Applications of First Order Equations
4.2 COOLING AND MIXING
319196x 109 Chloroquine pho.phote
• 1G.12<;, ot pho&phntc Chloroquine sol t The solution woG oad� in distill d w�ter.
PRT,', ,;Qtt':'" i': n:.: I' HCSPHA 'i' £
11oli,cul�r weight • 455. )9111
Base , • 455,3 - 2H3 P04
• �55.3 - 195.9,.
• 259.35
:. 455.lgui of Pri�aquinc diphoophatc contains 259.35 o! active aubs lonce.
Ratio·' of a11l t to octi ve i;\lbatanco '
•4S5.3 1 259.lS • 1.7S5:1
A solution ot 1°'Ja of rrtno1uine bane will contain 45�.1 10 o: colt 259. 3•.
• 17.55gn The aolutlon weti oa.Je in dit.Llllud W•lor ,
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-112-T.1:m.\CYC:.It:E l f lDROCHLc:RIO;;
Holccul.ir >eight • •180,9,y:i
•
,. bate ..-i • 480,9 -(HCJ) ,
• 480,9 -36.S
• 444,4
Ratio, or salt to ba:ic • t.00 .9 1 '1-!•l,4
a
1,802:1
A oolution of 109m of tctrocyclinc ha�e will cont�in l'.80,9,: 10
444.'1 1
Thus 10 ,82011 or t1:lr1Jcyclino hydrocholoride 1:: oq\1111al!!nt to 109� o! the b��c.
Th<! �lock: Golut on wbc maJe ln ,01t hydrochloric acid.
\-1!13306� !!Cl
�loloculo:- wcighl • S'19,4a:n
" b�GC •. � .. S49,4 -llcl
• 512,9
Ratio or 1elt lo bouc • 54�.( 1 512.9
• 1,07 1 1
So 549,<:Jm of liR3306l IICl conto.1na 519,2 oC beae .
A eolution cont•ining 109a ot .r.t33063 boa,: 1e oquivolont to
10,79m of lffl33063 Hcl Solution, The stock solution won �ade
in 0.1� Twcan 80. UNIVERSITY OF IBADAN LIBRARY
-:1.13-2 .e. Proparin:ion of cul turc l"edium Rr-11:1. :1.640
'l'ht RP!'l �640 used 1n thin work a11 .iuppl1ed by
Gr.:mc! Isl cll;l B 1olog!c11l :i, :;e.., Yorlc 1 U. S.A. in p ck� ti:
of balcncctl ingrcdi<:nt::i ,ufficicnt to r.i11ke onl• lit.::,.
10, i'J" of !ll.'�i: powacr w,,, o1sao1 v,:,d 1n 900 1 of dcubl -di�llllco -., t.r, t..dC:1..w al:o c,1, 'i.94'.Jr., of
N-�-llydro:1y c�yl,. p1pcro&1ne -r:-2- ethLno eulphonic acid (H!:I'-S) c.,d the totol volu=o w�s 1111de up to 960ml,
This solution was st�r111zcd through u 0,42�� �illi�orc filter and dl�cna d aucepticblly 1n 100ml vol��c ir.to
screw coped bo:tles (atcrili:�d by outocl oving). SS
eo:i1WI bic rbonete .., ,1 f'.ttp , d 11."1 11t-,r111:cd l)y th i:.nc process o� !1ltcret1on .:ind prca ·rv •d in to rile a::r.iw
ce:-e bottle:.. To u110 , 100111 bottle of Rfllt 1640, 1.2 l of St ao�1u:::: bicert'Gflole va3 a�dcd by ator1lc pipette
(th1a ?•vo n pH o! 7.(.), U:uolly 50 U?/�l o�
Gcntw:iycin v r addc� to •11 tnau b11Cter1 l ltitcct!on.
2,9 1ar.ri.- •10:: err' T. 19!1
IIOdJu::i (Burro-.1a lielc- l!cckcnhul woa dl111101v d .in 950 1 of dout,Jc dlatlllcd w tcr cni 1.4 GJ/1 ot ao11u.
bicarbon<1tc .,,., addod t.o �1 ,e • r-'1 et 7,4 a11J tt • C]l1.oc,
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-11<1-conccntrat1on wo:. inc:rroaed fco:si 1.00 to 3.009/1J The ·-,hole
•olution was atorilised by filtration (milliporo)1nd �torcd at 4 c. Gentamyc1n 0 was added o• in Rl'K1 1640 .
GLUCOSt ta:onn:.
20S 9luco10 �oa aGde by disaolvin� 20gn ot glucose per 100?11 of sterile di1tillc11: w ot<:r.
Different types of tr.ra wero used al differont 5tDJ�a of this •t:udy. �llen AB Lciznune serwn was needed, it was obtained frOfl u.c.H. blood bonk. AD non il:::lune sel"\LO was
obtained froa Rockefeller f oundation U.S,A. Hor.iologoun scrua wu obtained fr� the pr plc o f puro-itiaed blood by centrlfugation.
2.12 Prea�rvatlon ot "1 �s� �8rosit�·1 By rree:ing ,
• The freezing aolution con,iotc of 28S 9lyec-r.�1, J�
aorDitol ll!ld 0.6St aodius chlor1do.4.� ot 1orbitol wu prepared in 0,9' 1odiua chlori�e aolution, 100al of thia preparation w11 added t.Q 70al of glycerol (99-i purity) and this wee filtered through 0.4�u nill!pore filter,
PP.ctZill:i1 The 1ntecte1:I blood wa1 ccnt,Jfujcd at :?000 CJ>II for 10 cinutca, the aur,ern tent 1111� butty coat reaove l.
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-115-Th1o wao repe�tcd after adding 10ml of mcdiua without scru., and centrifuged l.(Jain . Blood from a palicnt wao normally
wei.hcd lwice with the medium. The cell� were rcauspendcd in e11uul wlul'\e or freezing solution 1111d dS i:itril>utE:d 1n
0.5ml - bill amounts 1n screw cap vials ond dropped by meens of e length of ro�e 1nlo liquid ni��ogl!ll r�fridg�rator(et
a temper�t:uro of -70 ° c), after cooling rapidly in olcohol
ice b,,th.
Preparation ot thowing �edium:- 3.si of soJium chloride (Hyptrtonic Saline) wa• prop.ired and 4teril1zed in autoclave • •
The�tew cop of the vial wes loosed olightly ond �ot upright in woj:er. The thawed suopena:ion was centrl i'u.51!d ,,l:
1500 r.p.m. for 7 l'linutcs ;md the t:upernat:ant r•,mo·,ed. The cell, were resuspended !n equal volume of hypertonic anlinc end c«:ntrifuged at 1500 rpm for 7 min. Agoin, utor re111oving the supernatant,, the cells >(ere resuspended 1n equal volu, 10
of culture medi\1111 ond w�·h�d twic e.
sri:nrLir'f: The cul turt ploteo wor,• stcril ised by or.po, J.no to bl traviolet Irrodiot1on, while th•• CJlo-.o wore a liltc
pippeto and univeraol bottles tlftur thom..gh clf!aning 1o1ere aterili .. cd in hot air ov�n or4ut o., 1'Gvt,• , 1'h• culture wa•
•
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-116-'l!he work1n') bench w411 r.tcr111scd with 70J ethanol and during the '
2. 15
process of culture, h�4t 1rrad1at1on fr0111 bunsen fl�ne was e111ployed.
ST.\TI::i':ICAL ,\Ui\LYSIS.
The re�ults were cx�ressed as 111ean - + standard e�ror of the mean. In animal experiments, th� changes in rellabilJ.ty
were accounted for by introducing a weighting coefficient (Sct11itzer and Hawking)- 1n Experim�ntal Chc�otherapy 1963 .
Student 't' test was applied in cor:ipnring effects
belween groups c •nd difrerence is taken to be significant when
P>o.os. Paired •t• test and a test of variance wer� used 11hcn necessary.
2.16 PRO-&DU�: Of TESTS.
The animal model employing the cyclical trann�ission of
!• berghei in mice provides a useful tool in drug evoluation.
It obviates the problem of chaG1n� test object• who often default, lnd allows unlillited nUlllber of repetitions of experimenta oince \he animals arr easily av4ilable. Dy
varying lh c tiJile at which the! drug treatment of 1poro�oite infected 111icto is 1nltl'ated,. prophylactic or 1chi::ontocidel activity can be determJned. Moreover the 1/R 33063 supplied
waa specified for uae 1n animal, or!!!. vitro modal• only.
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'
•
-117-Blool !khi::ontoc1!l11l Aetl•,ity in vlvo'4- DAY Tr.ST '
The J:1<:,thcd • ""' bosed 09 that described ny r 1:tcr ., r,ortert.?c tu::-19 .,
(&Pot�rs 196��. Tho blood schizontocidal activity of
Chloroquin!!, llit33063 1 T� tracyclinc .nd Pr!r.iaquinc dgala:�t the dt·u') scnr.iti,·e P. berahei br-rnh,.1 wes testecd on ct.:;1.,1no
-Sw1!:s rnlce · (�P<!rythro:oon free) wc,!.9h,ing, betwten 18 ,o
(229111. Ani.�&l• received o standard do�c o! 1c 7 pftrasilJsed red blood c,11& lntrevcnouclv fr().'11 one donor mouse. Tt� .
doy of inoculation �ar temed Oo. Al the a�e ticc the an'mius r.ceivcd once uaily tr tment at aevcr�l dos�
level• of antioolariol d::-ugs, 00 1 01, o
2 ond D3• Thin bloo1 films �ere •ade fr= lhc t,11 blood on o� and us�J in a,��ssin� the activity of the drugs,
Tht av ·raie p rcent ge suppression of por aitacrtla by varioua drugs wa, cal��l�t�d by the !ollowin9
fornula1-,, '
(Average i �r�1ito��1 in r • r 1 •
A·1cra��:: rarasita�m1c1
!n t r c! .. 1,.,,. l �) ) Average i rb�aoitotnia in control
Th control c.ni:l.sl:i received th<' vehicl,: only , 7he rc.:iulta wore oxtrapolot�d 9r�phicolly to �etenainc the EDSO.
Y.CTHO!> I:!: CV•lurt'cn of '.'•hi�rntor!1nl .\.:tivitv In vl'7o un½J • _ an �stebli�hnd 1n·�ct�on 1 I �9 - D\Y T:�T'
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-118-treatment woa withheld in this exper.1:nent until the
parasit�cmia was relatively high in order to ensure a nore reliable .lS6IIY of activit}'• The nethod employed w11:i that de1cribed by Ryley and Peters (1970).
Male albino S•1i!>� mice were inoculated with 10 par.i:.ites .,
in 0.2ml inoculum. The treatment was withheld for three
days tu Fer:nit the infection to become �ell established b�t not l.:ile enough to hove produced sufficient debility to alt•r
the responf:.11. of the host to the effects of the drugs. Thia experiment i� dependent on cr.mparing th · reopon�es of nice infected with .!!.· bernlwi berghei to ".:hlo:-oquinc, WR330G3 1 Tetracycline find Prilra1qui1'.e with • eurviv:il tioes of the
controls, untr.e.:ited \lith drugs. Seve-nty-t110 hours aft:r
parasite inoculation the mice were g!v�n �1ther o sinqle dose of the candidate compound or once doily dosecf!ive days
running. The drugs w�re ln•ectcd Gub-cutancously Qlld the
animals obsar,ed over a period of 2.: days , They wore recon!ed
cured if free fr0:11 infection on Do•28 days.
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Elementary Differential Equations with Boundary Value Problems
(Page 149-160)