs may cause a drop in the

WBC count, leading to increased susceptibility to infection. The infection usually comes

from the animal’s own body (e.g., bacteria normally found in the intestines, mouth, skin,

etc.). Severe infections may require hospitalization for intensive supportive care,

including iv fluid and antibiotics. When a chemotherapeutic drug is used that is known to

have a high potential for bone marrow suppression, a complete blood count (CBC) is




10

checked several days after the treatment. If the WBC count is low but the pet is feeling

well, the antibiotics are prescribed as a preventative measure. Subsequent doses of

chemotherapy are adjusted based on the results of the CBC. Anaemia (low red blood cell

count) is often a complication of cancer but is rarely caused by the chemotherapy drugs

used in veterinary medicine.

Hair follicle cell in dogs (and cats) that are wire-haired or non-shedding may be

particularly susceptible to the chemotherapy. Certain breeds of dogs, such as terriers and

poodles, will experience variable amounts of hair loss. The hair loss often is most evident

on the face and tail. Whiskers and the long hairs over the eyes often fall out in cats. The

hairs will regrow once the chemotherapy is stopped, but may initially have a modest

change in colour or texture.

There are many different types of chemotherapy agents, and each has a different

likelihood of causing side effects. If the pet is treated with drugs known to cause certain

side effects, prescribe the medications to help prevent these complications, such as

antiemetics (anti-nausea/anti-vomiting medication). In addition, give the instructions on

what to do if and when a problem arises. Severe side effects are seldom seen as described

above; it is estimated to be less than 5% of all pets receiving the chemotherapy. With

proper management, most animals recover uneventfully within a few days. Keep in mind

that any animal can have an unexpected reaction to any medication.

When people think of chemotherapy, they generally assume horrible side effects like

severe vomiting, diarrhoea, weight loss, lethargy and an overall poor quality of life.

However, in veterinary medicine, the majority of patients (80-85%) have minimal to no

side effects when receiving the chemotherapy. There are a couple of reasons for this

result. First, in veterinary medicine, we do not use the large doses and aggressive

chemotherapy protocols used in human medicine. Our primary goal in veterinary

oncology is to maintain and extend the pet’s quality of life. Therefore, we design our

chemotherapy protocols with this goal in mind. Second, pets generally do not experience

severe adverse effects from chemotherapy because they tolerate the drugs better than

people. In veterinary oncology, we are very proactive to prevent adverse side effects from

chemotherapy. If dogs or cats experience the side effects while going through

chemotherapy, they can usually be mitigated with protocol adjustments and supportive

medications at home. There have been great advances in antiemetic therapy, as well as

antibiotic therapy, which have been able to greatly reduce the number and severity of




11

adverse effects secondary to the chemotherapy.

In animals, the toxicities due to chemotherapy drugs are most often acute, but

chronic or delayed effects do occur. An index of suspicion for these problems is essential

for their diagnosis. The chemotherapy kills rapidly dividing cells. Unfortunately, the

chemotherapy drugs do not differentiate between the killing tumour cells and normal

cells. Thus, the general side effects of chemotherapy include bone marrow suppression,

gastrointestinal problems (nausea, vomiting and diarrhoea) and alopecia. However, in

addition to the general side effects seen, the specific side effects can result from certain

drugs, e.g., doxorubicin (cardiotoxicity) and cisplatin (fatal pulmonary edema in cats, and

renal toxicity in dogs). The most common toxicity associated with chemotherapy is the

bone marrow suppression. The bone marrow cells divide rapidly because there is

normally a high growth fraction in this tissue. Since activity of most anticancer drugs is

greatest in tissues with a high growth fraction, the bone marrow is a prime target. The

clinical result of myelosuppression is varying degrees of peripheral blood cytopenias. The

rate of disappearance of individual blood cell lines correlates with the life span of that

line. Granulocytopenia (specifically neutropenia) usually occurs first, and is most oftenly

followed by thrombocytopenia.

In dogs, the anaemia is rare and usually only mild to moderate. Neutropenia is

usually the most serious and dose limiting cytopenia associated with the

chemotherapeutic drug administration. The nadir (time of the lowest neutrophil count)

varies with individual drugs. Neutrophil counts usually rebound from the nadir. Immature

granulocytes are an indicator for return of granulocytic activity. Animals with < 1000

neutrophils/μl are at an increased risk for sepsis and require close monitoring. Neutrophil

counts < 500/μl (categorized as a grade-4 neutropenia) usually are associated with fever

and sepsis. In addition to lower neutrophil counts, the chemotherapy can result in

gastrointestinal epithelial desquamation, and the combination of these factors can lead to

increased opportunity for enteric bacteria to enter the circulation causing septicemia, with

fewer phagocytic neutrophils to effectively clear infection which can be life-threatening.

Chemotherapy works by killing rapidly dividing cells. In a dog or cat with cancer,

the most rapidly dividing cells in the body are the cancer cells. However, there are two

locations in the body where normally rapidly dividing cells are located: the bone marrow

(where our infection fighting white blood cells are produced); and the GIT. Sometimes

chemotherapy can damage the bone marrow cells to the point where the WBC count




12

decreases to a critical level allowing infection to take hold and resulting in fever,

lethargy, anorexia, etc., requiring medical intervention. This side effect of chemotherapy

is rare (less than 5%). The GIT is lined with little finger like projections called villi which

allow absorption of the nutrients we eat. The villi are constantly replaced by precursor

cells in the villi crypts. The villi crypt cells are rapidly dividing and can be affected by

the chemotherapy resulting in anorexia, nausea and diarrhoea. These effects are generally

mild, occurring in 15-20% of patients receiving chemotherapy and are usually responsive

to supportive medications and diet change. Rarely, the gastrointestinal side effects require

more advanced medical intervention.

OCCUPATIONAL PRECAUTIONS

The healthcare workers exposed to antineoplastic agents take precautions to keep

their exposure to a minimum. There is a limitation in cytotoxic dissolution in Australia

and the United States to 20 dissolutions per pharmacist/nurse, since pharmacists that

prepare these drugs or nurses that may prepare or administer them are the two

occupational groups with the highest potential exposure to antineoplastic agents. In

addition, the physicians and operating room personnel may also be exposed through the

treatment of patients. Hospital staff, such as shipping and receiving personnel, custodial

workers, laundry workers, and waste handlers, all have potential exposure to these drugs

during the course of their work. The increased use of antineoplastic agents in veterinary

oncology also puts these workers at risk for exposure to these drugs. The routes of entry

into the user’s body are skin absorption, inhalation and ingestion. The long term effects

of exposure include chromosomal abnormalities and infertility.

When administering the oral chemotherapy medications at home, it is very important

to follow the oncologist’s instructions regarding frequency of administration, whether to

administer the medication with food and whether other medications are contraindicated

with the chemotherapy agent. In addition, the chemotherapy pills or tablets should never

be opened or crushed. The latex gloves should always be worn when handling the

chemotherapy pills at home. The hands should be washed thoroughly after handling any

chemotherapy agent.




13

2

VARIOUS CHEMOTHERAPY DRUGS :

ANTIBACTERIALS (SULPHONAMIDES

AND NITROFURANS)

OBJECTIVE

To know the dosage and use of different sulphonamide and nitrofuran antibacterial

drugs (chemotherapeutic agents) given in animals.

SULPHONAMIDES (ANTIBACTERIAL DRUGS)

Various sulphonamides (sulpha drugs) with their dosage and use in different species

of animals have been mentioned in Table 1.

Table 1: Dosage and Use of Sulphonamides in Animals

Sulphonamides Animals with Doses and Routes of Drugs Uses

Sulphadiazine All species of animals: Initial dose- 200 mg/kg,

oral; maintenance dose- 100 mg/kg, oral, b.d.

Mixed bacterial

infections

Sulphamethazine All species of animals: Initial dose- 110 mg/kg,

sc or iv; maintenance dose (33.33% solution)-

110 mg/kg, oral, sc or iv

Mixed bacterial

infections

Sulphapyridine All species of animals: Initial dose- 132 mg/kg,

oral; maintenance dose- 66 mg/kg, oral

Mixed bacterial

infections

Sulphadimidine All species of animals: Initial dose- 200 mg/kg,

oral; maintenance dose- 100 mg/kg, oral, o.d.

Mixed bacterial

infections

Sulphadimethoxin All species of animals: Initial dose- 55 mg/kg,

oral; maintenance dose- 27.5 mg/kg, oral, o.d.

Mixed bacterial

infections

Sulphathiazole All species of animals: Initial dose- 66 mg/kg,

oral; maintenance dose- 33 mg/kg, oral

Mixed bacterial

infections

Sulphafurazole All species of animals: Initial dose- 200 mg/kg,

oral; maintenance dose- 100 mg/kg, oral, b.d.

Mixed bacterial

infections

Sulphamethoxy-

pyridazine

All species of animals: Initial dose- 55 mg/kg,

oral, sc or iv; maintenance dose- 27.5 mg/kg,

oral, sc or iv

Mixed bacterial

infections

Succinyl

sulphathiazole

All species of animals: 100-150 mg/kg, b.d. Mixed bacterial

infections

Phthalyl

sulphathiazole

All species of animals: 100-150 mg/kg, b.d. Mixed bacterial

infections

Sulphaguanidine All species of animals: 100-300 mg/kg, oral,

daily in divided doses

Mixed bacterial

infections




14

Sulphaquinoxaline All species of animals: 0.0125% in food (for

prevention); 0.05% in food or 0.04% in water

(for treatment)

Mixed bacterial

infections

Sulphacetamide

sodium

All species of animals: 30% solution or 10%

ointment, for eye

Mixed bacterial

infections

Silver sulphadiazine All species of animals: 1% cream, for surface

application

Mixed bacterial

infections

Mafenide All species of animals: 1% cream, for surface

application

Mixed bacterial

infections

Sulphadiazine +

Trimethoprim

Large animals: 15-30 mg/kg, oral, sc or im

Dogs: 15 mg/kg, b.i.d.

Infections of urinary,

genital, respiratory

and alimentary tracts

Sulphadoxine 200 mg

+ Trimethoprim 40 mg

All species of animals: 1 ml/10-15 kg, sc or im Mixed bacterial

infections

Sulphadiazine +

Trimethoprim

All species of animals: 30 mg/kg, oral and as

dispersible powder

Cows and mares: 2.4-4.0 g, as bolus

Sows and ewes: 1.2-2.4 g, intrauterine

Mixed bacterial

infections

NITROFURANS (ANTIBACTERIAL DRUGS)

Various nitrofuran compounds with their dosage and use in different species of

animals have been mentioned in Table 2.

Table 2: Dosage and Use of Nitrofurans in Animals

Nitrofurans Animals with Doses and Routes of Drugs Uses

Nitrofurazone All species of animals: 0.05% in feed for 7 days;

0.2% as cream

With feed- necrotic enteritis in

pig

As cream- bacterial infection

of wound, burn and ulcer

Nitrofurantoin Calves and horses: 10 mg/kg, oral

Dogs: 4.4 mg/kg, oral, t.i.d.; 3.3 mg/kg, im, b.i.d.

Urinary antiseptic

Furazolidone Poultry: 0.04% in feed for 7 days

Large animals: 10-12 mg/kg, for 5-7 days

Salmonellosis




15

3


ANTIBACTERIALS (ANTIBIOTICS)

AND ANTIFUNGAL DRUGS

OBJECTIVE

To know the dosage and use of different antibacterial antibiotics and antifungal drugs

given in different species of animals.

ANTIBACTERIAL ANTIBIOTICS

The dosage and use of various antibacterial antibiotics (viz., penicillins,

cephalosporins, tetracyclines, chloramphenicols, aminoglycosides, macrolides and

fluoroquinolones) prescribed in different species of animals have been described in

Tables 3 to 9.

Table 3: Dosage and Use of Penicillins in Animals

Penicillins Animals, Doses and Routes Uses

Penicillin G Large and small animals: 11000-22000 IU/kg,

im- every 4 hr for sodium or potassium salt in

water; every 4 hr for soluble salt in oil; every

24 hr for procaine penicillin G in aqueous

suspension; every 48-72 hr for procaine

penicillin G in oil with aluminium

monostearate; every 5-7 days for benzathine

penicillin G

Mostly in Gram positive (G+)

bacterial infections

Phenoxymethyl

penicillin

Small animals: 3.3-4.4 mg/kg, oral, 3-4 times

daily

Bacterial infections

Benzathine

penicillin

Large animals: 12000 IU/kg, im

Small animals (Dogs and cats): 40000 IU/kg,

im

Actinomycosis, anthrax,

tetanus, mastitis, metritis, black

leg and HS

Ampicillin

Large animals: 5-10 mg/kg, oral, im or iv,

q.i.d.

Small animals (Dogs and cats): 11-22 mg/kg,

oral, 2-3 times daily; 10 mg/kg, sc, im or iv,

q.i.d.

Infections of respiratory,

gastrointestinal and urinary

tracts, pneumonia, metritis, and

infections of skin

Ampicillin +

cloxacillin

All species of animals: As directed by the

doctor/veterinarian (as per prescription)

Septicaemia, pneumonia,

mastitis, metritis, chronic

wounds and post-surgery




16

Amoxycillin Large animals: 10 mg/kg, oral, im or iv, b.i.d.

Small animals (Dogs and cats): 10-20 mg/kg,

oral, im or iv, b.i.d.

Animals: Infections of

respiratory and urinary tracts,

mastitis, wounds, calf scours,

HS, metritis and otitis

Birds: Coryza, fowl cholera and

fowl typhoid

Amoxycillin +

clavulanate

potassium

Small animals (Dogs and cats): 12.5-25 mg/kg,

oral, b.i.d.

Infections of respiratory and

urinary tracts, skin and soft

tissues infections, and mixed

bacterial infections

Amoxycillin +

cloxacillin



Respiratory tract infections,

pneumonia, mastitis, metritis,

enteritis, septicaemia,

dermatitis and pyelonephritis

Cloxacillin and

oxacillin

Small animals: 4-10 mg/kg

Large animals: 1-2 mg/kg

Intramammary in large animals: sodium

cloxacillin- 200 mg/quarter; benzathine

cloxacillin- 500 mg/quarter


pneumonia, mastitis, metritis,

enteritis, septicaemia,

dermatitis and pyelonephritis

Carbenicillin All species of animals: 50-200 mg/kg,

parenteral, q.i.d.


Ticarcillin Horses and dogs: 40-50 mg/kg, im or iv Bacterial infections

Bacitracin All species of animals: As directed by the


Necrotic enteritis, streptococcal

infections and swine dysentery

Table 4: Dosage and Use of Cephalosporins in Animals

Cephalosporins Animals, Doses and Routes Uses

Cephalexin Large animals (Cattle and buffaloes): 5-10

mg/kg, b.i.d.

Small animals: 10-30 mg/kg, oral, t.i.d.

Respiratory and urinary

tracts infections, and skin

and soft tissues infections

Cefadroxil Small animals: 10-30 mg/kg, oral, t.i.d. Respiratory and urinary

tracts infections, and skin

and soft tissues infections

Cefachlor Calves: 3.5 mg/kg, oral, 12 hourly Bacterial infections

Cefalothin Dogs: 20-40 mg/kg, im or iv, 8 hourly Bacterial infections

Cefaloridin Small animals: 11-25 mg/kg, im, b.i.d. Bacterial infections

Cefotaxime Goats: 50 mg/kg, im, 12 hourly

Dogs: 20-40 mg/kg, im, 8 hourly


Cerftriaxone All species of animals: 5-10 mg/kg, sc, im or iv

BQ, HS, nephritis, cystitis,

mastitis and metritis

Table 5: Dosage and Use of Tetracyclines in Animals

Tetracyclines Animals, Doses and Routes (General dose

of tetracyclines: 4.4-11mg/kg, im or iv)

Uses

Chloretracyclines Large animals: 10-20 mg/kg, oral, 12 hourly

in 2 divided doses

Small animals: 25-50 mg/kg, oral, 12 hourly

in 2 divided doses

(Parenteral route is not recommended)

Mixed and secendory bacterial

infections, synovitis and blue

comb




17

Oxytetracycline Cattle, horses, sheep and pigs: 5-10 mg/kg,

parenteral

Calves and foals: 10-20 mg/kg, oral, b.i.d.

Pigs: 10-30 mg/kg, oral, b.i.d.

Dogs and cats: 27 mg/kg, oral, b.i.d.; 7-11

mg/kg, parenteral

Poultry: 0.1-0.3 g/L, oral

Genitourinary tract

infections, HS, BQ, anthrax,

pneumonia, enteritis, brucellosis,

mastitis and wound infections

Tetracycline Cattle, horses, sheep and pigs: 2.2-4.4

mg/kg, im

Dogs and cats: 4.4-11 mg/kg, im

Genitourinary tract infections,

HS, BQ, anthrax, pneumonia,

enteritis, brucellosis, mastitis

and wound infections

Minocycline Dogs: mg/kg, oral, 12 hourly Genitourinary tract infections,

HS and BQ

Rolitetracycline Horses: 2 mg/kg, iv, 24 hourly Bacterial infections

Methacycline Dogs and cats: 16.5 mg/kg, b.i.d. Genitourinary tract infections,

HS and BQ

Table 6: Dosage and Use of Chloramphenicols in Animals

Chloramphenicols Animals, Doses and Routes Uses

Chloramphenicol

palmitate

Small animals: 55-220 mg/kg, im or iv, in

3-4 divided doses

Respiratory and urinary tracts

infections, enteritis, mastitis,

metritis, meningitis, otitis, and

ocular infections

Chloramphenicol

sodium succinate

Cattle, buffaloes and horses: 2-4 mg/kg, iv

(10% solution), b.i.d.

Sheep, goats, calves and foals: 10 mg/kg,

iv (10% solution); 30-40 mg/kg, im (40%

solution)


infections, enteritis, mastitis,

metritis, meningitis, otitis, and

ocular infections

Lincomycin Bovines: l0 mg/kg, oral, im or iv, b.i.d.

Pigs: 11 mg/kg, im, b.i.d.

Dogs and cats: 20 mg/kg, oral, l2 hourly


mastitis, metritis, and infections

of skin, joints and soft tissues

Polymyxin B All species of animals: 5 mg/kg, oral, 12

hourly; 2.2 mg/kg, im, 12 hourly; 0.5-1.0

mg/g ointment, as topical

Ocular infections and systemic

Gram positive (G+) bacterial

infections

Tiamulin Calves, pigs and poultry: 8.8 mg/kg, with

feed or water, daily for 3-5 days

Pneumonia, enzootic

pneumonia, dysentery,

mycoplasmal arthritis and CRD

Sodium novobiocin Cattle: 250 mg/kg/quarter, intramammary Bovine mastitis

Norfloxacin Dogs and cats: 22 mg/kg, oral, b.i.d. Genitourinary tract infections

and prostatitis

Colistin All species of animals: 2.5-5.0 mg/kg, t.i.d. Bacterial infections

Table 7: Dosage and Use of Aminoglycosides in Animals

Aminoglycosides Animals, Doses and Routes Uses

Strptomycin Cattle, horses, sheep and pigs: 10 mg/kg, im,

12 hourly

Cattle: 100 mg/quarter, intramammary

Young animals and dogs: 20 mg/kg, oral, t.i.d.

Fowl: 0.1-0.2 g/bird, im, as a single dose

Pasterellosis, salemenallosis,

brucellosis, actinomycosis,

actinobacillosis, bovine

tuberculosis, leptospirosis and

abortion




18

Streptomycin +

penicillin



Mixed bacterial infections by

G+ and G- organisms

Gentamicin All species of animals: 2-4 mg/kg, im, 3-4

times daily; 0.3% solution, as topical eye/ear

drops

Infections of respiratory,

gastrointestinal and urinary

tracts, mastitis, metritis,

pyometra, otitis, ocular

infections, pneumonia, CRD,

and collibacillosis

Neomycin Cattle, sheep, goats and pigs: 2.5-5 mg/kg, im

or iv, b.i.d.; 10 mg/kg, oral in divided doses

Calves and dogs: 20 mg/kg, oral, 6 hourly

GIT infections and bacterial

enteritis

Neomycin +

doxycycline

All species of animals: 1 g/5-10 L of water,

for 4-5 days

Diarrhoea and mixed bacterial

infections

Kanamycin Cattle, sheep, pigs and dogs: 5-12 mg/kg, oral,

12 hourly; 1 g/10 ml distilled water, daily


infections, post-surgical sepsis,

ocular infections, and

secondary bacterial infections

with viral diseases

Amikacin Large animals: 7 mg/kg, im or iv

Dogs and cats: 10 mg/kg, sc, im or iv

Septicaemia, burns, post-

operative infections

Table 8: Dosage and Use of Macrolide Antibiotics in Animals

Macrolides Animals, Doses and Routes Uses

Erythromycin Dogs and cats: 6.6-8.8 mg/kg, oral, t.i.d.

Poultry: 1 part in 10000 parts of drinking water,

for 3 days

CRD and sinusitis

Erythromycin

lactobionate

All species of animals: 4-8 mg/kg, iv, 12 hourly Respiratory tract infections

Oleandomycin and

troleandornycin

All species of animals: 1.1-2.2 mg/kg, oral,

q.i.d.

Respiratory tract infections

Tobramycin Dogs: 1 mg/kg, oral, t.i.d. Septicaemia and urinary

tract infections

Tylosin Cattle: 4-8 mg/kg, im, daily

Pigs: 2-10 mg/kg, im, daily

Chicken and turkeys: 0.5 g/L in drinking water

Pneumonia, foot rot,

metritis, swine dysentery,

upper respiratory tract

infections and otitis externa

Table 9: Dosage and Use of Fluoroquinolones in Animals

Fluoroquinolones Animals, Doses and Routes Uses

Enrofloxacin Ruminants and pigs: 2.5-5 mg/kg,

im

Dogs and cats: 2.5 mg/kg, oral,

b.i.d.; 2.5 mg/kg, im, o.i.d.

Acute and chronic mastitis, metritis,

pyometra, respiratory and urinary tracts

infections, HS, BQ, wooden tongue,

mixed bacterial infections, colibacillosis,

salmonellosis, and pasteurellosis

Ciprofloxacin Cattle and sheep: 4-5 mg/kg, oral,

im or iv, b.i.d.

Dog: 5-15 mg/kg, oral, im or iv,

b.i.d.

Enterotoxacmia, CRD, respiratory and

GIT infections, mastitis, septic arthritis,

anthrax, and salmonellosis




19

ANTIFUNGAL DRUGS

The dosage and use antifungal drugs prescribed in different animals have been

described in Table 10.

Table 10: Dosage and Use of Antifungal Drugs in Animals

Antifungal Drugs Animals, Doses and Routes Uses

Grieseofulvin

(Antibiotic)

Small animals: 15-20 mg/kg, oral,

daily for 3-4 weeks

Cattle: 7.5-10 mg/kg, oral, daily for

30 days

Ringworm and Microsporum species

Amphotericin B

(Antibiotic)

Dogs: 0.25-0.5mg/kg, iv, every 48 hr

in systemic mycosis; 1.8 mg/kg, iv,

for 7 days in histoplasmosis

Histoplasmosis,coccidiomycosis,

blastomycosis and candidiasis

Nystatin

(Antibiotic)

All species of animals: Topical Dermatomycosis

Ketoconazole Dogs: l0 mg/kg, oral, daily for 2 days Systemic mycosis, histoplasmosis,

coccidiomycosis and blastomycosis

Clotrimazole All species of animals: Topical Dermatophycosis

Miconazole All species of animals: Topical Dermatomycosis




20

4


ANTHELMINTICS AND ECTOPARASITICIDES

OBJECTIVE

To know the dosage and use of different anthelmintics given in animals.

ANTHELMINTICS

The dosage and use of various anthelmintics (antihelmintic drugs) prescribed in

different species of animals have been mentioned in Table 11.

Table 11: Dosage and Use of Anthelmintics in Animals

Anthelmintics Animals, Doses and Routes Uses

Phenothiazine Cattle: 10 g/45 kg, oral

Sheep and goats: 25-30 g, oral

Horses: 3-5 g/45 kg, oral

Chicken: 1 g/bird in drinking water

Infestation of various

helminthes

Piperazine Calves, foals, pigs, dogs and cats: 200-300

mg/kg, oral

Poultry: 300-400 mg/kg in drinking water

Gastrointestinal

nematodes

Albendazole Cattle, horses, sheep, goats and pigs: 5-10 mg/

kg, single oral dose in nematodes and cestodes;

15-20 mg/kg, single oral dose in flukes

Dogs and cats: 25-50 mg/kg, oral, for 3-5 days

Nematodal, cestodal and

liver fluke (Fasciola sp.)

infestations (both adults

and larvae)

Fenbendazole Cattle, horses, sheep, goats and pigs: 5-7.5

mg/kg, single oral dose

Dogs: 50 mg/kg, oral, for 3 days

Cats: 30 mg/kg, oral, for 3 days




and larvae)

Triclabendazole Cattle and buffaloes: 12 mg/kg, oral

Sheep and goats: l0 mg/kg, oral

Fascioliasis (liver fluke

disease by Fasciola sp.)

Mebendazole Cattle, horses, sheep, goats and pigs: 10-15

mg/kg, oral, for 3 days

Dogs and cats: 22 mg/kg, oral, for 3 days

Poultry: 10 mg/kg, oral, for 2 days

Nematodal and cestodal


and larvae)

Cambendazole Cattle, horses and sheep: 20 mg/kg, oral, for 2

days

Pigs: 20-40 mg/kg, oral, for 2 days

Pigeons: 75-150 mg/kg, oral, for 2 days



infestations

Metronidazole Cattle: 20 mg/kg, iv in divided doses

Dogs: 25-50 mg/kg, oral in divided doses;

20 mg/kg, iv in divided doses

Amoebiasis, giardiasis,

trichomoniasis and anae-

robic bacterial infection




21

Metronidazole 100 mg

+ diloxanide l25 mg +

furazolidone 50 mg

Large animals: 60 ml, oral (suspension)

Small animals: 3-5 ml, oral (suspension)

Specific and non-

specific diarrhoea and

mixed infections

Metronidazole 300 mg

+ furazolidone l00 mg

Dogs and cats: 1 tab, t.i.d. Specific and non-

specific diarrhoea and

mixed infections

Metronidazole + fura-

zolidone + atropine

Large animals: 1 bolus/50 kg, oral Specific and non-

specific diarrhoea

Tinidazole Dogs and cats: 30-50 mg/kg, oral, b.i.d. Amoebiasis, giardiasis,

trichomoniasis and anae-

robic bacterial infection

Norfloxacin 1200 mg +

tinidazole 1800 mg

Large animals: l-2 boli, oral, b.i.d.

Small animals: 1 / 2 - 1 bolus, oral, b.i.d.

Diarrhoea, dysentery,

mixed infections and

bacterial enteritis

Praziquantel Dogs: 5-7.5 mg/kg, oral, im Cestode infection (adult

and immature stages);

avoid in pups

Dichlorophen Cattle, sheep and goats: 0.5 g/kg, oral

Dogs and cats: 0.2 g/kg, oral

Poultry: 0.2 g/kg, oral

Cestodiasis

Hexachlorophen Cattle, sheep and goats: 10-20 mg/kg, oral Trematodal infestations

(liver flukes and

paramphistomes)

Rafoxanide Cattle, sheep and goats: 7.5 mg/kg, oral Infestations of Fasciola,

Hemonchus and

nematodes, nasal bots,

and bunostomiasis

Levamisole Cattle, sheep and pigs: 7.5 mg/kg, sc (18.2%)

Dogs: 5 mg/kg

Poultry: 25-50 mg/kg

Filaroides and other

nematodal infestations

Tetramisole Cattle, horses, sheep, goats and pigs: 15 mg/kg,

single dose

Dogs: 2 mg/kg for 2-3 days

Filaroides and other

nematodal infections

Oxfendazole Cattle, sheep and goats: 5mg/kg, single dose Nematodal, cestodal and

trematodal infections

(both adults and larvae)

Niclosamide Cattle, horses, sheep and goats: 50-100 mg/kg,

oral, o.i.d.

Dogs and cats: 75-100 mg/kg, oral, o.i.d.

Poultry: 175 mg/kg, oral, o.i.d.

Cestodes,

paramphistomes and

Monezia infestations

Oxyclozanide Cattle and buffaloes: 10-15 mg/kg, oral

Sheep and goats: 15 mg/kg, oral

Fascioliasis, amphi-

stomiasis and moneziasis

Oxyclozanide (3%) +

levamisole (1.5%)

Cattle and buffaloes: 5 ml/10 kg, parenteral

Sheep and goats: 1 ml/2 kg, parenteral



Oxyclozanide (3%) +

levamisole (3%)

Cattle, buffaloes, sheep and goats: 1 ml/3 kg,

oral



Diethyl carbamazine Dogs: 6.6 mg/kg, oral, daily for 3-4 weeks Canine heart worm

disease and dirofilariasis

Milbemycin oxime Dogs: 0.5 mg/kg, oral Canine heart worm dise-

ase and ancylostomiasis

Morantel citrate Cattle, horses, sheep and goats: 10 mg/kg, oral

Pigs: 15 mg/kg, oral

Dogs and cats: 7.5 mg/kg, oral

Nematodal infestation




22

Pyrantel pamoate or

pyrantel embonate

Horses: 6.6 mg/kg, oral

Dogs: 5-10 mg/kg, oral

Nematodal infestation

ECTOPARASITICIDES AND ECTOPARASITICIDES

The dosage and use of various ectoparasiticides and ect-endoparasiticides are given

mentioned in Tables 12 and 13, respectively.

Table 12: Dosage and Use of Ectoparasiticides in Animals

Ectoparasiticides Animals, Doses and Routes Uses

γ-benzene hexa-

chloride (BHC)

All species of animals: Topical/surface application Mange infestation

(follicular mange)

Malathion All species of animals: Topical/surface application Ectoparasitic infestation

Diazinon Cattle, buffaloes, sheep and goats: 60 ml/20 L of

water, as spray

Dogs: 2-3 L/1000 L of water, as spray

Ectoparasitic infestation,

especially flies

Dichlorvos All species of animals: Topical/surface application Ectoparasitic infestation

Coumaphos All species of animals: Topical/surface application Ectoparasitic infestation

Fenvalerate All species of animals: Topical/surface application Ectoparasitic infestation

Carbaryl All species of animals: Topical/surface application

(5% solution)

Ticks, lice, fleas and

ked infestations

Amitraz All species of animals: 2-4 ml/L of water, as spray Ticks, lice, fleas, ked,

mites and mange

infestations

Monosulfiram All species of animals: Topical/surface application Ectoparasitic infestation

Almethrin All species of animals: Topical/surface application Ectoparasitic infestation

Cypermethrin All species of animals: 1 ml/L of water, as spray Ectoparasitic infestation

Deltamethrin All species of animals: 2-4ml/L of water, as spray Ectoparasitic infestation

Flumethrin All species of animals: 1 ml/10 kg, topical/surface

application

Ectoparasitic infestation

Permethrin All species of animals: Topical/surface application Ectoparasitic infestation

Table 13: Dosage and Use of Ect-endoparasiticides in Animals

Ect-endoparasiticides Animals, Doses and Routes Uses

Ivermectin All species of animals: 200

mcg/kg or 10 mg/50 kg, sc or

im

Infections of nematodes (GIT, eye and

lung worms), cestodes, and ectoparasites

(cutaneous myiasis, mange and larval

stage of canine heart worm disease)

Moxydectin All species of animals: As

stated (as per prescription)

Ectoparasites (cutaneous myiasis, ticks,

lice, mites and nasal bots)

Doramectin All species of animals: 200

mcg/kg or 10 mg/50 kg, sc or

im

Infection of nematodes (GIT, eye and lung

worms), cestodes, and ectoparasites

(cutaneous myiasis, mange and larval

stage of canine heart worm disease)

Closantel Cattle, sheep and goats: 7.5-

10 mg/kg, sc or im

Infections of nematodes, cestodes, flukes

and ectoparasites




23

5


ANTIPROTOZOANS

OBJECTIVE

To know the dosage and use of different antiprotozoans given in animals.

ANTIPROTOZOANS

The dosage and use of two kinds of antiprotozoal drugs, viz., antihaemoprotozoans

and anticoccidials, have been mentioned in Tables 14 and 15, respectively.

Table 14: Dosage and Use of Antihaemoprotozoans in Animals

Antihaemoprotozoans Animals, Doses and Routes Uses

Suramin

Cattle: 10-12 mg/kg, iv

Horses: 6-10 mg/kg, iv

Dog: 30-50 mg/kg, iv

(avoid sc/im, local reactions may occur)

Trypanosomiasis and

dourine

Oxytetracycline All species of animals: 10-20 mg/kg, iv Anaplasmosis, Ehrlichiosis

and theileriosis

Quinapyramine All species of animals: 0.25 ml/kg, iv with

15 ml of distilled water

Trypanosomiasis

Buparvaquone Cattle and buffaloes: 2.5 mg/kg, im

(avoid sc/iv, local reactions may occur)

Theileriosis

Diaminazine aceturate All species of animals: 0.8-1.6 g/100 kg, iv Trypanosomiasis and

babesiosis

Lithum antimony

thiomalate

All species of animals: 15-20 ml, 3 doses

im at 7 days interval; 15-20 ml, im on

alternate day; 1.0-2.5 ml, im on alternate

day

Nasal granuloma,

papailomatosis, filariasis

and leishmaniasis

Table 15: Dosage and Use of Anticoccidials in Animals

Anticoccidials Animals, Doses and Routes Uses

Amprolium Cattle: 10 mg/kg, oral; prophylactic- 5

mg/kg, oral

Sheep and goats: 25-45 mg/kg, oral

Caecal and intestinal

coccidiosis (thiamine

utilization may be inhibited,

producing toxic symptoms)

Amprolium +

furaltadone

Poultry: 0.6 g/L of water, oral for 10-15 days Caecal and intestinal

coccidiosis




24

Furazolidone Cattle, sheep, goats and poultry: As stated Coccidial infection

Clopidol Cattle, sheep, goats and poultry: As stated Prevention of coccidiosis

Sulphadimidine Cattle, sheep, goats and poultry: 200 mg/kg,

oral

Caecal and intestinal

coccidiosis

Sidium sulpha-

dimethyl pyrimidine

Cattle, sheep, goats and poultry: As stated Coccidial infection

Lasalocid Cattle, sheep, goats and poultry: As stated Prevention of coccidiosis

Dinitolmide Cattle, sheep, goats and poultry: 500 g/tonne

of feed

Coccidial infection

Maduramicin Cattle, sheep, goats and poultry: 500 g/tonne

of feed

Prevention of coccidiosis

Salinomycin Cattle, sheep, goats and poultry: 500 g/tonne

of feed

Prevention of coccidiosis

Monensin Cattle, sheep, goats and poultry: As stated Prevention of coccidiosis

Robenidine Cattle, sheep, goats and poultry: As stated Prevention of coccidiosis




25

6


ANTINEOPLASTIC (CYTOTOXIC) DRUGS

OBJECTIVE

To know the dosage and use of some important antineoplastic drugs given in

animals.

ANTINEOPLASTIC/ANTICANCER DRUGS

The dosage and use of some important antineoplastic/anticancer (cytotoxic) drugs

prescribed in different species of animals have been mentioned in Table 16.

Table 16: Dosage and Use of Antineoplastic/Anticancer Drugs in Animals

Antineoplastic Drugs Animals, Doses and Routes Uses

Cyclophosphamide Dogs: 2.2 mg/kg, im, for 2-4

days in a week

Mammary and pulmonary carcinoma,

and lympho-reticular neoplasia

Methotrexate Dogs and cats: 0.3-0.8 mg/kg,

iv, weekly

Lympho-reticular neoplasia,

transmissible veneral tumour,

osteosarcoma and rheumatoid arthritis

Chlorambucil Dogs: 0.1-0.2 mg/kg, im, for

2-4 days in a week

Chronic lymphocytic leukaemia and

macroglobulinaemia

Vincristine Dogs and cats: 0.025 mg/kg,

iv, once weekly

Transmissible veneral tumour and

lymphosarcoma

Busulphan Dogs: 0.1 mg/kg, oral, daily Chronic mylocytic leukaemia

Doxorubicin All species of animals: As

stated

Lymphosarcoma, feline mammary

cancer and non-functional thyroid cancer

Fluoro-uracil Dogs: 5-10 mg/kg, im, weekly Carcinoma of GIT, lung and liver

Melphalan All species of animals: 0.1 mg/

kg, im, for 10 days; followed

by 0.05-0.1 mg/kg, im, daily

Lympho-reticular neoplasia and multiple

myeloma

Mercaptopurine All species of animals: 2 mg/

kg, im, once daily

Acute lymphocytic leukaemia,

granulocytic leukaemia and

lymphosarcoma




26

7

SOME CHEMOTHERAPEUTIC PREPARATIONS :

SOLUTIONS

OBJECTIVE

To prepare the solutions of some drugs useful in veterinary chemotherapy.

POTASSIUM PERMANGANATE SOLUTION

Composition:

Potassium permanganate- 50 mg

Distilled water to make- 50 ml

Method:

Weigh/measure the above chemicals/ingredients and put them in mortar and pestle,

and triturate. Then, dispense the solution in a dispensing bottle/vial with the labeling

instruction of composition, use and dosage.

Fig. 2: Mortar and Pestle Fig. 3: Dispensing Bottles Fig. 4: Dispensing Vials

(Source: Google websites, gratefully acknowledged)

Action and Use:

1) Potassium permanganate (KMnO4) solution is antiseptic due to oxidation.

2) It is used in wound, stomatitis, snake bite and in sterilizing hand and instrument.




27

LUGOL’S IODINE SOLUTION

Composition:

Iodine- 2.5 g

Potassium iodide- 5 g


Method:




Action and Use:

1) Lugol’s iodine solution (Strong solution) is antiseptic as an iodine supplement.

2) It is applied on wounds.

3) In anoestrous cases, Lugol’s paint is used as intrauterine and vaginal irrigation

after dilution to 0.2% or less.

TINCTURE IODIDE (TINCTURE OF IODINE)

Composition:

Iodine- 2.5 g

Potassium iodine- 2.5 g

Distilled water- 2.5 ml

Alcohol to make- 100 ml

Method:




Action and Use:

1) Tr. iodide is an antiseptic.




28

2) It is used in abrasion, minor cuts and to sterilize the skin before surgical incisions.

TRYPAN BLUE SOLUTION

Composition:

Trypan blue- 1 g


Method:




Action and Use:

1) It is an antibabesial drug, effective against Babesia parasites. It is given at the

dose of 100-200 ml, iv in large animals and 5-10 ml, iv in dogs as a single dose.

2) As an antibacterial agent, it is given in septicemia, and foot and mouth disease.

GENTIAN VIOLET SOLUTION

Composition:

Gentian violet- 100 mg


Method:




Action and Use:

1) Gentian violet is an antiseptic.

2) It can be used in abraded skin, wounds, eczema and ulcers.

3) As an antifungal agent, it is used in superficial infections.




29

8

SOME CHEMOTHERAPEUTIC PREPARATIONS :

OINTMENTS

OBJECTIVE

To prepare the ointments of some drugs prescribed for various uses in veterinary

chemotherapy.

ZINC OXIDE OINTMENT

Composition:

Zinc oxide- 1.5 g

Petroleum jelly- 8.5 g

Method:

Weigh the above chemicals/ingredients and put them in mortar and pestle, and

triturate. Then, dispense the ointment in a dispensing bottle/vial with the labeling


Action and Use:

1) It is an antiseptic.

2) It is used in superficial wounds and ulcers.

WHIT’S FLUID OINTMENT (OINTMENT OF SALICYLIC ACID WITH

BENZOIC ACID)

Composition:

Salicylic acid powder- 1.5 g

Benzoic acid- 3.0 g

Petroleum jelly- 50.0 g




30

Method:




Action and Use:

1) It is an antiseptic agent.

2) It can be used in abraded skin, wounds, eczema and ulcers.

3) As an antifungal agent, it is used in superficial infections.

BORIC ACID OINTMENT

Composition:

Boric acid- 1 g

Petroleum jelly- 9 g

Method:




Action and Use:

1) Boric acid ointment is an antiseptic, antipruritic and soothing agent.

2) It is used in skin affections, and over the wounds and burnt areas.




31

9

SPECTROPHOTOMETRY

OBJECTIVE

To study the principle and mechanism of spectrophotometry.

PRINCIPLE

As the ‘colour’ of solution becomes darker and darker, the concentration of the

substance in the solution becomes higher and higher. This is a basic principle of the

“spectrophotometric assay”. ‘Intensity of colour’ is a measure of the amount of a material

in solution. The quantitative measurement of colourless materials is usually achieved

based on the principle that they will be converted to coloured substances in certain

chemical or biological reactions.

Fig. 5: Spectrophotometer (Source: Google website, gratefully acknowledged)

To understand the Spectrophotometry or to know the mechanism/principle of

“Spectrophotometer” (Fig. 5), the following descriptions must be known:

A. Relationship of Colour and Light:

When two colours are mixed in appropriate proportions, a white colour will be

produced. These two colours are called ‘complementary colour’ (Fig. 6).




32

Green

Yellow Cyan

Ultraviolet Visible light Infrared

Orange Cyanine

400 nm 760

Red Blue

Purple

Fig. 6: Map of Complementary Colour Fig. 7: Spectrum

Colours of a sample solution (Fig. 6) are due to selective absorption of certain

wavelengths (WLs) of visible light and transmittance of remaining light. If the sample

absorbs all WLs in the visible region, it will appear black; if it absorbs none of them, it

will appear white or colourless. Different colours are seen when particular WLs of the

radiant energy strike eyes. Assuming that we shine a beam of white light at lactoflavin, it

absorbs the blue light. Since the blue component of white light gets absorbed by

substance, the light which is transmitted is mostly yellow (complementary colour of

blue). This yellow light reaches our eyes, and we ‘see’ the substance as a yellow coloured

substance. The visible range is only a very small part of the electromagnetic spectrum.

Ultraviolet (UV) and infrared (IR) spectophotometric methods are suitable for many

colourless substances which absorb strongly in the UV or IR spectral regions (Fig. 7).

B. Lambert-Beer’s Law:

When monochromatic light (light of a specific wavelength) passes through a colour

solution, there is usually a quantitative relationship between the solute concentration and

the intensity of transmitted light, that is (Fig. 8),

I0 I

L

Fig. 8: Diagrammatic Presentation of Lambert-Beer’s Law

C Where:

I0 = Intensity of incident light;

I = Intensity of transmitted light;

C = Concentration of solute being measured;

L = Path length, i.e., length of radiation path

through the sample.

White light




33

“Transmittance” (T) is defined as the ratio of the amount of transmitted light to the

amount of incident. Thus,

Transmittance = I / I0 = Intensity of transmitted light / Intensity of incident light

“Absorbance” (A) is defined as the negative logarithm (-log) of the transmittance;

and the absorbance and transmittance bear an inverse relationship. Thus,

Absorbance = -log I / I0

The absorbance of a solution is proportional to the number of absorbing molecules,

that is the concentration (C) and the distance or length (L) that the light passes through.

This relationship is called the “Lambert-Beer’s law”. So,

A = KCL

where “K”, is the proportionality constant, which goes by any names like ‘extinction

coefficient’, ‘absorption coefficient’ or ‘absorptivity constant’. This is a characteristic of

each solute. It is dependent on the wavelength (WL) of light and on the conditions of

solvent. The unit for the extinction coefficient depends on the units used to express the

concentration (C) and the path length (L). A molar extinction coefficient (expressed in

“ε”) has units of M-1

cm-1

. Sometimes, the “K” is given as the absorbance of a 1% w/v

solution in a cell with a path length of 1 cm. In this case, the constant in the Lambert-

Beer’s equation becomes ε l%.

CALCULATION OF CONCENTRATION OF UNKNOWN SUBSTANCE

1. Standard Comparison Method:

In practice, the concentration of a solute in a sample with unknown concentration or

sample (u) can be determined directly by comparing the A of the unknown sample (u) to

the A of a standard solution (s) whose concentration is known or named as standard

sample (s) provided that such compounds obey the Lambert-Beer’s law, and all

conditions under which standard and unknown are prepared should be kept identical.

Standard sample (As) = Ks x Cs x Ls

Unknown sample (Au) = Ku x Cu x Lu

Since the L (path length) and the K (extinction coefficient) will be constant, that is,

Ls = Lu; Ks = Ku

Thus,

As / Au = Cs / Cu; Cu = Au / As x Cs




34

2. Standard Curve Method:

According to the Lambert-Beer’s law, there is a linear relationship between the

absorbance and concentration of a solute when all conditions under which standard(s) and

unknown(s) are prepared should be kept identical. So, a plot of absorbance versus (vs.)

concentration of absorbing solute yields a straight line passing through the origin. This is

usually done by preparing a series of standard solutions, each with a known concentration

of a given compound, measuring its absorbance value and plotting absorbance vs.

concentration to construct a curve. The concentration of the unknown sample can be

located by drawing a straight line from point of absorbance of the unknown until it

intersects with concentration curve, and then draw perpendicularly to the X-axis to

identify the concentration of unknown sample.

The Lambert-Beer’s law implies that when concentration is equal to zero (C = 0), the

absorbance must also be zero (A = 0). On the other hand, the standard curve must pass

through the origin.

GENERAL PROCEDURE

I. Parameters:

a) Choice of wavelength- The plot of absorbance (A) of a sample vs. wavelength

(WL) is called the ‘absorption spectrum’. For example, a plot gives the absorption

spectrum of potassium permanganate (KMn04). You will see that the A will be

changed with WL. Theoretically, one may choose any WL for quantitative

estimation of concentration. However, the magnitude of the A is important,

particularly when it is tried to detect very small amount of material. Because of

this, the WL of maximum A for a given sample is usually selected, and it is used

in the absorbance measurements.

b) Choice of absorbance- It is strongly recommended to measure the absorbance in

the range 0.05 to 1.0 because: (i) when we are trying to detect very small amounts

of material, the magnitude of the absorbance is important; (ii) when the

absorption band has a ‘flat’ top, the rate of change in the absorbance with WL is

smaller than that on the rising and falling shoulder of the peaks.

c) Blank reference solution (Blank)- Since transmittance is a relative

measurement, the light transmitted by the sample should be compared to the light




35

transmitted by a ‘reference solution’ (Blank). The reference solution is usually the

solvent in which the coloured compound is dissolved. A reference is necessary

because the solvent itself might absorb some light at the used WL, and one must

correct for that absorbance. Assume that the blank transmits 100% of the light

entering it, that is the scale is set to read zero absorbance. Now, use the full scale

of the spectrophotometer.

II. Instrument Components of Spectrophotometer:

The instrument used in spectrophotometic assay is called “spectrophotometer”. All

spectrophotometers have the following fundamental parts-

(1) a ‘source of light’;

(2) a ‘prism or grating’ to separate the light into narrow wavelength regions;

(3) a ‘device’ for holding the sample;

(4) a ‘photoelectric cell’ for measuring the light intensity.




36

10

TESTING OF BACTERIAL SENSITIVITY

OBJECTIVE

To study about the ‘in vitro testing’ for microbial sensitivity to antimicrobial agents.

METHODS OF BACTERIAL SENSITIVITY TESTING

The degree of sensitivity/resistance of different bacteria to different concentrations of

antimicrobial agents is known as ‘in vitro testing’ for microbial sensitivity.

There are two methods used for microbial sensitivity to antimicrobial agents:

1. Disc Diffusion Technique:

“Disc diffusion or Kirbey Baur technique” is generally used in lab. In this, the discs

(Fig. 9) with known amount of antibacterial drugs are placed on the surface of an agar

plate (Fig. 10) which has been streaked or swabbed with bacteria. The bacterial culture is

grown in broth incubated for 12 to 24 hr, and the organism growth or lack of growth is

seen. Lack of growth around the disc (‘zone of inhibition’) indicates the sensitivity of

microorganism to drug in the disc; while, the growth of microbes up to the disc, no zone

of inhibition shows the high susceptibility of microorganism. Larger zone of inhibition

indicates the high susceptibility of bacteria. It provides only semiquantitative or

qualitative information on the susceptibility of microbes to a given antibiotic.

2. Broth Dilution Technique:

In this technique, the isolated organism is inoculated in a tube (Fig. 11) or plate

having liquid medium with increasing concentration of drug. The tubes are inoculated

with standard number of organisms for a standard period of time. Each tube is then

observed for the growth. The tube with the lowest concentration of drug with no

observable growth contains the ‘minimum inhibitory concentration’ (MIC) of drug. This

method provides the quantitative data regarding the amount of drug necessary to inhibit

the bacterial growth. It can also show the ‘minimum bactericidal concentration’ (MBC).




37

Fig. 9: Glass Discs Fig. 10: Glass Plates Fig. 11: Glass Tubes


DETERMINATION OF MINIMUM INHIBITORY AND BACTERICIDAL

CONCENTRATIONS

Determination of Minimum Inhibitory Concentration (MIC):

The lowest concentration of antimicrobial agent which prevents the visible growth of

a bacterium after 18 to 24 hr of incubation is known as the ‘MIC’. It gives a quantitative

measure of sensitivity of specific bacteria to a specific antibiotic.

Determination of Minimum Bactericidal Concentration (MBC):

‘MBC’ is the lowest concentration of an antibiotic which kills 99.9% of the bacteria.

It is essential to know whether an antibiotic is bactericidal or bacteriostatic. To do this,

the tube with no bacterial growth is further plated, and the smallest concentration which

produces no bacterial growth is the MBC.

Inference:

i. If no bacterial growth in any subculture from the tubes with no visible growth

occurs, or if there is a small difference between MIC and MBC, it means that the

antibiotic is primarily ‘bactericidal’.

ii. If the growth in all subcultures from the tubes with no visible growth occurs, or if

there is a large difference between MIC and MBC, it shows that the antibiotic is

primarily ‘bacteriostatic’.




38

11

ASSAY OF ANTIBIOTICS

OBJECTIVE

To perform the assay of antibiotics.

BIOLOGICAL ASSAY OF ANTIBIOTICS

The standard and unknown dilutions of antibiotics (Fig. 12) are placed in cups or

wells having agar, and are incubated at 37°C. The antibiotic diffuses from the cup into

the surrounding agar, so producing a ‘zone’ where no bacterial growth occurs. The size of

the zone of inhibition is proportional to the concentration of the antibiotic present. It is a

sensitive and accurate method of assessment of most antibiotics. This method is mostly

used for penicillins where the amounts are low (9-10 g/ml).

PHYSICOCHEMICAL ASSAY OF ANTIBIOTICS

Spectrophotometer, spectrofluorimeter (Fig. 13) and high performance liquid

chromatography (HPLC; Fig. 14) are the physicochemical methods for measuring the

antibiotics. These methods are less sensitive than the biological assay method, and

require more extensive preparation and purification of samples.

Fig. 12: Some Antibiotics Fig. 13: Fluorimeter Fig. 14: HPLC





39

12

ESTIMATION OF SULPHONAMIDES

OBJECTIVE

To estimate the sulphonamides in blood/plasma/urine by the spectrophotometric/

colorimetric and paper impregnation methods.

PRINCIPLE

Estimation of sulphonamides by spectrophotometric or colorimetric method is based

on the diazotization reaction and its subsequent coupling with naphthyl ethylene diamine

dihydrochloridc (NEDD) to form a purple red colour complex. When an acidic aqueous

solution of a primary aromatic amine is treated with sodium nitrite, there is formation of a

corresponding diazonium salt by diazotization reaction. The excess nitrite is destroyed

with ammonium sulphamate solution. The diazonium salt so formed is then coupled with

N-1 NEDD to yield a stable purple red dye, and the intensity of both unknown and

standard solutions is measured at 540 nm WL. The concentration of unknown solution is

estimated by comparing its optical density (OD) with the OD of standard sulphonamide

solution, or by preparing and using a standard curve for sulphonamide.

REQUIREMENT

Equipments:

1. Spectrophotometer or colorimeter (Fig. 15) with appropriate filter;

2. Cuvettes (Fig. 16);

3. Centrifuging machine (Fig. 17);

4. Whatman filter paper number 42.

Drugs/Solutions:

1. Whole blood, plasma, serum, urine or cerebrospinal fluid (CSF);




40

2. 15% trichloroacetic acid (TCA) solution- 15 g of TCA is dissolved in distilled water

to make the volume 100 ml;

3. 0.05% sodium nitrite solution- 50 mg of sodium nitrite is dissolved in distilled water

to make the volume 100 ml and stored in the refrigerator;

4. 0.5% ammonium sulphamate solution- 500 mg of ammonium sulphamate is dissolved

in distilled water to make the volume 100 ml;

5. 0.5% NEDD solution- 500 mg of naphthyl ethylene diamine dihydrochloridc (NEDD)

is dissolved in distilled water or in 95% ethyl alcohol to make the volume 100 ml;

6. Hydrochloric acid 4N;

7. Stock standard sulphonamide solution (1 mg/ml or 1000 µg/ml)- 100 mg of

sulphonamide is dissolved in distilled water to make the volume 100 ml;

8. Working standard solution of sulphonamide (100 µg/ml).

Fig. 15: Colorimeter Fig. 16: Cuvettes Fig. 17: Centrifuging Machine


METHOD

A. Estimation of Sulphonamides by Spectrophotometric/Colorimetric Method:

1. Take 1 ml of sample in a 50 ml test tube and add 14 ml of distilled water

and 5 ml of 15% TCA solution. Shake and mix the contents thoroughly.

2. Keep the tube at room temperature for 15 minutes for deproteinization and

centrifuge in centrifuging machine for 15 minutes at 3000 rpm (rate per minute)

or filter the contents through Whatman filter paper to obtain protein free sample.

3. Take 5 ml of this protein free filtrate in a test tube and add 0.5 ml of sodium

nitrite solution (0.05%). Mix the contents thoroughly and wait for 5 minutes.

4. Add 0.5 ml of ammonium sulphamate solution (0.5%). Mix it properly and wait

for 3 minutes.




41

5. Add 0.5 ml of 0.5% NEDD solution and shake the tube thoroughly. Wait for 5

minutes.

6. Read the OD in spectrophotometer/colorimeter at 545 nm WL against the ‘blank’

within 30 minutes of development of colour. The ‘blank’ is prepared by adding all

the ingredients but instead of test sample 1 ml of distilled water is added.

The observation/calculation should be done as per the following example (Table 17)-

Table 17: Estimation of Sulphonamide by Spectrophotometric/Colorimetric Method

Test Tube No. Concentration

of Standard

Optical Density

(OD)

T-l (Sodium nitrite solution) 5 0.014

T-2 (Standard: sulphonamide solution) 10 0.02

T-3 (Ammonium sulphamate solution) 20 0.04

T-4 (NEDD solution) 30 0.08

T-5 (Test/unknown sample) ??? 0.13

T-6 (Blank) 0 0

Therefore, from the above Table, the concentration of unknown (test sample) can be

calculated as under-

OD of unknown / OD of standard x concentration of standard

= 0.13 / 0.02 x 10 = 65 µg/ml

On the basis of the samples of different known concentrations (5, 10, 20, 30, 40 µg,

etc.), we can prepare a ‘standard curve’ of the drug by plotting the concentration of drug

on X-axis against the respective OD in the Y-axis. Read the exact value of test sample

from the ‘standard curve’.

B. Estimation of Sulphonamides by Paper Impregnation Method:

Impregnate the strips of filter paper in saturated alcoholic solution of P-dimethyl

aminobenzaldehyde and dry them. Dip one of these test papers in hydrochloric acid and

then dip in urine sample. A yellow colour is indicative of the presence of sulphonamide.




42

13

ESTIMATION OF OXYTETRACYCLINE

OBJECTIVE

To estimate the oxytetracycline in plasma by the spectrophotometric/colorimetric

methods.

PRINCIPLE

Estimation of oxytetracycline is based on the colorimetric/spectrophotometric

method. When a solution of oxytetracycline is treated with paranitroaniline colour

reagent and then incubated at 65°C for 45 minutes, it forms a yellow colour complex. The

amount of colour formed is directly proportional to the concentration of oxytetracycline

in the plasma. The intensity of colour of both unknown and standard is measured in

colorimeter or spectrophotometer at 435 nm of WL, and the concentration of

oxytetracycline in unknown solution is estimated by comparing its OD with the OD of

standard oxytetracycline solution, or derived directly from a standard curve of

oxytetracycline.

REQUIREMENT

Equipments:

1. Spectrophotometer or colorimeter with appropriate filter;

2. Cuvettes;

3. Centrifuging machine.

Drugs/Solutions:

1. Plasma;

2. 15% trichloroacetic acid (TCA) solution- 15 g of TCA is dissolved in distilled

water to make the volume 100 ml;

3. Paranitroaniline colour reagent;




43

4. Glacial acetic acid;

5. Stock standard oxytetracycline solution (1 mg/ml or 1000 µg/ml)- 100 mg of

oxytetracycline is dissolved in distilled water to make the volume 100 ml;

6. Working standard solution of oxytetracycline (100 µg/ml).

METHOD

1. Take 1 ml of plasma sample and add 2 ml of 15% TCA in a test tube for

deproteinization. Mix it thoroughly and centrifuge at 4000 rpm for 15 minutes,

and collect the supernatant.

2. Take 2 ml of supernatant, and add 0.2 ml of paranitroaniline colour reagent and 2

ml of glacial acetic acid. Mix it properly and incubate at 65°C for 45 minutes.

3. Measure the resultant colour intensity of unknown/test sample at 435 nm in a

spectrophotometer/colorimeter against the ‘blank’.

4. Prepare the ‘blank’ in plasma and run along with the unknown sample.

5. Prepare the working standard solution of oxytetracycline from the stock solution

to obtain the concentrations in the range of 5 to 60 µg/ml in plasma and run along

with the sample.

6. Derive the concentration of oxytetracycline in the sample using standard formula

or from the ‘standard curve’.

If only one ‘standard’ concentration is taken, then the concentration of unknown is-

OD of unknown / OD of standard x concentration of standard

For example, OD of a standard 10 µg is 0.02 and OD of unknown sample is 0.12.

So, concentration of unknown sample = 0.12 / 0.02 x 10 µg = 60 µg/ml

If we take the samples of different known concentrations (5, 10, 20, 30, 40 µg, etc.)

in different test tubes marked as T1 to T5, then to each test tube, follow the same

procedure as for single sample and note the OD. We can prepare a ‘standard curve’ of

the drug by plotting the concentration of the test drug on X-axis against the respective

OD in the Y-axis. Read the exact value of test sample from the ‘standard curve’.




44

14

ESTIMATION OF TETRACYCLINE

AND DOXYCYCLINE

OBJECTIVE

To estimate the tetracycline and doxycycline in plasma or urine by the fluorimetric

method.

PRINCIPLE

Tetracyclines have the chelating property with divalent metal ions like calcium, and

form the fluorescent complex which is extracted into ethyl acetate in the presence of

barbital and is fluorimetrically measured.

REQUIREMENT

Equipments:

1. Fluorimeter;

2. Centrifuge tubes;

3. Centrifuging machine.

Drugs/Solutions:

1. Plasma or urine;

2. TCA 1.8M solution;

3. Calcium chloride 0.16M solution;

4. Ethyl acetate;

5. Barbital sodium 0.9M solution;

6. Stock standard solution of drugs- 100 µg/ml (dissolve 10.8 mg of tetracycline

hydrochloride and doxycycline hydrochloride, separately in 100 ml of water);

7. Working standard solution of tetracycline hydrochloride or doxycycline

hydrochloride, separately.




45

METHOD

1. Take 0.5 ml of plasma or 0.1 ml of urine in a centrifuge tube and make up the

volume to 5 ml with distilled water.

2. Add 1 ml of protein precipitant and mix well (protein precipitant is prepared by

taking 1.8M TCA and 0.16M calcium chloride).

3. Centrifuge at 3000 rpm for 15 minutes. Take 4 ml of clear supernatant in another

test tube, and add 4 ml of ethyl acetate and 3 ml of barbital sodium solution.

4. Mix thoroughly for 1 minute on vertex mixture and allow the phases to separate.

5. Take the upper layer and measure its fluorescence on a fluorimeter using 400 nm

as excitation and 520 nm as emission WL.

6. Prepare the ‘reagent blank’ by taking distilled water in place of test sample

(unknown) and process like the test sample.

7. Dilute the stock standard solution and prepare the working standard solutions of

0.5, 1, 2, 4, 8, 10 µg/ml, etc. of tetracycline or doxycycline. Make the final

volume of 5 ml and process like the test sample. Plot the fluorescence of the

‘standard’ graphically.

8. Finally, calculate the concentration from ‘standard curve/graph’ and multiply the

result obtained by appropriate factor depending on the volume of test sample

(unknown) used.




46

15

ESTIMATION OF CHLORAMPHENICOL

OBJECTIVE

To estimate the chloramphenicol in plasma/serum by the spectrophotometer method.

PRINCIPLE

Chloramphenicol is an aromatic nitro compound, which undergoes quantitative

reduction of the aromatic nitro group and forms a primary amine determined by the

diazotization and subsequent coupling with NEED.

REQUIREMENT

Equipments and Other Materials:

1. Spectrophotometer with appropriate filter;


3. Centrifuging machine;

4. Water bath;

5. Whatman filter paper No. 42.

Drugs/Solutions:

1. Plasma or serum;

2. 25% TCA solution;

3. 70% hydrochloric acid (HCl);

4. 0.8% stannous chloride in 70% HCl (to be prepared freshly);

5. 0.1% aqueous solution of sodium nitrite (to be prepared freshly);

6. 0.5% ammonium sulphamate solution;

7. 0.2% aqueous solution of NEED;

8. Stock standard chloramphenicol solution (1 mg/ml, 1000 µg/ml or 0.1%)- 100 mg

of chloramphenicol is dissolved in distilled water to make the volume 100 ml;

9. Working standard solution of chloramphenicol.




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METHOD

1. Take 1 ml of test sample (blood plasma or serum) in a centrifuge tube and make

up the volume to 4 ml by adding distilled water.

2. Add 1 ml of TCA to deproteinize the sample and allow to stand for few minutes.

3. Centrifuge at 2500 rpm for 30 minutes. Filter it by using the Whatman filter paper

No. 42 and separate the supernatant.

4. Take two tubes and label as ‘B’ (blank) to account for aryl amines and ‘T’ (test),

and add 1.5 ml of the supernatant in each of the two tubes.

5. In tube ‘B’, add 0.5 ml of 70% HCl; and in tube ‘T’, add 0.5 ml of 0.8% stannous

chloride solution.

6. Place both the tubes in boiling water bath for 20 minutes. Remove the tubes and

cool in room temperature.

7. Add 0.5 ml of 0.1% sodium nitrite solution to each tube and wait for 5 minutes.

8. Add 2 ml of 0.5% ammonium sulphamate reagent. Mix well by vertex mixture

and wait for 5 minutes.

9. Add 0.2% solution of NEDD (coupling reagent) for development of the colour.

10. Keep the tubes at room temperature for approximately 1 hour and measure the

optical density at 555 nm in spectrophotometer against the ‘reagent blank’.

11. The ‘reagent blank’ is prepared in the same for the test sample is prepared but

instead of test sample, 1 ml distilled water is added in this.

12. The working ‘standard’ is prepared from the stock standard solutions of

chloramphenicol to get the concentrations in the range of 5 to 50 µg/ml.

Difference between the ‘T’ (test) and ‘B’ (blank) gives the actual value for the total

nitro group present and the value is calculated from the ‘standard curve’. Read the exact

value of test sample from the ‘standard curve’.




48

16

ESTIMATION OF PEFLOXACIN/OFLOXACIN

OBJECTIVE

To estimate the pefloxacin/ofloxacin in plasma/serum by the spectrophotometer

method.

REQUIREMENT


1. Ultraviolet (UV) spectrophotometer;



Drugs/Solutions:

1. Plasma- Blood is collected and centrifuged at 4000 rpm for 15 minutes. The

supernatant colourless fluid is ‘plasma’;

2. 0.05% formic acid in isopropyl alcohol- Mix 99.95 ml of isopropyl alcohol in

0.05 ml of formic acid;

3. Stock standard solution of pefloxacin/ofloxacin (100 µg/ml);

4. Working standard solution of pefloxacin/ofloxacin.

METHOD

1. Take 1 ml of plasma sample in a centrifuge tube and add 3 ml of 0.05% formic

acid containing isopropyl alcohol.

2. Shake well for 5 minutes and centrifuge the contents at 3000 rpm for 15 minutes.

3. Decant the supernatant of above test sample (unknown) in a separate test tube and

read the absorbance of solution (test sample) at 260 nm in UV spectrophotometer

against the ‘blank’.

4. The ‘blank’ is prepared by the same method of test sample except that in place of

plasma, distilled water is added.




49

5. Working ‘standard’ solution of pefloxacin/ofloxacin is prepared from the stock

standard solution in different concentrations of 5, 10, 20, 30, 40 and 50 µg/ml.

Take 1 ml of these solutions and follow the same procedure as for the test sample.

Thus on the basis of the samples of different known concentrations (5, 10, 20, 30,

40, 50 µg, etc.), the ‘standard curve’ of pefloxacin/ofloxacin is prepared by

plotting the concentration of this drug on X-axis against the respective OD in the

Y-axis.

6. Read the exact value of test sample from the ‘standard curve’.




50

17

ESTIMATION OF CEFOTAXIME

OBJECTIVE

To estimate the cefotaxime in plasma by the spectrophotometer method.

REQUIREMENT


1. UV spectrophotometer;



Drugs/Solutions:

1. Plasma- Blood is collected and centrifuged at 4000 rpm for 15 minutes. The

supernatant colourless fluid is ‘plasma’;

5. 0.05% acetic acid in isopropyl alcohol- Mix 99.95 ml of isopropyl alcohol in 0.05

ml of acetic acid;

6. Stock standard solution of cefotaxime (100 µg/ml);

7. Working standard solution of cefotaxime.

METHOD

1. Take 1 ml of experimental plasma (test sample) in a centrifuge tube and add 3 ml

of 0.05% of acetic acid in isopropyl alcohol.

2. Shake thoroughly for 5 minutes and centrifuge the contents for 15 minutes.

3. Decant the supernatant of above test sample in a separate test tube and read the

absorbance of solution at 295 nm in UV spectrophotometer against the ‘blank’.

4. The ‘blank’ is prepared by the same method of test sample except that in place of

plasma, distilled water or 1 ml control plasma is added.

5. Working ‘standard’ solution of cefotaxime is prepared from the stock standard

solution in different concentrations of 5, 10, 20, 30, 35 and 40 µg/ml. Take 1 ml




51

of these solutions and follow the same procedure as for the test sample. Thus on

the basis of the samples of different known concentrations (5, 10, 20, 30, 35, 40

µg, etc.), the ‘standard curve’ of cefotaxime is prepared by plotting the

concentration of this drug on X-axis against the respective OD in the Y-axis.

6. Read the exact value of test sample from the ‘standard curve’.




52

ABOUT AUTHOR : DR. GOVIND PANDEY

Dr. Govind Pandey, “Professor/Principal Scientist” of Pharmacology

& Toxicology, possesses about 33 yr. of experience in ‘Research/Teaching/

Extension/Administration’. He is an able academician, scientist, veterinarian

and administrator; a Hindi literalist and eloquent speaker endowed with

strong writing flair. Dr. Pandey is probably “Only person in Madhya Pradesh

and alone veterinarian in India with maximum academic qualifications” (20

Degrees/Diplomas/Certificates). He obtained PhD degree with Honours in

Veterinary Pharmacology & Toxicology from the Jawaharlal Nehru Krishi Vishwa

Vidyalaya (JNKVV), Jabalpur, MP in the year 1990. Presently, he is doing DSc. He has

been ‘awarded/honoured’ by eminent persons; and also ‘published/broadcasted’ in

different media for great contribution in education, science, research, Hindi literature and

culture, public, governmental and social works. His “Biography” is included in the

famous directory/book of the world, “Who’s Who in the World 2011” (28th

edition,

America). Dr. Pandey is honoured with 3 prestigious national and international

‘Fellowship Titles’, viz., “FASAW, FSLSc and FISCA”.

Dr. Pandey started his career as “Veterinary Assistant Surgeon/Lecturer” on 7th

August, 1980 at Artificial Insemination Training Institute, Mandla, Animal Husbandry

(AH) Department, Government of MP. Under this Department, he also worked as

“Veterinary Surgeon/Senior Veterinary Surgeon” in different offices at Jabalpur,

including “Officer-In-Charge cum Drawing Disbursing Officer (DDO)” of Rinder Pest,

Jabalpur Division, Jabalpur till 19th

April, 2012. During this tenure, he also served as

“Chief Executive Officer/Block Development Officer cum DDO” of some Janapad

Panchayats under the Panchayat & Rural Development Department, Govt. of MP; and as

“Assistant Professor & Head, and Professor/Principal Scientist & Head” of

Pharmacology in Pharmacy colleges. On 20th

April, 2012, he joined as “Deputy Director

of Research/Associate Professor/Senior Scientist” at the Directorate of Research

Services, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur. On 26th

November, 2012, he has resumed the post of “Professor/Principal Scientist & Sectional

Head”, Department of Pharmacology & Toxicology, College of Veterinary Science &

AH, Rewa (NDVSU, Jabalpur).

Dr. Pandey is working in different areas of Life Science, with specialization in

Pharmacology & Toxicology. He has investigated some “Antihepatotoxic and anticancer

herbal drugs, and experimental hepatotoxic and cancer models” in animals. He has also

made a good contribution in Fishery Science, Hindi literature, Human Resource

Management, Political Science, Sociology, Public Administration, Law and Astrology.

He has published more than 225 scientific papers and delivered many speeches in a

number of platforms. Dr. Pandey has ‘supervised/guided/co-guided’ many PhD/PG/UG

students, and carried out some ‘Research Projects’. His “1 e-Book and 2 e-Manuals” have

been published by the International E - Publication, ISCA (2013). His “9 scientific

Books/Manuals” are under publication. He is the recipient of “30 Awards/Fellowships/

Sponsorships/Honours/Recognitions” (including “ICAR Senior Research Fellowship”

and “Sri Ram Lal Agrawal National Award”) in science, research and Hindi literature. In

Hindi literature, he has published “5 Books”, released “2 Audiocassettes” of own lyrics

and edited “1 Book”. His several poems, lyrics, dramas, or stories have been published/

broadcasted through various media. He is the “Life Member” of ‘25 scientific,

professional, literary and cultural associations/societies/journals’. He has acted as the

“Chairperson/Chief Guest/Judge/Expert” in many ‘conferences/seminars/projects/

committees/programmes’. He has also acted as the “Editor/Mentor/Editorial Board




53

Member/Reviewer” of some books, journals or magazines. Dr. Pandey has successfully

organized many academic, official, literary, cultural and social programmes in an

exemplary manner. He was also the “Captain of Badminton”, “Sergeant of NCC”,

“Literary Secretary” and “Hostel Prefect” in the College of Veterinary Science & AH,

Jabalpur. He has also passed “C Certificate of NCC”, and done “2 years’ course of

National Service Scheme” (NSS).

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