PROPOSAL FOR A MULTICENTRE RANDOMISED CONTROLLED TRIAL

OF REFINED NEEM LEAF EXTRACT IN THE TREATMENT OF

UNCOMPLICATED Plasmodium falciparum MALARIA

AUTHORS and AFFILIATION:

Dr C.P. Chijioke MA MD FWACP FRCP, Dept of Pharmacology & Therapeutics1
Dr U.B. Anyaehie MB BS, Dept of Physiology1
Dr C.U. Chukwu MB BS, General Outpatients Dept2
Dr E.N. Shu PhD, Dept of Pharmacology & Therapeutics1
Dr P. Nwonu PhD, Dept of Pharmacology & Therapeutics3
Mr T. Nubila MSc AIMLT, Dept of Medical Laboratory Sciences1
Rev. Sister Dr A. Muobuike FWACS, Annunciation Specialist Hospital, Emene,
Enugu State
Professor Okwuasaba PhD, Dept of Pharmacology & Therapeutics4
Dr U. Inyang PhD, National Institute for Pharmaceutical Research & Development,
Abuja
1
College of Medicine, Univesity of Nigeria Enugu Campus
2
University of Nigeria Teaching Hospital
3
College of Medicine, Enugu State University
4
College of Medicine, Jos University Teaching Hospital

BACKGROUND
The favourable safety and efficacy data from in vitro studies and open trials of
fractionated neem leaf extract (irab)1-7, combined with the long history of traditional
use and efficacy of neem extracts in malaria, indicate the need to conduct a
randomized clinical trial of irab in malaria. This need is underscored by the increasing
incidence of resistance to, and/ or the lack of tolerability/ safety of the currently
available antimalarial drugs.
Rationale
The development of a fractionated, refined neem leaf extract offers the prospect of
harnessing the therapeutic potential of an ancient traditional medicine, while avoiding
the hepatotoxicity of the crude traditional formulations4,6,7.
Apart from schizonticidal and gametocytocidal effects in malaria6,7, irab also
blocks the adhesion of parasitized erythrocytes to the capillary endothelium in
malaria4,5. This multipronged mode of action in malaria would make it difficult for
Plasmodium falciparum to evolve resistance: in spite of millennia of traditional use
for “fevers” including malaria, neem preparations have retained their efficacy without
the need to resort to combination chemotherapy.
By blocking cell surface adhesion receptors, irab offers the prospect of
antineoplastic and antiviral actions (i.e. viral entry blockade) as well5. A particular
advantage of irab, suggested by in vitro and clinical observations, is combined
antiretroviral and antimalarial efficacy. This would thwart the synergistic comorbidity
of malaria and HIV infection9,10.

STUDY OBJECTIVES
Primary Objective:
The primary objective is to confirm the hypothesis that irab is non-inferior
to artemether-lumefantrine, in the treatment of symptomatic, uncomplicated malaria
due to P. falciparum.
Secondary Objectives:
To assess:
1 Efficacy (asexual parasite clearance rate) of irab at Day 28;
2 Safety and tolerability for both treatment regimens;
3 % Early treatment failures;
4 % Late treatment failures;
5 % Late clinical failure
6 % Late parasitological failure (LPF)
7 % Adequate clinical and parasitological response (ACPR)
8 Clinical cure rates at 3, 7, 14 & 28 days;
9 Asexual parasite clearance rate at 7,14, 21 & 28 days;
10 Gametocyte clearance rate at 7, 14, 21 & 28 days;
11 Fever clearance time;
12 Asexual parasite clearance time.
13 To compare the clinical efficacy of irab to that of standard artemisinin
combination therapy (ACT, artemether-lumefantrine).

METHODS
STUDY DESIGN
General organization
The clinical study will be carried out in 360 eligible subjects with proven P.
falciparum malaria infection. They will be treated with irab formulated into capsule
form and given orally at a dose of 20 mg/kg (750 mg bd for 75 kg adult) daily for four
days. Daily thick and thin smears shall be made on Days 0, 1, 2, 3, 7, 14, 21 and 28 to
detect the presence of P. falciparum parasites. Safety of the product shall be
monitored clinically, haematologically and biochemically. 120 volunteers shall be
recruited for the study per centre: 60 on irab and 60 on artemether-lumefantrine. The
design for the trial is double blind, three centres, randomized, comparative, parallel
group study using patients with acute but uncomplicated malaria.

Inclusion criteria
Adults i.e. 18 years and above, who present at the study centres with a recent history
of fever and a positive smear (parasitaemia of 1,000 to 100,000 per mm3)
Both Male and Female
Uncomplicated P. falciparum malaria, i.e. no evidence of danger sign
[neurological involvement, haemolysis or renal impairment].
Not taken any antimalarial drug in the past 7 days
Haemoglobin greater than 5 grammes per dl
PCV > 15%
Willingness to participate in the study as will be indicated by informed written
consent.
Ability to take drug orally.
Serum glucose > 40 mg/dL (fingerprick test or peripheral blood)

Exclusion criteria
Age range: less than 18 years
Complicated malaria i.e. evidence of danger sign [cerebral involvement (meningitis or
encephalitis), vomiting, haemolysis, dehydration, renal involvement].
Severe underlying disease requiring specific therapeutic intervention.
Haemoglobin less than 5 grams per dl or PCV < 15%
Parasitaemia greater than 200,000 per mm3 of blood.
No written consent to participate in the study
Serious medical conditions such as hypertension, diabetes and AIDS.

Serum Chemistry and Haematology

This will include liver function test (bilirubin, transaminases, alkaline phosphatase),
electrolytes (Na+, K+, Cl-, HCO3-), urea, and creatinine and blood sugar. A full
haemogram and total differential white cell count will be done.

Treatment phase
The sequence of contact period for symptomatic assessment and adverse effects
monitoring shall be on Days 0, 1, 2, 3, 7, 14, 21 and 28. Each subject will be admitted
for a period of three days as an in-patient in the hospital. Recruitment and registration
of volunteers for the study will vary at the different study centres due to logistics and
availability of eligible subjects. Thus, the duration for the study may span 6-12
months. This will include follow up and all necessary assessments as the need may
arise.

Randomization
The total number of subjects to be enrolled per centre in the study is 120. All study
drugs will be administered as blinded therapy. Subjects will be randomized in equal
numbers to one of the following two regimens:

IRAB 20mg/kg/day PO (three 250 mg caps two times daily for four days:
Days 0,1,2,3)
and matching placebo of artemether-lumefantrine four (20/120 mg tabs) stat, repeated
after 8h, 24h and 48h (Days 0, 1, 2)
(Irab will be supplied as formulated capsules to be administered orally in doses of 20
mg/kg daily one hour before meals morning and evening for four days.)

OR
Matching placebo for IRAB 20mg/kg/day PO (three 250 mg caps two times daily for
four days: Days 0, 1, 2, 3)
And artemether-lumefantrine four (20/120 mg tabs) stat, repeated after 8h, 24h and
48h (Days 0, 1, 2)

PATIENT FOLLOW-UP AND SAFETY MONITORING

Parasitaemia: Daily thick and thin blood smears obtained by finger pricks will be
done on Days 0, 1, 2, 3, 7, 14 and 28 in duplicates

Serum Chemistry and Haematology: This will be repeated on Days 7 and 28 and will
include the same tests as listed above.

Physical examination and vital signs

Patients will be examined physically on a daily basis while temperature, pulse
rate, blood pressure and respiration shall be recorded 8 hourly when the
patients are in the hospital, and more frequently if the condition of the patient
warrants. These parameters will be repeated at each follow up visit on Days 7,
14 and 28.
Adverse events
All adverse events affecting study participants will be documented, as well as
the likelihood that any such events were caused by drugs administered to the
participants. Severe adverse events will be reported to the coordinating
Investigator urgently on Tel. Nos. 07038680668, 07084496385,
cpchij@yahoo.com

Treatment failures
All treatment failures shall be treated with either quinine or an artemisinin
(amodiaquine-artesunate combination, or artemether-doxycycline if pregnancy
excluded).

DISCONTINUATION FROM STUDY

Patients who show partial response or no response will be offered alternative
treatment. Subjects will be withdrawn from the study if they manifest adverse
reactions. Patients have the right to withdraw from the study at any time and
for any reason without affecting the subject’s right to treatment by the
investigator.

DATA MANAGEMENT
Assessment of Efficacy
Determining the time taken to achieve negative blood slide and to be
asymptomatic will assess efficacy. The following definitions will be used:
(a) Complete cure = Complete aparasitaemia by day 7;
(b) Partial cure = Malaria parasite count dropped by at least 25% of
baseline by day 7;
(c) No cure/treatment failure = No change in parasite count from baseline
or increase is observed by day 3, or whenever withdrawn from the trial.

Assessment of Safety
This will be done by comparing the baseline haematology parameters,
biochemistry parameters and vital/physical signs with those obtained at
subsequent follow-up visits. The following definitions of adverse effects will be
used:
(a) Mild - Tolerable, patient active and able to conduct his normal
business efficiently
(b) Moderate - Causes discomfort, but still able to conduct normal
activities with decreased efficiency.
(c) Severe - Interferes with the patient's activities.

RESULTS FROM EARLIER STUDIES

A standard pharmaceutical approach is to identify and purify those extracts from a
herbal medicine which retain the desired therapeutic efficacy, while hopefully
discarding those fractions responsible for toxicity. This approach has been applied to
neem leaves by extracting them in a mixture of acetone and water (Udeinya 1993).
The residue from the bottom layer of the crude extract is then fractionated by standard
HPLC. This yields two fractions (dubbed IRDN-A and IRDN-B). Fractionated neem
leaves extract (also known as irab) is a complex molecule with functional groups that
include sodium salts of carboxylic acid and a non-aromatic dialcohol (Udeinya,
unpublished). In assays conducted with P. falciparum cell cultures, these fractions
exhibited schizonticidal activity in vitro superior to that of chloroquine, as well as
gametocytocidal activity which would help interruption transmission of malaria
(Udeinya et al 2006; Udeinya et al 2008).
In-vitro studies with malaria-infected erythrocytes and metastatic cancer cells have
shown that IRAB has broad-spectrum anticytoadhesion activity. It inhibited the
adhesion of malaria-infected erythrocytes and cancer cells to endothelial cells and the
invasion of human CD4 T-lymphocytes by HIV (Udeinya et al 2004). In phase-1
clinical trials lasting 30 days, it was found to be safe and well tolerated, without any
drug-related adverse events.
Fractionated neem leaf extract (irab) has potent antimalarial activity, arising
from both direct toxicity to malaria parasites and cytoadhesion blockade of parasitised
erythrocytes (Ekanem 1971; Badam et al 1987; Udeinya et al 1993; Udeinya et al
2006; Udeinya et al 2008). This is of particular interest in view of the synergistic co-
morbidity of HIV infection and malaria (Kamya et al 1975; Kublin & Steketee 1975):
PLWHA are at increased risk of new malarial infection; hence there would be added
benefit from an antiretroviral which is also antimalarial, as in the case of irab.

Effect of Neem leaf extract (IRAB) on

Cultured P. Falciparum malaria
parasites
Study of 60 patients (34 male, 26 female) with HIV infection
median age 30 yrs (23-50)
Median weight 58kg (41-74), median Hb 10 g/dl (6.6-14.3),
median WBC 4600/cmm (2800-8900)

Incidence of clinical features before and after 12 weeks’ irab treatment

with nutritional supplements (dosage 500 mg bd)

CLINICAL PRE- POST- CLINICAL PRE- POST-

CONDITION IRAB IRAB CONDITION IRAB IRAB
Weight loss 17 1 anorexia 3 0
Fever 27 0 Oral thrush 10 0
Diarrhoea 13 0 Skin rash 10 0
Herpes zoster 1 0 headache 11 0
Generalized 15 3 dizziness 1 0
lymphadenopathy
Cough 13 1 Chest pain 1 0
pallor 7 0
 Week 0 Week 4 Week 8 Week 12
CD4+ 167 220 305 411
count/ ul (30-331) (107-434) (109-589) (262-610)
ESR 59 43 22 16
(mm/hr) (4-120) (12-98) (12-47) (12-22)

PARAMETER MEAN CHANGE at week 12

Neutrophil count -21%
Lymphocyte count +20%
Hemoglobin conc’n (g/dl) +24%
Body weight (kg) +12.3%

Friedman’s nonparametric ANOVA for repeated measures confirms

all these changes to be highly statistically significant (p<0.0001). For body
weight, p<0.05

Concomitant monitoring of malaria parasite density in patients with HIV infection

shows consistent suppression of malaria parasitaemia within three months of starting
irab treatment.

DISCUSSION/ CONCLUSIONS
Thus preliminary studies indicate the following advantages of irab (as compared to
other currently available antimalarial drugs), which need to be confirmed in a
randomized controlled clinical trial:

a) triple mode of action in malaria (from the in vitro studies quoted above):
schizonticidal, gametocytocidal and anticytoadhesion. Such a multipronged attack on
Plasmodium would make the likelihood of development of resistance less than with
conventional drugs which have a single mode of action. Hence there would be less
need for combination therapy with irab as compared to the single-mode-of-action
drugs.

b) no serious toxicity and good tolerability observed on clinical and laboratory

monitoring: this contrasts markedly with some of the conventional drugs which have
poor tolerability and the potential for serious toxicity

c) convenient twice daily oral dosing regimen: this would help promote patient
adherence to treatment regimens.

d) affordability: being a neem leaf extract, irab is relatively cheap to produce, and this
represents a distinct advantage over relatively expensive imported conventional drugs.

e) combined antiretroviral and antimalarial efficacy, unlike the conventional

antiretrovirals. This is an important advantage in view of the synergistic interaction
between HIV infection and malaria.

f) a regimen which could be easily adapted for pediatric use (neem leaf preparations
have been used extensively in children), and
g) a potentially efficacious and safe therapy with relatively few side effects, for
pregnant women with chloroquine-resistant P. falciparum.

Irab has a particularly promising role to play in pregnant women both for the
prophylaxis and treatment of malaria. Unlike the antifolate prophylactics, there would
not be the worry of an adverse effect on nucleotide metabolism in the embryo, which
might influence the likelihood of neural tube defects. The anticytoadhesion efficacy
makes irab a drug of choice to combat the sequestration of malaria parasites in the
placental microcirculation, which mediates much of the morbidity and fetal mortality
in malaria. In a few case reports of pregnant women with HIV infection who have
relied on irab treatment, there was effective prevention of malaria during pregnancy,
and prevention of mother to child transmission of HIV (Udeinya, personal
communication).

BIBLIOGRAPHY

1. Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U.

Biological activities and medicinal properties of neem (Azadirachta indica).
Current Science 2002; 82: 1336-1345

2. Ekanem OJ. Has Azadirachta indica (dogonyaro) any antimalarial activity?

Niger. Med. J. 1971; 8: 8-11

3. Badam L, Deolankar RP, Kulkarni MM, Nagsampgi BA, Wagh UV. In vitro
antimalarial activity of neem (Azadirachta indica A. Juss) leaf and seed extracts.
Indian J. Malaria 1987; 24: 111-117

4. Udeinya IJ. Antimalarial activity of Nigerian neem leaves.

Trans Roy Soc Trop Med Hyg 1993; 87: 471

5. Udeinya I.J., Mbah A.U., Chijioke C.P., Shu E.N. An antimalarial extract from
neem leaves is antiretroviral. Trans Roy Soc Trop Med Hyg 2004; 95: 435–437.

6. Udeinya IJ, Brown N, Shu EN, Udeinya FI, Quakeyie I.

Fractions of an antimalarial neem-leaf extract have activities superior to
chloroquine, and are gametocytocidal. Ann Trop Med Parasitol. 2006; 100(1): 17-22.

7. Udeinya JI, Shu EN, Quakeyie I, Ajayi FO.

An antimalarial neem leaf extract has both schizonticidal and gametocytocidal
activities. Am. J. Therap. 2008; 15: 108-110.

8. Shu EN, Mbah AU, Udeinya IJ, Chijioke CP, Nubila T, Udeinya F, Muobuike A,
Mmuobieri A, Obioma MS. Fractionated neem leaf extract is safe and increases CD4+
cell levels in HIV/AIDS patients. Am. J. Therap. 2007; 14: 369-374.

9. Kamya MR, Gasasira AF, Yeka A et al.

Effect of HIV-1 infection on antimalarial treatment outcomes in Uganda: a
population-based study. J. Infect. Dis. 2005; 193: 9-15

10. Kublin JG, Steketee RW. HIV infection and malaria: understanding the
interactions. J. Infect. Dis. 2005; 193: 1-3