Synthesis of primaquine glyco-conjugates as potential tissue schizontocidal antimalarial agents

Article abstract of DOI:10.1111/cbdd.12944

Primaquine (PQ) is the only drug used to prevent relapse of malaria due to P. vivax and P. ovale, by eradicating the dormant liver form of the parasite (hypnozoites). The side-effects associated with PQ limits is uses in treatment of malaria. To overcome the premature oxidative deamination and to increase the life span of drug in the biological system, the novel glyco-conjugates of PQ were synthesized by coupling of primaquine with hexoses in phosphate buffer. The saccharide part of the hybrid molecules thought to direct the drug to the liver, where hypnozoites resides. All the synthesized compounds were fully characterized and evaluated for their radical curative activities. The three compounds viz glucoside (15a), galactoside (15b) and mannoside (15c) with high activity were tested for their activity in rhesus monkeys where the most active compound 15b showed twofold activity (100% radical curative activity at 1.92?mmol/kg) than the standard drug PQ diphosphate (3.861?mmol/kg). It is proposed that results from these studies may be advantageous to develop a new potent tissue schizonticide antimalarial compound.

Full text of DOI:10.1111/cbdd.12944

DR. ANIL KUMAR SAXENA (Orcid ID : 0000-0002-0212-7246)
Received Date : 13-Nov-2016
Revised Date : 03-Jan-2017
Accepted Date : 05-Jan-2017
Article type : Research Article
Synthesis of Primaquine Glyco-conjugates as potential tissue-schizontocidal
antimalarial agents
Chandra S. Azada, Mridula Saxenab, Arif J. Siddiquic, Jyoti Bhardwajc, Sunil K. Puric, Guru
P. Duttaa, Nityananda, Anil K. Saxena*a
(a) Division of Medicinal and Process Chemistry CSIR-Central Drug Research Institute Lucknow- 226 031, U.P., India.
(b) Department of chemistry Amity University (Lucknow Campus) Gomti Nagar Scheme, Lucknow- 226028, U.P., India
(c) Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow- 226 031, U.P., India.
To whom correspondence should be addressed; E‐mail: anilsak@gmail.com
ABSTRACT
Primaquine (PQ) is the only drug used to prevent relapse of malaria due to P. vivax
and P. ovale, by eradicating the dormant liver form of the parasite (hypnozoites). The side
effects associated with PQ limits is uses in treatment of malaria. To overcome the premature
oxidative deamination and to increase the life span of drug in the biological system the novel
glyco-conjugates of PQ were synthesized by coupling of primaquine with hexoses in
phosphate buffer. The saccharide part of the hybrid molecules thought to be direct the drug to
the liver, where hypnozoites resides. All the synthesized compounds were fully characterized
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12944
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and evaluated for their radical curative activities. The three compounds viz glucoside (15a),
galactoside (15b) and mannoside (15c) with high activity were tested for their activity in
rhesus monkeys where the most active compound 15b showed two-fold activity (100%
radical curative activity at 1.92 mmol/kg) than the standard drug PQ diphosphate (3.861
mmol/kg). It is proposed that results from these studies may be advantageous to develop a
new potent tissue schizontocide antimalarial compound.
KEYWORDS
Primaquine diphosphate, Phosphate buffer, Tissue-schizontocidal activity, Radical curative
activity, 8-amino quinolones.
Malaria in human is caused by five plasmodium species, viz. P. malariae, P. ovale, P.
vivax, P. falciparum and occasionally P. knowlesi. It is the most prevalent among the top 10
diseases in the world.1 It occurs in tropical and subtropical countries lying between latitude
30oS-40oN.2 Malaria affecting nearly 200 million persons across the globe and led to 429
thousand deaths in 2015.3 Most deaths in 2015 are estimated to have occurred in the WHO
African Region (92%), and most deaths were in children under 5 years of age which is
equivalent to 70% of the total deaths.3 The global burden of malaria is estimated to have
declined by 60% worldwide between 200-2015 and 26 % between 2010 and 2015,
nevertheless this situation would be better if there is no presence of anti-malarial drug
resistance.3 In addition to the chloroquine resistance, resistance to the combination of
sulfadoxine-pyrimethamine which was already present in South America and in South-East
Asia is now incipient in East Africa.4a Recently, concern has been raised towards the
resistance to artemisinins, which could endanger the artemisinin combination therapies
(ACTs).4b Intensive research has been done for the discovery of malaria vaccine; and no
effective vaccine is introduced in the clinical practice. Till date, GSK RTS,S/AS01 (trade
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name Mosquirix) is the only recombinant protein-based malaria vaccine approved for use by
European regulators in July 2015, with the limitation of its use in the region specific
children.5
The most currently used antimalarials are potent blood schizontocidals i.e., they act
rapidly against the parasite forms that invade erythrocytes and cause the well-described
malaria symptoms. Among the plethora of antimalarial drugs, 8-aminoquinolines are the most
potent class of anti-malarial drugs with radical curative activity, having properties to
eliminate the hypnozoites from the liver.6 Considering the historical perspective of
aminoquinolines, 8-aminoquinolines are the successor of quinine (1) and methylene blue (2)
(Figure 1).7 Among the important 8-aminoquinoline derivatives Primaquine (PQ) is the most
effective being the only drug drug against P. vivax hypnozoites.8 PQ acts specifically on the
pre-erythrocytic schizonts which are concentrated predominantly in the liver and causes
relapse after multiplication.
If PQ is not administered to patient with proven P.vivax and P.ovale infection, there is
a very high likelihood of relapses within weeks or months (sometimes years).6b PQ is also
effective against mild to moderate Pneumocystis pneumonia (PCP) and is co-administered
with clindamycin to AIDS patients.10 It is the toxicity of the PQ which limits its clinical
utility in therapeutic applications. The most serious side effect of the PQ is the haemolysis
particularly in individuals with heredity deficiency of G6PD (glucose-6-phophate
dehydrogenase).11 The other significant side effect associated with PQ and 8-aminoquinolines
derivatives are methemoglobinemia, leucopoenia, abdominal cramps, and epigastric
distress.12 In addition of its toxicity PQ is readily absorbed metabolized and eliminated, so
prolonged doses are required which amplify its undesirable effect thus reduces the interest in
the PQ adjunct therapy.13 So there is a persistent need for safe, long lasting tissue
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schizontocide active agent against latent exoerythrocytic forms with prolonged action for the
treatment of liver stage of malaria.
To decrease the undesirable side effect of the PQ, modification of primary amine
group also play important role because altering this amino group shows tremendous
improvement in terms of the bioavailability of PQ. These types of modification at primary
amino group involve (i) coupling the N-cycteinyl–PQ to carrier protein, e.g. lactosaminated
serum albumin,14 (ii) N-acylation of PQ with amino acids or oligopeptides,15 (iii)
introduction of an imidazolidin-4-one ring over amine of the PQ,16 (iv) dimerization of the
PQ with N-acylated PQ with amino acids,17 (v) dihydro-furan-2-one linked PQ.18 The
explanation why the side-chain modified derivatives of PQ have enhanced curative properties
is not clear. The most accepted explanation for these modification may be significantly
reduce the extent of PQ conversion into carboxyprimaquine which is the important metabolite
of the PQ and have been identified in mice, monkeys and humans.19 PQ is promptly
converted into carboxyprimaquine (4) attaining 10 times higher plasma level than those of
parent drug, but carboxyprimaquine is significantly less potent than parent drug PQ (Figure
2). These modifications ongoing as a way to avoid premature PQ inactivation by oxidative
deamination to carboxyprimaquine (4). To overcome the two major challenges first inhibit
the premature inactivation and second targeting the drug towards the liver where latent
tissues exist and responsible for the relapses, we are keen interested to synthesize glyco-
conjugate of PQ (7) and then evaluate its biological activity (Figure 3). In continuation of our
efforts to design and synthesize new antimalarial agents we designed these molecules on the
basis of drug delivery and prodrug approach with improved life span of the drug in the
biological system.20 The saccharide derivative easy hydrolyzed in the biological system to
free primaquine at the slow rate, so lesser doses are required and simultaneously inhibit the
premature oxidative deammination. The foremost idea is based on the drug delivery system.
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It is well known that receptor on plasma membrane of liver parenchymal cells and
liver von Kupffer cells recognize hexose like galactose and mannose respectively. From this
it would appear that title compound with hexopyranose part as galactose or mannose may
direct the PQ to the liver where the latent tissue forms exist. This may result in substantial
decrease in its systemic circulation which may reduce the hematological toxicity and increase
in its selectivity. Bhadra et al designed a drug delivery system in which they intended to
deliver primaquine phosphate directly to liver cells using polypropyleneimine (PPI)
dendrimers-coated peripherally with galactose. The galactose coating of PPI dendrimers can
therefore make the PPI systems more efficient and appropriate for targeted delivery of PQ
phosphate to liver.21
Experimental
General chemistry
Reagent grade solvents were used for the extraction and flash chromatography. All
the reagents and chemicals were purchased from Sigma Aldrich Chemical Co., Lancaster and
were used directly without further purification. The progress of reactions was checked by
analytical thin-layer chromatography (TLC, Merck silica gel 60F-254 plates). The plates
were visualized first with UV illumination followed by charring with 10% H2SO4 in CH3OH.
Flash column chromatography was performed using silica gel (230-400 mesh). The solvent
compositions reported for all chromatographic separations are on a volume/volume (v/v)
basis. All glassware’s were dried in an open flame before use in connection with an inert
atmosphere. Solvents were evaporated under reduced pressure. Tetramethylsilane (0.0 ppm)
1
13
was used as an internal standard in H NMR and CDCl3 (77.0 ppm) was used in C NMR.
The abbreviations used to indicate the peak multiplicity were; s, singlet; bs, broad singlet; d,
doublet; dd, double doublet; t, triplet; q, quartet; m, multiplet; Hz, Hertz. FAB MS was
recorded on Jeol (Japan)/SX-102. Infrared spectrum was taken with KBr on Perkin-Elmer
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RX-1. Melting points were determined on a Buchi 535 digital melting point apparatus and
were uncorrected. Elemental analysis was performed on a Perkin-Elmer 2400 C, H, N
analyzer and values were within 0.4% of the calculated values. In this study all the
compounds were synthesised in dark.
Bioevaluation methods
For all the experimental studies, laboratory bred Albino Swiss mice of female sex
(weight 25 g) were used. All the animals were housed in animal rooms under standard
conditions of temperature 22 10C and humidity (50-65%) and maintained on commercially
available pellet diet supplemented with soaked grains.
Activity in the rodent model
For the radical curative test, compounds were administered orally in three doses on
days -1, 0, +1 and animals were challenged intravenously with 1 X 104 P. yoelii nigeriensis
sporozoites on day 0. The thin blood smear was prepared from each animal on day 4. The
degree of infection was microscopically recorded in terms of number of P. yoelli infected
cells per 100 RBC (i.e. percentage Parasitemia) the mean value determined for group of 5
mice was used to calculate the percentage suppression of parasitemia with respect to the
untreated control group. Blood smears from experimental animals were observed until day 28
to record the day of patency. The animals which did not show any parasites on day 4 were
subsequently monitored twice weekly till day 28 post-infection. The animals which did not
develop any infection till day 28 were recorded as cured.
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Drugs
The aqueous suspension of each test agent was freshly prepared on the day of initial
use and the required drug concentrations were administered orally, once daily in 0.5 ml
volume. Pure samples of PQ, were obtained from Sigma, USA.
Radical curative Activity in Rhesus monkeys
Anopheles stephensi mosquitoes were fed on Plasmodium cynomolgi bastianelli
infected rhesus monkey during the declining phase of secondary peak parasitaemia when
mature gametocytes were in circulation (male to female gametocyte ratio of 1:3 is ideal). The
mosquitoes were maintained in the insectary under controlled condition of 27
1oC
temperature and 75-80 % relative humidity. The monkeys were left untreated till patency.
When parasitaemia ranging between 50-100 parasites per 10,000 erythrocytes was reached,
the monkeys were treated with the orally administration of the test compounds along with
chloroquine phosphate 5 mg/kg (base) per day for 7 days. Monkeys were followed thereafter
up to 90 days for reappearance of blood parasites. The absence of any relapse till 90 days
after end of treatment indicates complete cure, and partial activity was indicates by delay in
appearance of relapse as compared to the control monkeys which were treated with
chloroquine only. Primaquine 1mg/kg for 7 days was used as the standard drug for radical
curative activity.
The animal studied and experimental protocol were approved by the institutional
ethics and usage committee of CSIR-Central Drug Research Institute (CDRI), Lucknow
which is specifically designed to review animal research and strictly follows the guidelines of
the Committee for the Purpose of Control and Supervision of Experimental on Animals
(CPCSEA).
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Chemistry
The desired PQ was prepared by classical Skraup’s synthesis starting from 4-
methoxy- 2-nitroaniline (8) which yielded the 6-methoxy-8-nitroquinoline (9) , followed by
reduction to offer the desired 8-amino-6-methoxyquinoline (8A6MQ, 10). The reaction of
8A6MQ with phthalimido-2-bromopentane followed by hydrolysis, gave the primaquine (3)
(Scheme 1).22 The reactivity of the hexoses towards PQ in yielding the PQ-glyco-conjugate
may depend on the stereochemistry and reactivity of the hexoses. This may influence the
formation of mixture of α/β epimers. Thus optimization of the N-glycosylation reaction in
terms of yield, reaction time, and diastereoselectivity was a significant challenge. Several
attempts were made for the synthesis of glyco-conjugate e.g. condensing the halide donor of
hexose with PQ base or PQ diphosphate in DCM or acetone using variety of bases, but non-
separable mixture was obtained.
It is well recognized that the reaction between an amine and reducible sugar such as
D-glucose in water, at high temperature and in slightly low pH first proceeds through the
unstable corresponding N-glycoside, which undergoes the Amadori rearrangement to produce
the Amadori product. When the reaction of PQ-base and D glucose in C2H5OH was carried
out a new compound containing hexose and PQ was formed, this led to the selection of EtOH
as a solvent of choice for the model reaction. The formation of the compound containing both
PQ and D-glucose was confirmed by the UV absorption and charring (10% H2SO4 in
CH3OH) test for the hexose counterpart. This compound also showed the required (ES-MS
1
(M+H) 422.2 m/z.), however its H NMR analysis revealed that product was not the N-
glycoside, because there was no signal of anomeric proton in the expected region (range 4.5-
6.2 ppm) and instead there was two double multiplets at 4.04-4.26 ppm and at 2.66-3.02 ppm
of CH2 which is the indication of formation of Amadori product 13a-c (Figure 4).23 This was
further confirmed by the reported spectra corresponding Amadori products and 13C NMR.
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The reaction was performed with different hexoses using variety of Lewis/Brϕnsted
acid catalysts to achieve the desirable N-glycoside (Table 1) but in each cases we ended up
with the Amadori rearranged products 13a-c (chromatographed using neutral alumina)
(Figure 4). Afterward, in the pursuit of good and efficient method for the synthesis of N-
glycoside a contemporary green methodology reported by Maugard et al was used for the
synthesis of N-glycosides in phosphate buffer. This novel synthetic modification afforded a
new route for the synthesis of N-glycosides of PQ.24
Therefore, in the first experiment the condensation between the PQ (3, 1.0 mmol) and
a D-glucose (14, 1.1 mmol) was carried out in aqueous phosphate buffer pH 6.5 and at 50 °C.
It resulted in the formation of product which on tlc analysis, showed a bright yellow spot (in
iodine) having Rf 0.4 in 30% CH3OH:CHCl3 suggesting the formation of a product different
from the Amadori products 13b, thus indicating to be possibly the required N-glucoside. This
product formed was purified by column chromatography using neutral alumina and the
13
analysis of this product by state of the art techniques like 1H NMR, C NMR and mass
1
spectra confirmed it to be the desired N-glycosides. In H NMR the anomeric potons
appeared as a doublet having coupling constant 7-9 Hz suggesting axial-axial configuration
of H1 and H2 and therefore in sugar 15a-b, and 15e-h, the glycosidic linkage is β one. In
compounds 15c and 15d the anomeric potons appeared as a doublet having coupling constant
3.2, 3.6 Hz repectively suggested axial-equatorial configuration of H1 and H2 and therefore
glycosidic linkage is α one. The preference for formation of the β isomer in 15-b, and 15e-h,
can be rationalized in terms of the competition between substitution at the sterically least
hindered position (β) against the substitution at a position which sustains stabilization from
the anomeric effect (α). The substitution is driven predominantly by the steric factor thus the
reaction is find to be stereoselective for the β configuration.
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Although the reaction conditions gave the preferred products (15a-h, Figure 5), but in
order to optimize them the effect of pH and temperature on the N-glycosylation reaction yield
were investigated. When the reaction was carried out at 40oC for 5 h with the same
concentrations of D-glucose (14) and PQ (3) base at different pH, the best yield (60%) of the
product was obtained at pH 6.5. The effect of the temperature was also studied with the same
concentrations of the reactants at pH 6.5, where the reaction was favored at below 50 oC. So
several N-PQ-glycosides, using D-glucose (15a), D-galactose (15b), D-mannose (15c), D-
arabinose, (15d), L-rhamnose (15e), D-ribose (15f), D-xylose (15g) and N-acetyl-D-
glucosamine (15h) were prepared at 40oC and a pH of 6.5. In all reactions hexose was taken
in 1.2mol % with respect to PQ-base, acceptable yields from 50-60% were obtained.
Due to the complex requirements for complete parasite development of Plasmodium
vivax, no in vitro model exists for routine screening of causal prophylactic and radical
curative compounds against human relapsing malaria, hence the target compounds were
evaluated for their tissue schizontocidal (radical curative) activity in Swiss mice and the
promising compounds were than tested on rhesus monkeys. The rhesus monkey relapsing
malarial model (P. cynomolgi) has been used more than four decades and is the most
trustworthy model for the screening of antimalarial compounds for both prevention (causal
prophylaxis) and treatment of relapse (radical cure).
Radical Curative (RC) Activity in the rodent model
Promising activity was exhibited by synthesized molecules in tissue schizontocidal test in
swiss mice.25 Where glycosyl (15a, Table 2, entry 1), galactosyl (15b, Table 2, entry 2) and
mannosyl (15c, Table 2, entry 3) derivatives were 100% curative even at a dose of 30
mg/kg, while the rhamnoside (15e, Table 2, entry 5) was only 60% curative indicating that
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the synthesized compound 15a-c are as almost as active as PQ. The corresponding Amadori
products (13a-c) were inactive (Table 2).
Radical Curative Activity in rhesus monkeyes.
The promising activity of the compounds 15a-c fortified for their evaluation in rhesus
monkey, since the PQ is effective in Primate Model at 1mg/kg dose which is equivalent to 3.8
mmoles/kg of PQ, and hence all the compounds have been tested in equimolar doses i.e. 3.8,
1.9 and 0.9 mmoles, where the compounds 15a-c exhibited excellent radical curative activity
in rhesus monkey. The galactosyl derivative (15b, Table 3, entry 2) was 100% curative even
at a dose of 0.81 mg/kg (1.9 mmmol/kg), while at this dose the glucosyl derivative (15a,
Table 3, entry 1) was approximately 90 % curative. The mannoside 15c (Table 3, entry 3)
was 83% curative at 0.8 mg/kg (1.9 mmol/kg) while at lower dose 0.4mg/kg (0.9 mmol/kg), it
was inactive. The comparison of the radical curative activity of these compounds with PQ
indicates that the compound 15b (1.92 mmol/kg) is two times more effective than PQ (3.861
mmol/kg). The higher activity of 15b may be attributed to the targeted delivery of the drug at
the liver where the tissue forms exist and to its action as prodrug because of its easy
conversion to PQ in biological System.
The highest activity of galactoside 15b may be attributed to the higher concentration
of galactoside in hepatocytes due to the high binding affinity of galactose with
asialoglycoprotein receptors localized in parenchymal liver cells.26 The lesser activity of
mannoside 15c as compared to 15a and 15b may be due to non-recognition of mannose by
the asialoglycoprotein receptors on parenchymal liver cells but its moderate activity due to
interaction with the receptor generally present on nonparenchymal liver cells and by Kupffer
cells. The higher activity of glucoside 15a than 15c and lesser than 15b may be because of
the recognition of glucose sugar by the glucose transporter 2 (GLT 2) protein which is found
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in cellular membrane of liver which facilitates the bidirectional glucose transport across the
hepatocyte plasma membrane.27 It is interesting to note that these three sugars present in 15a-
c have primary alcoholic group (-CH2OH) while all other hexoses 15d, 15f, and 15g have
except 15h neither primary alcoholic group nor the extra carbon at this position. Thus
indicating the importance of primary alcoholic group which may be phosphorylated in the
biological system for further biological transformation.28 Some activity of rhamnoside 15e
devoid of primary alcoholic group may be due to the presence of –CH3 group at C6 position,
which may be necessary for favorable interaction with receptors nevertheless other hexoses
15d, 15f, and 15g missed this type of substitution at C6 position. The inactivity of 15h with
primary alcoholic group at C6 position and acyl-amino at C2 position can be attributed to the
importance of free hydroxyl group at C2 position.
Conclusion
To conclude, glyco-conjugates of PQ possessing high tissue-schizontocidal
antimalarial activity have been synthesized by the coupling between the primary amino
groups of PQ with hexoses in phosphate buffer. Among the three PQ conjugated hexoses
15a-c, the galactoside conjugate 15b is twofold active than the PQ. This prominent in vivo
efficacy of 15b against P. cyanomolgi infected rhesus monkey is indicative of the potential
utility of this compound in the therapy of P. vivax malaria infection. This enhanced activity
can be justified by its targeted delivery to the liver and its action as prodrug. It is intended
that results from these studies may be useful to develop a new potent tissue schizontocide
antimalarial.
SUPPORTING INFORMATION
Synthesis of compounds and analytical data (1H and 13C) are given in the Appendix S1.
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ACKNOWLEDGEMENTS
The authors also acknowledge SAIF-CDRI for providing spectral and analytical data. CDRI
communication number: XXXX.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
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Figure legends
Figure 1. Precursors of 8-aminoquinoline.
Figure 2. Proposed pathways for side-chain metabolism of Primaquine.
Figure 3. Prototype of the designed Glyco-conjugate of Primaquine.
Figure 4. Amadori products synthesized from the PQ and corresponding hexose.
Figure 5. N-Glycosides of PQ synthesized from the direct coupling of PQ with hexose.
Scheme 1.
Scheme 1. Synthesis route for the synthesis of PQ phosphate; Reagent and condition: (i)
As2O5/H3PO4, Glycerine, 100oC,1 h (ii) Raney-Ni,50 psi,4-5h (iii) phthalimido-2-
bromopentane, TEA, 150 oC, 6h (iv) NH2NH2, EtOH, reflux 72h.(v) ethereal phosphoric acid.
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Table 1. Effect of catalyst on synthesis of N-glycosyl PQ. a
Entry
Catalyst
Time (h.)
10
Yield (%)
1
-
~
2
Cuprous Bromide
Iron(III) chloride
Copper(II) sulphate
Nickel(IV) chloride
Zirconium(IV) chloride
Zinc(II) triflates
Copper(II) triflates
Zinc acetate
12
25
30
25
20
20
25
28
25
26
25
28
2
12
3
12
4
12
5
12
6
10
7
10
8
16
9
Chloro acetic acid
TFA
Acetic acid
10
10
11
10
12
a. The reaction was conducted with PQ-Base (1 mmol), D-glucose (1.1 mmol) and 15 mol% of catalyst in EtOH at room temperature
Table 2. Tissue schizontocidal activity table of Synthesised compound.*
Test
sample
Dose
mg/kg
30
Percent
protection
S. no.
1
100
0
0
100
0
0
100
0
0
0
60
0
0
0
0
15a
15b
15c
22.5
15.0
30
22.5
15.0
30
22.5
15.0
30
30
30
30
30
2
3
15d
15e
15f
15g
15h
13a
13b
13c
4
5
6
7
8
13
14
15
30
30
30
0
0
30
15.0
100
0
PQ
17
0
Control
--
0
18
0
* Swiss mice model, in a group of 4-5 mice.
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Table 3. Radical Curative activity in rhesus monkey.*
Entry
Test
sample
Dose
mg/kg
Percent
protection
(mmol/kg)
0.82 (1.9)
0.42 (1)
3.25 (7.7)
1.62 (3.8)
0.81 (1.9)
0.51 (1.2)
0.4 (0.9)
1.62 (3.8)
0.8 (1.9)
0.4 (0.9)
1.0 (3.8)
0.6 (2.3)
1
2
15a
15b
~90
~72
100
100
100
50
Nil
100
~83
Nil
100
40
3
4
15c
PQ
* Rhesus monkey model, in a group of 3-11 monkies for each dose.
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