Drug Resistance as a Therapeutic Opportunity

8/13/2019 Drug Resistance as a Therapeutic Opportunity

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MULTITECCHEMICAL & ENGINEERING PROJECTS

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Turning Drug Resistance On Its Head Therapeutic Opportunity

Drug resistance is a major cause of cancer drug failure.

However where we can turn this resistance on its head it may offer therapeutic

opportunities. With the intriguing prospect that the more the resistance the moreeffective

may be our therapeutic response.

Bacteria are globally emerging resisting all known antibiotics which have helped extend our

lives and allowed major surgery. Because governments have been slow to step in to protect

us where pharmaceutical companies have for a number of reasons generally scaled back

on research for new antibiotic categories, we may be exposed to these infections for a

number of years before new antibiotic categories are clinically available.

There are good indications that our methods can be extended to bacteria and major

modifications under consideration make this more likely. Since clinically accepted drugs can

be used this can be expected to become quickly and economically available to fill the

therapeutic gap. Therefore a short preliminary paper is here brought forward.
mailto:[email protected]:[email protected]


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Drug Resistance as a Therapeutic Opportunity

Bernard M. Turner

SUMMARY

The wall of resistance to cancer and bacterial drugs may to some extent be an artefact of

prevailing chemotherapy. This comprehensive adaptive response to drugs is fortunately largely

selective to cancer and many infectious diseases. Here we show in vitro that this may give us an

opportunity for a corresponding on-going chemotherapy where the resistance to a drug is used

to set up the target cells to activate within the cells a subsequently delivered counterdrug.

This may be of comparable if not more selectivity and efficacy than the drug resisted and can

sometimes use clinically accepted drugs for a quicker and more economical development as

well as increase the cost-effectiveness of targeted drugs. Moreover, this may be modified to

set up conditions favouring the delay or even reversal of resistance to a drug by codosing it

with the counterdrug. This can be empirical in not necessarily requiring the resistance

mechanism to be known. The prevailing cancer chemotherapy is becoming increasingly

unsustainable and bacteria resistant to almost all known antibiotics are globally emerging, so

that this alternative strategy should be a timely challenge to consider clinically developing and

extending.

Major causes of cancer drug failure are drug resistance, toxicity limiting the dosage and metastases.

A dearth of major new drugs to replace those coming off patents despite a steady rise in R&D

expenditure1has been attributed to a lack of sufficient innovation.2This with genetic drug

competition has caught up with the pharmaceutical companies3leading to downsizing, redundancies


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and a scramble for M&A as well as efforts to reduce the enormous cost and time of drug

development.4

Antibiotics have an additional built-in obsolescence (Fig 1) discouraging R&D. For while cancer

drug resistance terminates with the death of a patient from whatever cause, bacterial drug resistance

can be cumulative, spreading rapidly horizontally in hospitals and other communities by clonal

expansion as well as plasmids between bacterial species, accelerated by global travel. This combined

with the misuse of antibiotics in humans and farm animals and the lack of leadership by governments

in regulating this and promoting R&D where pharmaceutical companies are lacking is a recipe for

the global incubation of bacteria now emerging. Resistant to almost all know antibiotics, e.g.

NDM-1-metallo-beta-lactamase plasmids, know for some thirteen years,5now spreading globally in

pathogenic bacteria.

The completion of the Human Genomic Project has given a great impetus to the search for new

anticancer targets for Ehrlichs magic bullets.6However, this is becoming increasingly

unsustainable with the Wests ageing population as currently emphasised.7Cancer care and

treatment alone is predicted to bankrupt the UKs NHS by 20258and is causing serious concern in

USA. There is an increasing clash between biomedical progress and equity yet to be confronted.9

It would therefore appear timely to pause and consider whether a reorientation of the prevailing trend

to complexity largely generic diagnostically and therapeutically is possible without sacrificing drug

efficacy. This article briefly outlines one such alternative approach with in vitro example of each as a

step towards the end objectives: 1) to overcome drug resistance, 2) use the latter as an opportunity

for delivering a more effective counterdrug, 3) where possible with clinically accepted drugs for

quicker and more economical development to the clinical stage equivalent to late-stage drug

development, 4) more sustainable to the Wests health services financial constraints and 5) more

accessible to the low-income economies where some 70% of cancer and most infectious diseases


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occur. 6) Where possible an empirical method for simplicity, economy as well as speed of response

when used. 7) Adding these advantages to targeted drugs, including those selected by personalized

cancer treatment to make these more sustainable.8-9

Drug resistance as a selective target.

As well as disclosing many new potential drug targets genomic projects have also shown the

complexity of interacting networks and feed-back loops within10and between11the genomic,

transcriptional and translational levels and with epigenetics. Blocks one target with a silver [costly]

magic bullet and alternative pathways resist this restoring the initial or an alternative equilibrium.

Like squeezing a balloon.12

This robust13adaptive response is perceived as a wall of resistance. Differing from organismal

evolution, neoplastic cells revert to asexual, unicellular organisms in expanding clonally. Evolving in

a time-scale of years and bacteria even of weeks instead of millennia. There are over 200 cancers

with clonal heterogeneity within each and of course many infectious diseases, with many

mechanisms of drug resistance. However, in this complexity we should not lose sight that drug

resistance is fortunatelylargely selective to cancers and infectious organisms and much rarer in

normal human tissues over shorter time spans and then quite likely epigenetic.

Since Nature has will but cannot see,14drug resistance may therefore offer us a favourable

opportunity to set up the target cells to a simpler, wide-spectrum chemotherapy. Based more upon

this adaptive response than molecular complexity, for comparable if not more efficacy and

selectively than the drug resisted. As well as increasing the cost-effectiveness of complex targeted

cancer drugs allowing us to use where possible lower cost, clinically accepted drugs.

This turns drug resistance on its head in the sense that the more the resistance the more effective can

be the counterdrug. Depending upon a number of factors including the therapeutic gain (tg) of


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counterdrug 2 (C.Dr 2) when it is activated by the resistance R1 to C.Dr 2* (Fig 2). Tg is here

defined as the LD50ratio C.Dr 2*: C.Dr 2.

Overcoming drug resistance (Fig 2) Cancer.

A drug 1 (Dr 1) develops or is further induced to develop a resistance R1. This in turn sets up the

target cell to activate a subsequently delivered C.Dr 2 (prodrug). Since this is of lower toxicity it can

be dosed at higher concentrations, generating endogenously (or at the cell wall) a higher

concentration of activated C.Dr 2* than can usually be given and with substantially lower side-

effects.

Where the gene induced to express R1 is also widely distributed in evolution this may allow a wide,

therapeutic spectrum based upon the foregoing specificity. Cytidine deaminase (CD) is one such

gene in our prototype formulation (Fig 3). With homologies throughout evolution from archaic

bacteria to humans. As a family of metabolic and activation induced (AID) members15-18which can

reach a high level of expression in ALL, AML and CML

19-20

and often other cancers when

activated. Deaminating to inactivate the long-established clinical drug 1-beta-D-arabinofuranosyl

cytosine (AraC) and activating the subsequently delivered, clinically well-established 5-fluocytosine

to 5-fluorouracil (5FU) (Fig 3). 5FU has only a narrow therapeutic window (TW) difficult to control

in vivo between effective dose and high toxicity. Where the CD expression developed or further

induced is high, this can generate endogenously a relatively high 5FU concentration equivalent to

high dose chemotherapy (HDC) with 5FU 21but with much reduced side effects expected. Which

may approach a knock-out (KO) dose important to prevent time for further acquired resistance R3

such as thymine synthase (TS) resistance developing, A modified method of refs22-23is also being

considered if required to reduce toxicity further and rescue normal host cells.


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Alveolar lung adenocarcinoma established cell line A549 gave a favourable response in vitro (Fig 3),

The CD resistance assayed as24increases steeply with multiple doses of 0.5mg/ml (net) AraC, which

has at this suboptimal dose a reduced toxicity.

Where a gene such as CD already exists in the human genome, gene transfection by injection into the

tumours25for selectivity would be unnecessary and miss metastases. Thus the thymidine kinase gene

in a replication-deficient adenoviral carrier has been transfected into a highly malignant brain glioma

to sensitize it to gangliovar.26Whereas this cancer and other primary and secondary brain tumours

would be potentially favourable candidates for the prototype formulation (Fig 3). Since AraC is

tolerated by the CNS,27the brain has a particularly low background CD28and analogues are under

consideration to reduce cerebral damage.

Bacteria

Serious problems of bacterial antibiotic resistance may also be by-passed by resorting to the

AraC/5FC formulations or analogues particularly if lower toxic modifications are successful or these

are incorporated into antibiotic formulations (below).

Streptococcal and faecal cells were made resistant to a serial dilution of AraC applied x1-2 daily

mostly in log growth, followed by 1-2 doses of 120 ug/ml 5FC (net) and assayed by a relative

turbidity (RT) method (Fig 5). Streptococcus showed a marked inverse relationship between RT and

5FC concentration. 5FC was also under these conditions bacteriocidal to faecal cells, increasing

linearly with a serial dilution of the AraC administered earlier to set up the cells.

Methicillin induces beta-lactamase overproducted29in MRSA. This can spread to other bacterial

species by plasmids and secreted beta-lactamases in infected or absess fluids30. A remarkably wide

range of beta-lactamases can be induced in resistance31. Counterdrugs to this cephalosporin-3-R2

derivatives, corelease the substituent R2 upon beta-lactamase cleavage of the beta-lactam bond,


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usefully also for beta-lactamase assays32. Considering this had a fairly low tg quite satisfactory in

vitro results were obtained (Fig 6).

Drug and counterdrug schedules.

A number were tested in vitro to approach stepwise a KO dose33with cancer or bacterial cells. 1)

The resistance R1 (Fig 2) may be built up be a succession of doses of Dr 1 to activate one or a few

doses of C.Dr 2 (Figs 4-6). 2) The cell platform burden reduced stepwise by a succession of Dr 1 and

C.Dr 2 interspersed with other drugs (Fig 7). 3) Dr 1 and C.Dr 2 can reciprocally set up for each

other the raising or lowering respectively of R1 (Fig 2). Thus when AraC and 5Fc were closely

alternated for two successive cycles (Fig 3) over several hours under log growth conditions changing

the medium at each step, there was a stepwise reduction to 85% bacteriocidal (Fig 8). 4) Of

particular note formulations intended to delay drug resistance may reach towards a KO for reasons

suggested below. It will be interesting to see how far these schedules can be adapted to in vivo

conditions, such as Fig 8 as an effective alternative to HDC of 5FU21by continuous intravenous

infusion with lower toxicity.34

Delaying drug resistance (Fig 9).

The success in codosing two or more HIV antiviral drugs has encouraged extension to anticancer

drugs with only limited success. However, where R1 and Dr 1 and tg of C.Dr 2 are sufficiently high

and the C.Dr 2 is codosed with a drug 3 (Dr3), resistance R2 to Dr 3 may be suppressed. For there

would be a steep rise in C.Dr 2* activity when cell activity resumes as R2 develops. Since this may

be close to if not at a KO (Fig 10), the target cells may have become trapped between Dr 1 and a

steeply rising C.Dr 2* activity (to be confirmed). Coupling a C.Dr2 with a Dr3 (Fig 9) may also

come to have the following advantages for cancer and bacterial applications:-


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(i) Delays passive cancer or bacteria cells resuming activity and (with cancer) metastasising, (ii) This

can be empirical, not always requiring Dr 3 resistance R2 mechanism to be identified at least

initially. (iii) Which may be particularly useful where there are complex resistances e.g. to EGF-R or

PDGF-R inhibitors or (iv) a rebound of resistant cancer cells after a successful course of treatment or

(v) against a histologically confirmed cancer metastasis from cancers of unknown primary sites (a

substantial cause of cancer death). (vi) Where a rapid response by an antibacterial drug to an

infection is required. e.g. to B.anthracis, haemolytic strains of E.Coli such as 0104:H4, or an initially

unknown bacterial infection.

Reversing drug resistance.

Drug resistance can sometimes be induced (Fig 2) or reversed (Fig 11) across the perceived wal l of

resistance. Reversed by varying the formulation delaying drug resistance (Fig 9). In either direction

selecting for minimizing the generation of high tg C.Dr 2* by drug resistance R1 or R2 respectively.

Thus the resistance of a Streptococcus to penicillin G was reversed by first incubating the bacteria

with suboptimal concentrations of penicillin G codosed with a full concentration if 5FC, followed by

assaying penicillin G activity with an optimal dose of penicillin G in fresh medium (Fig 12). The

reversion of resistance might expect to sometimes overrun weak original drug efficiency, increasing

this by selecting for e.g. increased drug influx, reduced efflux or even increased conformation of the

target to Dr 3. Increasing Dr 3 efficacy and widening its useful spectrum (to be confirmed).

Discussion.

The perceived wall of resistance and dose limits to cancer drugs may sometimes be overcome and

this extended to bacteria. Not only as a simpler alternative to prevailing methods but also to seek to

increase the efficacy of targeted cancer drugs. Where these are selected by personalized cancer

treatment it may help to increase sustainability which may come to limit application. 8-9


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For reasons given these methods may be timely to develop quite quickly and economically and

consider how far they may be adapted to clinical practice. For where we can turn drug resistance on

its head, the more the resistance the more effective may be the ongoing chemotherapy. Turning the

resistance we fear to advantage. Rapidly resisting organisms such as HIV and the flu retroviruses,

malignant melanomas and brain gliomas becoming candidates and antiangeniosis drugs35reaching a

fuller application.

Acknowledgments: I thank Mrs P. J. Turner for unstinting and skilled collaboration; Mr J. M.

Turner for ongoing IT support; Mrs E. M. Kiddle for IT assistance; Mr R. Sharp and Mr A.

Williams, Gigstream plc, Oxford UK for computer research advice; Mrs J, Collard for assistance

with photomicrography and photography. Competing interests: Patent applications.


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15. Laliberte J & Momparler RL. Human cytidine deaminase purification of enzyme, cloning and

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myclosuppression by Prolonged Administration of High-Dose Uridine. J.Nat.Cancer Inst. 81(2),

157-162 (1989).

23. Leyva et al. Phase 1 & Pharmacokinetic studies of High-Dose Uridine intended for rescue from

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24. Anderson L. et al. Cytidine deaminase essay Arch. Microbiol. 152, 115-118 (1989).

25. Harris JD, Guttierrez AA, Hurst HC, et al. Gene Therapy for Cancer Using Tumour-specific

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26. Sandmair AM. et al. Thymidine kinase gene therapy for human glioma Using replication

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29. Keseru JS, Gal Z, Barabas G.et al. Investigation of beta-lactamases in Clinical Isolates of

Staphylococcus aureus for Further Explanation of Boderline Methicillin Resistance.

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30. Brook I. Beta-lactamase-producing bacteria and their role in infection. Rev. in Microbology 16,

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31. Patterson JE, Reck M & Jorgensen JH Extended-spectrum beta-lactamases: dilemmas in

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33. Nyce JW. Drug-induced DNA hypermethylations. A potential mediator of acquired drug

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LEGENDS.

Fig 1. Resistance to antibiotics develops quite rapidly. The beginning of the arrows when a given

antibiotic was clinically introduced, the tip of the arrow when the first clinical case of

resistance to it was reported.

Fig 2. Overcoming drug resistance. When resistance R1 to drug 1 (Dr 1) develops or is further

induced it activates a subsequently delivered lower toxic counterdrug 2 (prodrug) (C.Dr 2) to

a cytocidal (or apoptotic) counterdrug 2 (C.Dr 2*).

Fig 3. Prototype formulation of Fig 2. One or more doses of AraC (Dr 1) are given until resistance

R1 (cytidine deaminase) resistance develops. 5FC (C.Dr 2) is then added at relatively high

concentration where it becomes deaminated by R1 to cytocidal 5FU (C, Dr 2*).

Fig 4. CD resistance to AraC sets up (sensitizes) cancer cells to counterdrug 5FC (Fig 3). Alveolar

type 2 lung adenocarcinoma cell line A549 dosed daily for 7d with serial dilution of AraC

changing medium daily. On +8d one dose of 5FC in fresh medium added. Residual cells

assayed as described in Methods. 1,0125. 3,025. 4,05 g/ml (net) AraC. 2,cells, medium

only. 5, resistance overcome by 5FC.

Fig 5. Overcoming bacterial resistance to AraC with counterdrug 5FC (Fig 3).

A serial dilution of AraC was added daily for four days with reinoculation into fresh medium

daily. 1,faecal. 3,a Streptococcus. Counterdrug 5FC was then added to 2,faecal and

4,Streptococcous and assayed by relative turbidity~19h later. Resistance overcome by the

5FC to 5,faecal and 6,Streptococcus. All concentrations within clinically accepted limits.

Fig 6. Overcoming penicillin resistance (Drug 1) with a counterdrug 2 (Fig 2). A penicillin-G

resistant Streptococcus was incubated with a beta-lactamase counterdrug and assayed 19h


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later. 3,penicillin-Gsensitive, 1-resistant bacteria. 5, penicillinresistant cells are sensitive

to counterdrug, but 4,penicillin sensitive bacteria are not.

Fig 7. Discrete, short multiple step dosing reduces the cell burden towards zero. A Streptococcus

inoculated successively into fresh medium each time in 25cm2cell culture flasks and

incubated 36-37C.(a)-(a*) AraC, (c)-(c) penicillin G, (d)-(d) 5FC, (e)-(e) AZT. Penicillin

G resistance 1> 2>3. 1,High AraC, low 5F activity, 2, very high AraC, fairly low 5FC

activity. 3,AraC and 5FC fairly low activity. All high AZT activity. Assayed relative turbidity

as inverse of time to reach a standard turbidity. Clinically accepted concentrations.

Fig 8. Discrete rapid alternation of drug 1 and counterdrug 2 (Fig 2) reduces cell load towards zero.

A Streptococcus model. Innoculi successively into fresh medium in 18-well plates incubated

at 36-37C for 2 or 3h alternatively with 2,5FC and 1,AraC in two cycles Fig 3. Relative

turbidity as for Fig 7.

Fig 9. Delaying drug resistance.

A. Dr 1* is codosed with its counterdrug C.Dr 2. Deactivating Dr 1* to Dr 1 by R1 is delayed

because the cancer or bacterial cells renewed activity activates the C.Dr 2 of high therapeutic

gain (as defined) to C.Dr 2*.

B. Codosing C.Dr 2 with a drug 3 can also for the same reasons delay the resistance R3

deactivating another drug Dr 3* becoming deactivated to Dr 3 by another resistance R3.

Fig 10. Delaying cancer drug resistance. Alveolar type 2 human lung adenocarcinoma cell line A549

incubated with 5 successive doses over nine days with change of media each time. 3,AraC,

challenged at +39d with 5FC. 4, 5FC. 1,AraC + 5FC. 7,cells in medium only. All

concentrations within clinically accepted concentrations. The absence of recovery of 1 after

44d+ in fresh medium no agents indicated an approach to a KO dose.


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Fig 11. Reversal of bacterial or cancer drug resistance. Fig 9B can be reversed when the cells are

resistant to a Dr 3. A suboptimal concentration 1/n x Dr 3 is codosed with an optimal

concentration of C. Dr 2. Dr 3 can develop activity to Dr 3* to inhibit the cell resuming

activity and activating the high therapeutic gain C. Dr 2 to C. Dr 2*. Such that an optimal

dose of Dr 3 alone (or other combination) subsequently delivered is cytocidal.

Fig 12. Reversal of penicillin-G resistance (Fig 11). 1, A Streptococcus presensitized to activate 5FC

(Fig 3) was incubated with a serial suboptimal concentration of penicillin-G. codosed with a

full dose of 5FC and then assayed with an optimal dose of penicillin-G. 2,as 1 but the bacteria

not presensitized to 5FC. a~70% reversion of penicillin activity, b before resistance

developed. 3, As 1 but not presensitized and no 5FC added.


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METHODS.

Materials. Sigma-Aldrich (Poole, Dorset, UK), Calbiochem (Merek Bio-Sciences Ltd, Beeston,

Notts, UK), Molecular Probes Europe BV (Leiden, The Netherlands), Upstate Ltd (Milton Keynes,

UK).

Microscopy.Tungsten (Tenstile,Prior), Mercury (intense UV lamp) with filters. X1-x100

transmission (Koeller), incident, phase contrast. Microdensitometer (Evans Electroselenium, UK) or

35mm camera (Leica/Leitz) in vertical tube of a binocular microscope. UV/visible Unicam

spectrophotometer. The latter and microscope stage with temperature control cell holders and

chambers respectively, circulated by water from Haake F3 Digital water bath (Seven Sales, Bristol,

UK).

Cancer cell culture and procedures. The cells were incubated at 37C in treated 75cm2or 25cm2

cell culture flasks or 6-96 well-plates with sealed lids (Corning Inc. Corning, NY, USA) in media

recommended by cell suppliers, usually with 10% foetal bovine serum supplemented with L-

glutamate, penicillin G, streptomycin sulphate and sometimes amphotericin B. Cells trypinised

(0.25% tyrpsin (1,250) / 0.02% EDTA) to detach. PC-3 cells also had 1% neaas and 0.01%

collagenase or no collagenase for monolayer or soft agar (for microspheres)47cultivation,

respectively.

Cells were incubated with 0.5mg/ml MTT or less / 15-60 min for measuring viability and metabolic

activity. For bulk assay of 25cm2flasks the medium was decanted off carefully, 3ml of 16% Triton

X-100/0.01 NHCI added, shaken vigorously/1 min, standing for 30min48. Residual bubbles could be

most simply removed with a trace only of n-octanol before measuring with the microphotometer.

Cell counts were also obtained directly by detaching monolayers (trypsin/EDTA), neutralizing with

10-20% FBS in buffer, measuring cells per unit area (number of graticule eyepiece squares) or in a


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hemacytometer or a 300l aliquot of cell suspension in a 24 well-plate, with or without trypan blue

viability test. In other cell viability counts with adherent cells 25cm2flasks were lightly scratched

with 3x3 lines lengthways and widthways (9 cross-reference points) using the finest (size 0) sewing

needle in a holder held at an oblique angle to the outside surface. Each reference point could be

followed from 3 days to 1 month of an experiment with periodic changes of medium. Initial and

subsequent cell counts were made of 4-6-8 eyepiece graticule squares aligned around cross-points,

following the usual cell counting procedure of a hemacytometer. Following MTT incubation, alpha-

formazan crystal deposit were estimated and the mean cell density estimated relative to controls (no

drugs), the insolubility of the crystals being here an advantage. 5-10 reference point readings per

concentration of drug etc. A similar procedure was followed closely on 6-12-24 well-plates. Faster

and giving more information, cells were counted visually in a given area as above as total number

(TN), living (TLN) dead (TDN), blebbing, vacuoles etc (apoptotic) or necrotic, mitotic figures where

N-cell counts normalized to 100. This could be followed by lightly incubating with 25-50g

MTT/15-30 min incubation and scoring crystal density in each cell relative to a 0-10 sketched scale,

0 (no alph-formazan crystals)10 (cell outline obscured by massive crystal deposit). Despite the

subjectivity of the latter, the precision was estimated at 5% sufficient for our purpose with 5+

counts per flask and when the MTT concentration/incubation time was adjusted to give the most

sensitivity on the 0-10 formazan crystal density scale.

Bacteria: Culture and procedures.The bacteria were incubated in 5ml liquid medium in closed

25cm2cell culture flasks at 36-37C. The bacterial suspensions were discarded daily except for 0.1-

0.5ml retained in a 1.0ml disposable syringe with a 20GA/2in needle (to prevent clumping) and used

in the inoculum in 5ml fresh medium in the same flasks and the various drugs were added. This

exposed the bacteria to a partly logarithmetic growth over 4-6 days. This was followed by a

reinoculating with fresh medium and withholding the drug and adding 120g/ml (net) of counterdrug

(5FC). The total bacteria was assayed by measuring the relative turbidity by the apparatus described,


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using 0.1-0.25ml cell suspension in 5ml fresh medium. By diluting the bacterial suspension before

assaying the turbidity the growth could be measured beyond two orders of magnitude if necessary.

The persistence of the resistance (important to indicate presensitizing in the hospital) was also

measured by reinoculating as above without additives over a period of time, then assaying the

counterdrug.


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Fig 1

Fig 2

Fig 3


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Fig 4


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Fig 5


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Fig 6


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Fig 7


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Fig 8


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Fig 9

Fig 10


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Fig 11


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Fig 12

ERRATUM, Fig 12

Omit Curve 3, since this refers to another test. The curve to legend 3, Fig 12 is fairly similarto curve 2.

Drug Resistance as a Therapeutic Opportunity

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