WO2001011962A1

WO2001011962A1 - Topical treatment for insect pests in companion animals

Info

Publication number: WO2001011962A1
Application number: PCT/US2000/019556
Authority: WO
Inventors: Daniel Earl Snyder
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 1999-08-12
Filing date: 2000-08-02
Publication date: 2001-02-22
Anticipated expiration: 2002-02-12
Also published as: AU6606400A; CO5221063A1

Abstract

The invention provides single-dose topical formulations for controlling an ectoparasite infestation on a companion animal for a prolonged time comprising a spinosyn, or a physiologically acceptable derivative or salt thereof, and a physiologically acceptable carrier. It also provides methods for controlling such infestations for a prolonged time comprising topically administering these formulations to the animal.

Description

TOPICAL TREATMENT FOR INSECT PESTS IN COMPANION ANIMALS

Companion animals, including but not limited to dogs, cats, and

horses, are an increasingly important part of today's society. They provide pleasure

and companionship to human friends, which leads to what has been termed the

human-animal bond. Unfortunately, a number of insect pests and parasites can infest

or infect these animals. Such pests include, for example, fleas, lice, mosquitoes,

mites, ticks and certain fly species. Safe, effective ways to eliminate these pests are

desired, both for the animal's well-being and for the comfort of its human associate.

The most common ectoparasites of cats and dogs world- wide are the

cat and dog fleas, Ctenocephalides felis felis and Ctenocephalides canis, respectively.

Interestingly, the cat flea very commonly infests dogs. Fleas annoy the animal it

infests and the pet's owner. Frequently, fleas cause more serious problems by

inducing flea-allergy dermatitis. It has been estimated that flea-related diseases

account for over 50% of the dermatological cases reported to veterinarians [D. E.

Bevier-Tournay, "Flea and Flea Control" Curr. Vet. Therapy 10: 586-592 (1989)]. In

addition, the cat flea is known to transmit tapeworms in dogs and has been implicated

in the transmission of cat scratch disease and murine typhus. Other pests of

companion animals, such as ticks and mosquitoes, are also known to transmit disease.

For example, ticks are known to transmit bacterial and viral diseases; and mosquitoes

can infect dogs and cats with the filarial nematode that causes heartworm disease.

Furthermore, economic expenses involved in flea control are high. In

the United States, for example, pet owners spend over $1 billion dollars for flea

control products annually [R. Conniff, "When It Comes to Pesky Flea, Ignorance is

Bliss," Smithsonian: 26: 76-85 (1995)]. Treatments currently available achieve varying degrees of success.

Most treatments involve chemicals applied to indoor and outdoor surfaces, as well as

to the pet. The chemicals used include a variety of carbamates, organophosphates,

pyrethrins and pyrethroids. These compounds often have toxic side effects that are a

problem for both the pet and its owner. For example, concentrated forms of

pyrethroids available for use on dogs are extremely toxic and lethal to cats and thus

cannot and should not be used on cats. In addition, there is evidence that the use of

these chemicals has led to multiple category insecticide resistance [N. K. Rust and M.

W. Dryden, Ann. Rev. Entomol. 42: 451-473 (1997)]. Thus, there continues to be a

need for relatively safe, effective agents for controlling ectoparasites on companion

animals, such as cat and dog fleas.

The spinosyns (also known as A83453 factors) are agricultural

insecticides that have shown activity against southern armyworm and other insects in

the order Lepidoptera and cotton aphid and other members of the order Homoptera. .

(See, for example, U.S. Patent No. 5,571,901).

The spinosyns were also reported to have some ectoparasiticidal

activity, i.e., in vitro activity against mosquito larvae, black blowfly larvae and adult

stable flies , which are members of the insect order Diptera, and transient systemic

activity against larval blowfly and adult stable fly in guinea pigs and sheep (see U.S.

Patent No. 5,571,901, Col. 26-32). Although it was suggested that the spinosyns

would be active against a number of ectoparasites in a number of animals by a variety

of routes, there have been no subsequent reported studies to support these suggestions.

This invention came about by the discovery that spinosyns, such as

spinosyn A, can provide prolonged residual control of an ectoparasite infestation on companion animals when a single dose of a spinosyn is applied topically to the

animal. Thus, the invention provides a method for prolonged control of the

ectoparasite in a safer manner than that achieved with previously known treatments.

One aspect of this invention is a long-acting, single-dose topical

formulation for controlling an ectoparasite infestation on a companion animal, said

formulation comprising an ectoparasiticidal amount of a spinosyn, or a

physiologically acceptable derivative or salt thereof, and a physiologically acceptable

carrier, in topical dosage form.

In another aspect, the invention relates to the use of a single, long-

acting topical formulation of a spinosyn, or a physiologically acceptable derivative or

salt thereof, for controlling an ectoparasite infestation on a companion animal.

This invention also relates to a method of controlling an ectoparasite

infestation on a companion animal for a prolonged time, comprising topically

administering a single dose of an effective amount of a spinosyn, or a physiologically

acceptable derivative or salt thereof, to the animal. An especially useful method of

this invention is a method for controlling a cat or dog flea infestation on a companion

animal for a prolonged time comprising topically administering a single dose of an

effective amount of a spinosyn, or a physiologically acceptable derivative or salt

thereof, to the animal.

The invention further relates to an article of manufacture, comprising

packaging material and a formulation for controlling an ectoparasite infestation on a

companion animal contained within said packaging material, wherein said

formulation comprises a long-acting topical unit dose of a formulation of this invention, i.e.,

an ectoparasiticidal amount of a spinosyn, or a physiologically

acceptable derivative or salt thereof, and a physiologically acceptable

carrier; and

wherein said packaging material comprises a label or package insert with instructions

for topically administering the dose to the animal.

This article of manufacture, or kit, is particularly appropriate when the

companion animal is a dog or a cat. The label or package insert will indicate the

number of unit doses to be applied to the dog or cat and the timing of such

administration. The timing of doses will generally be every 30 days. The contents of

each kit would typically be sufficient to control the ectoparasite infestation for a

period of several months.

The invention also relates to the use of a spinosyn, or a physiologically

acceptable derivative or salt thereof, for the manufacture of a long-acting single-dose

topical medicament for controlling an ectoparasite infestation on a companion animal.

Spinosyns are naturally derived fermentation products. They are

macro lides produced by cultivation of Saccharopolyspora spinosa. The fermentation

produces many factors, including spinosyn A and spinosyn D (also called A83543A

and A8354D). Spinosyn A and spinosyn D are the two spinosyns that are most active

as insecticides. A product comprised mainly of these two spinosyns is available

commercially under the trade name "spinosad". The major spinosyn factor, spinosyn

A, is known to have an excellent human and animal safety and toxicological profile.

Each spinosyn has a 12-membered macrocyclic ring that is part of an

unusual tetracyclic ring system to which two different sugars are attached, the amino- sugar forosamine and the neutral sugar 2N,3N,4N-(tri-O-methyl)rhamnose. This

unique structure sets the spinosyns apart from other macrocyclic compounds.

Spinosyn A was the first spinosyn isolated and identified from the

fermentation broth of Saccharopolyspora spinosa. Subsequent examination of the

fermentation broth revealed that S. spinosa produced a number of spinosyns that have

been called spinosyns A to J (A83543A to J). The primary components are spinosyns

A and D. Additional spinosyns, lettered from K to W, have been identified from

mutant strains of S. spinosa. The various spinosyns are characterized by differences in

the substitution patterns on the amino group of the forosamine, at selected sites on the

tetracyclic ring system and on the 2N,3N,4N-(tri-O-methyl)rhamnose group.

The term "spinosyn or a derivative thereof as used herein refers to an

individual spinosyn factor (spinosyn A, B, C, D, E, F, G, H, J, K, L, M, N, O, P, Q, R,

S, T, U, V, W or Y), an N-demethyl derivative of an individual spinosyn factor, or a

combination thereof. For convenience, the term "spinosyn component" will also be

used herein to mean an individual spinosyn, or a physiologically acceptable derivative

or salt thereof, or a combination thereof.

Boeck et al. described spinosyns A-H and J (which they called A83543

factors A, B, C, D, E, F, G, H and J), and salts thereof, in U.S. Patent Nos. 5,362,634

(issued Nov. 8, 1994); 5,496,932 (issued March 5, 1996); and 5,571,901 (issued

Nov. 5, 1996). Mynderse et al. described spinosyns L-N (which they called A83543

factors L, M and N), their N-demethyl derivatives, and salts thereof, in U.S. Patent

No. 5,202,242 (issued Apr. 13, 1993); and Turner et al. described spinosyns Q-T

(which they called A83543 factors Q, R, S and T), their N-demethyl derivatives, and

salts thereof, in U.S. Patent Nos. 5,591,606 (issued January 7, 1997) and 5,631,155 (issued May 29, 1997). Spinosyns K, O, P, U, V, W and Y are described, for

example, by Carl V. DeAmicis, James E. Dripps, Chris J. Hatton and Laura I. Karr in

American Chemical Society's Symposium Series: Phytochemicals for Pest Control,

Chapter 11, "Physical and Biological Properties of Spinosyns: Novel Macrolide Pest-

Control Agents from Fermentation", pages 146-154 (1997).

The spinosyns can react to form salts that are also useful in the

methods and formulations of this invention. The salts are prepared using standard

procedures for salt preparation. For example, spinosyn A can be neutralized with an

appropriate acid to form an acid addition salt. The acid addition salts of spinosyns are

particularly useful. Representative suitable acid addition salts include salts formed by

reaction with either an organic or inorganic acid such as, for example, sulfuric,

hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic,

pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, formic,

lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic,

cinnamic and like acids.

The formulations of this invention may further include, in combination

with the spinosyn component, one or more other compounds that have activity against

the specific ectoparasite or endoparasite to be controlled, such as, for example,

synthetic pyrethroids, natural pyrethins, organophosphates, organochlorines,

carbamates, foramidines, avermectins, milbemycins, insect growth regulators

(including chitin synthesis inhibitors, juvenile hormone analogs, and juvenile

hormones), nitromethylenes, pyridines and pyrazoles.

The methods and formulations of this invention have several

advantages. First, the spinosyns are naturally derived fermentation products, and spinosyn A has excellent human and animal safety profile, in contrast to the profiles

of the currently used synthetic organically derived compounds, such as synthetic

pyrethroids or permethrins, organophosphates, organochlorines, and carbamates. For

example, some of the currently used products such as pyrethroids are very toxic to

cats and can be lethal.

Another advantage is that spinosyns are very effective against fleas,

mites, ticks, lice and flies with post-treatment residual protection, depending upon the

dosages used. Furthermore, spinosyns have no cross resistance to compounds

currently used to treat these insects. Thus, spinosyns can be used on companion

animals against insect populations that have existing levels of resistance to currently

used products. Spinosyns can be used, therefore, in integrated pest management

(IPM) programs to extend the lifeline of commonly used products where resistance is

not developed or has not yet developed.

All ratios, percentages, and parts discussed herein are "by weight"

unless otherwise specified.

The term "topical formulation" means a formulation that is suitable to

be applied to the external surface of the animal so the ectoparasites will be exposed to

lethal levels of the spinosyn component of the formulation. The external surface

consists of epidermis, dermis, hair, skin secretions and oils, and skin appendages of

the animal. Topical application to the external surface of the animal can be diffuse, as

in a spray, dip or dust, or localized as in a pour-on or concentrated as in a spot-on.

Topical formulations can be applied on any part of the animal's body, but are

typically applied on the head, neck or dorsal midline of the body. The spinosyn component or components, either alone or in

combination with one or more of the other types of compounds listed supra, can be

formulated into a number of topically applied end-use products or formulations

(topical dosage forms). These products or formulations include, but are not limited to,

spot-ons, pour-ons, sprays, dips, dusts, lotions, gels, ointments, salves, dressings,

ready-to-use towels or towelettes, face masks, cremes, sticks, soaps, shampoos,

mousses, collars, medallions, and tail bands. Another example of a topical dosage

form is a "tag" that contains a sustained-release formulation with diffusion holes or

openings that afford contact with the external surface of the animal

The term "single-dose formulation" or "single-dose medicament"

refers to a one time application of a sufficient amount of the formulation to control the

ectoparasite infestation.

The term "controlling an ectoparasite infestation" refers to preventing,

minimizing or eliminating an infestation by an ectoparasite. The term "ectoparasite"

refers to insect or acarine insect pests that commonly infest or infect companion

animals. Examples of such ectoparasites include the egg, larval, pupal, nymphal and

adult stages of fleas, lice, mosquitoes, mites, ticks and blood-sucking, biting or

nuisance fly species.

The term "companion animals" includes dogs, cats, horses, rabbits and

other pets owned and maintained in close association with humans as part of the

human-animal bond.

The term "prolonged time" comprises a period of at least 7 days,

preferably a period of at least two weeks. The term "long-acting" means the activity

lasts for a prolonged time. The methods of this invention are carried out by topically

administering the spinosyn component to the animal. As discussed supra, topical

application may be carried out in a number of ways known in the art. Especially

useful methods of topically applying the spinosyn component are spot-ons, sprays and

pour-ons. Spot-on treatments are ones in which the active agent is applied once as a

single spot in an area that is not accesible to the animal's mouth. In cats and dogs, for

example, the area at the base of the neck between the shoulder blades is suitable for

spot-on administration. When the spinosyn component is applied as a pour-on, spray,

dip, dust, or lotion, it is important to apply a sufficient amount to wet the animal's

hair so that the spinosyn component reaches the skin.

In carrying out a method of this invention, an effective amount of a

spinosyn or a physiologically acceptable derivative or salt thereof, is applied topically

to the companion animal. The terms "effective amount" and "ectoparasiticidal

amount" refer to the amount needed to control the particular ectoparasite infestation.

As those in the art will understand, this amount will vary depending upon a number of

factors. These factors include, for example, the type of companion animal being

treated, its weight and its general physical condition and the type of ectoparasite to be

controlled.

In general, an effective amount is from 1 to 100 mg of the spinosyn per

kg of body weight of the companion animal. More commonly, the effective amount is

from 10 to 50 mg/kg of body weight of the animal. In spot-on formulations the

spinosyn component will typically comprise from 10 to 60 percent by weight of the

formulation.

The following examples illustrate the methods of this invention: EXAMPLE 1

Prolonged Topical Control of Cat Fleas in Dogs

Dogs of the beagle breed were separated into 5 groups, with two dogs

in each group. The dogs were housed indoors. Unfed adult cat fleas (200) were

applied to each dog pre-treatment (to assess therapeutic knock-down efficacy) and

again at 7, 14, 21 and 28 days post-treatment (to assess post-treatment residual

efficacy). Each dog was sprayed once on day 0 with a total volume of 53 mL per dose

of a spinosad spray solution. The spray solutions were prepared by making a

concentrated solution of spinosad in isopropyl myristate (IPM). The IPM solutions

were diluted in water to the desired concentration. One group received only diluted

vehicle (control) spray; the remaining 4 groups received a diluted spray solution

containing 1, 10, 100 or 1000 ppm spinosad prepared from 0.0429, 0.430, 4.307 and

44.07 mg/mL IPM concentrates, respectively. On days 1 and 7 post-treatment, the

dogs sprayed with 100 and 1000-ppm spray solutions showed more than 90% control

of the fleas. At days 14 and 21 post-treatment, the dogs treated with the 1000-ppm

spray solution showed more than 90% control of the fleas. At day 28 post-treatment,

the dogs receiving the 1000-ppm spray solution continued to show about 89% control

of the fleas. No adverse reactions were seen or reported during the study with any of

the dogs. EXAMPLE 2

Efficacy of Spinosad in Spot-on and Spray Formulations for the Treatment of Ctenocephalides felis on Dogs

In this study dogs of the beagle breed were divided into two control

and five treatment groups (3 dogs per group). Each group was infested with fleas as

described in Example 1. The treatments were applied either as a spot-on or a spray.

Spot-on treatments were applied once as a single spot (1 to 4 mL total volume) placed

onto the skin/hair at the base of the neck between the shoulder blades. Spray

treatments were administered to cover the entire animal, wetting the hair down to the

skin. For spot-on application, spinosad was dissolved in IPM; for spray application,

an IPM solution of spinosad was diluted in water to the desired concentration. Two

control groups (one housed inside and one housed outside) were treated spot-on with

vehicle only. The treatment groups were treated with different doses of spinosad and

handled as follows:

The dogs were observed at days 1, 7, 14, 21, 28 and 35. The results

observed with the different treatment groups are summarized in Table 1. Table l: Percent Reduction in Adult Flea Counts in Dogs Treated with Spinosad

Compared to Untreated Control Group

No adverse reactions were seen.

EXAMPLE 3

Efficacy of Spinosad in Spot-on Formulations for the Treatment of Ctenocephalides felis on Dogs

In this study dogs were experimentally infested with fleas as in

Example 1 and separated into groups (3 dogs per group). Spinosad was applied as a

spot-on as in Example 2. In two groups, it was administered in an aqueous

suspension (45%). In two other groups, it was administered in an IPM concentrate

(17%). In the control group vehicle without spinosad was applied. The doses were

applied as follows:

The results of this study are summarized in Table 2.

No adverse results were seen in this study.

EXAMPLE 4

Efficacy of Spinosad in Spot-on and Spray Formulations for the Treatment of Ctenocephalides felis on Cats

In this study, cats were divided into a control and five treatment groups

(3 cats per group). Each group was infested with fleas as in Example 1 and treated

with either a spot-on or spray formulation. Spot-on and spray treatments were carried

out as described in Example 2. Spinosad was formulated in IPM solutions or as an

aqueous suspension. The control group was treated with a spot-on with IPM vehicle

only. The various treatment groups were:

The cats were observed after 8 hours and at days 1, 7, 14, 21, 29, 35/36

and 42. The results observed with the different treatment groups are summarized in

Table 3.

Table 3: Percent Reduction in Adult Flea Counts in Cats

Treated with Spinosad Compared to Untreated Control Group

Treatment Group T3 and the control group were observed for an additional 42 days. The results observed at days 49, 56, 63, 70, 77 and 84 are summarized in Table 3a.

Table 3a: Percent Reduction in Adult Flea Counts in Cats

Treated with Spinosad Compared to Untreated Control Group

Cats were bathed at this point in the trial.

No adverse reactions were seen in this study. EXAMPLE 5

Efficacy of Spinosad against Adult Stable Fly (Stomoxys calcitrans) and House Fly (Musca domestica) Infestations on Horses

Five horses were used in this study with one horse per group. The

control group was untreated, and 4 groups were treated with an aqueous suspension of

spinosad (25 g/L) that was diluted in water just prior to use and applied as a spray to

each horse. Each horse was treated with a pressurized spray apparatus by spraying

each diluted spray over the dorsum and each side (barrell) of the body from the

shoulders to the hips. Each horse received approximately 120 mL of each diluted

spray as follows:

Horse # Treatment³

1 untreated

2 500 ppm spinosad

3 1,000 ppm spinosad

4 5,000 ppm spinosad

5 10,000 ppm spinosad Spinosad sprays were prepared by diluting a 25 g/L spinosad suspension concentrate in an appropriate amount of water to make up each ppm concentration.

After the applied spray had dried, 6 petri dish cages (3 per side) of

unfed stable fly and 6 petri dish cages (3 per side) of house fly, each containing 10

adult flies per dish, were placed under a screened belt that was tied around the animal.

The bottom of the petri dishes had a mesh that allowed the stable flies to probe

through and obtain a blood meal and the house flies to probe through with their mouth

parts with both being exposed to the treated hair and skin. The flies in the petri dishes

were exposed to the treated surface of each horse for 20 minutes, after which the plates were removed and taken to the laboratory to evaluate percent kill at 4, 8 and 24

hours post-exposure. Petri dishes were positioned on and exposed to each treated

horse immediately after treatment and again on days 1, 3, 5 and 7 post-treatment to

evaluate residual activity.

Results: Stable fly control exceeded 90% on the first day in the 5,000 and

10,000 ppm-treated horses. Stable fly control remained above 80% for the 10,000

ppm-treated horse on day 7. House fly control was generally better than stable fly

control. The highest percent control was seen on day 3 post-treatment. No adverse

reactions were seen.

Claims

CLAIMS1 A long-acting single-dose topical formulation for contiolhng anectoparasite infestation on a companion animal, said formulation comprising anectoparasiticidal amount of a spinosyn, oi a physiologically acceptable derivative orsalt thereof, and a physiologically acceptable caπiei, m topical dosage form2 A formulation of Claim 1 wherein the spinosyn is spinosyn A3. A formulation of Claim 1 oi 2 wherein the ectopaiasiticidalamount is fiom 1 to 100 mg of the spinosyn per kg of body weight of the animal4 A formulation of Claim 1 or 2 wherein the spinosyn comprisesabout 10 to 60 percent by weight of the formulation5 A formulation of Claim 1 , 2, 3 or 4 wherein the animal is a dog,a cat, a horse, or a rabbit6 A formulation of Claim 1 , 2, 3, 4 or 5 wherein the ectoparasiteis a cat flea, a dog flea, a biting or nuisance fly, a mosquito, a tick or a louse7. An article of manufactuie, comprising packaging material and aformulation for controlling an ectoparasite infestation on a companion animalcontained within said packaging material, wherein said formulation comprisesa long-acting topical unit dose of a formulation of Claim 1, 2, 3, 4, 5 or6, andwherein said packaging material comprises a label or package insert with instructionsfor topically administering the dose to the animal8. The use of a formulation of Claim 1 , 2, 3, 4, 5 or 6 for topicallycontrolling an ectoparasite infestation on a companion animal 9 The use of a spinosyn, or a physiologically acceptabledeπvative or salt thereof, for the manufacture of a long-acting single-dose topicalmedicament for controlling an ectoparasite infestation on a companion animal10 A use of Claim 9 wherein the spinosyn is spinosyn A1 1 A method of controlling an ectoparasite infestation on acompanion animal for a prolonged time, comprising topically administeiing a singledose of an effective amount of a spinosyn, or a physiologically acceptable derivativeor salt thereof, to the animal12 A method of Claim 1 1 wherein spinosyn is spinosyn A13 A method of Claim 1 1 or 12 wherein the animal is a dog, a cat,a horse or a rabbit14 A method of Claim 1 1, 12 or 13 wherein the ectoparasite is acat flea, a dog flea, a biting or nuisance fly, a mosquito, a tick or a louse15 A long-acting, single-dose topical formulation of a spinosyn, ora physiologically acceptable derivative or salt thereof, for topically controlling anectoparasite infestation in a companion animal for a prolonged time substantially ashereinbefore described with leference to any one of the Examples AMENDED CLAIMS[received by the International Bureau on 04 December 2000 (04.12.00); original claims 1-15 replaced by new claims 1-9 (2 pages)]CLAIMS

1. A topical formulation for controlling an ectoparasite infestation on a companion animal, said formulation compπsing 10-60% by weight of spinosyn A, or a physiologically acceptable deπvative or salt thereof, and a physiologically acceptable earner, wherein said topical formulation is administered to a companion animal in need of treatment at from 1 to 100 mg of spinosyn A per kg of companion animal body weight once every 7 days to two weeks.

2. A formulation of Claim 1 wherein the animal is a dog, a cat, a horse, or a rabbit.

3. A formulation of Claim 1 wherein the ectoparasite is a cat flea, a dog flea, a biting or nuisance fly, a mosquito, a tick or a louse.

4. An article of manufacture, compπsing packaging mateπal and a topical formulation for controlling an ectoparasite infestation on a companion animal contained within said packaging mateπal, wherein said formulation compπses a unit dose of a formulation of Claim 1, 2 or 3; and wherein said packaging mateπal compπses a label or package insert with instructions for topically administeπng the dose to the animal.

5. The use of spinosyn A, or a physiologically acceptable deπvative or salt thereof, for the manufacture of a topical medicament compπsing 10- 60% by weight of spinosyn A for controlling an ectoparasite infestation on a companion animal.

6. A method of controlling an ectoparasite infestation on a companion animal, compπsing topically admimstenng a single dose once every 7 days to two weeks of a topical formulation containing from 1 to 100 mg of spinosyn A, or a physiologically acceptable deπvative or salt thereof, to the animal.

7. A method of Claim 6 wherein the animal is a dog, a cat, a horse

or a rabbit.

8. A method of Claim 6 wherein the ectoparasite is a cat flea, a

dog flea, a biting or nuisance fly, a mosquito, a tick or a louse.

9. A topical formulation of spinosyn A, or a physiologically

acceptable deπvative or salt thereof, for topically controlling an ectoparasite

infestation in a companion animal substantially as hereinbefore descnbed with

reference to any one of the Examples.