PHARMACEUTICAL TOXICOLOGY RESEARCH DIVISION

The Pharmaceutical Toxicology Research Division, established under NPCC, involves 3 major area activities. To (1) conduct research projects on drug-related poisoning and toxicity, (2) provide information and analytical services to the health sector on prevention, control and management of drug poisoning, and (3) conduct education and training to health personnel concerning poisoning and toxicology. Provision of services includes drug screening and identification in cases of unknown poisoning, quantification of drug levels to support treatment in cases of acute poisoning and provision of poison information to doctors and health care professionals in selected major hospitals for poison control and management.

RESEARCH PROJECTS

1. NON-COMMUNICABLE DISEASES

1.1. MALARIA

1.1.1. Pharmacokinetics of piperaquine and clinical outcome of acute, uncomplicated malarial patients after administration of Piperamisinin, a locally manufactured ACT in Myanmar

Uncomplicated P. falciparum malaria is mainly treated with artemisinin- based combination therapy (ACT) and Piperamisinin, a locally manufactured Dihydroarremisininpiperaquine (DHA-PPA) co-formulation. Eighteen acute, uncomplicated falciparum malarial patients admitted to Loikaw General Hospital were recruited in the study. They were given 3 tablets of Piperamisinin once a day for 3 consecutive days. Temperature, parasite count and plasma PPQ concentrations were investigated. Plasma PPQ concentration were analyzed at Oh, 0.5h,1h,2h,3h,4h,6h,8h,24h,48h,168h,336h,504h, and 672h by HPLC-UV method. Pharmacokinetic parameters of PPQ were concentration at time 0(Co)1029.20 ± 1250.25μg/L, maximum concentration(Cmax) 356.03 ± 176.83 μg/L, time to reach maximum concentration (tmax)4.56±1.9 h, absorption rate constant (kab)0.62 ± 0.40h-1,absorption half life (t1/2ab)1.16 ± 0.99h, elimination rate constant (kel)0.002 ± 0.0008 h-1,elimination half life (t1/2el) 446.90 ± 224.85h(18.62 ± 10.20 days), area under the concentration-time curve (AUC) 620290 ± 1062390 μg/L. h, volume of distribution (Vd ) 40.26 ± 26.92 L/kg and clearance (CL)0.0643 ± 0.0341L/h/kg. Wide inter-individual variation of pharmacokinetic parameters of PPQ were found among the patients. Mean Fever Clearance Time (FCT) and Mean Parasite Clearance Time (PCT) were 22.67 ± 17.04h and 6.78 ± 3.0 h respectively. Adequate Clinical and Parasitological Response (ACPR) was 100%. All patients were well tolerated with very few minor side effects. No significant cardiovascular, hematological and biochemical abnormalities were detected. Piperamisinin is an efficacious, tolerable and less expensive ACT with wide margin of safety and is also responsible for prevention of transmission and recrudescence. This is a collaborative project done with the University of Pharmacy (Yangon).

1.1.2. Determination of piperaquine concentrations in red blood cells and plasma of Myanmar healthy volunteers and uncomplicated falciparum malarial patients.

Piperaquine, a bisquinoline antimalaria drug and a structural analogue of chloroquine, has been produced by the Myanma Pharmaceutical Facrory as a partner drug for fixed-dose artemisinin-based combinations (ACTs) called Piperamisinin (containing 50mg dihydroartemisinin & 350mg piperaquine. With the aim to understand the relationship between the pharmacokinetics of piperaquine and its concentrations within the red blood cells (RBC), 18 healthy volunteers receiving a single dose of 3 tablets Piperamisinin and 18 uncomplicated falciparum malarial patients from State General Hospital, Loikaw, receiving 3 tablets Piperamisinin daily for 3 days were studied. Results indicated piperaquine to have a very long half life, reaching the maximum concentration (Cmax) of 839.9±513,5g/L and 356.1±176.8g/L at 2.7±0.8 hours and 4.6±1.9 hours respectively, in healthy volunteers and malaria patients. Cmax of RBC was significantly higher 1616.6±1041.9g/L and 525.6±270.8g/L, respectively, in both healthy volunteers and malaria patients. The RBC to plasma partition ratio (RBC:plasma) was 2.54±1.35 and 2.73±2.28, respectively, for healthy volunteers and malaria patients. No significant difference was seen between males and females volunteers. The study indicated that piperaquine was 2-3 times more concentrated in the RBCs and although its concentration was higher in healthy volunteers than malarial patients, its RBC:plasma ratio was not significantly different. Both the adequate bioavailability (Cmax) and prolong half-life indicated piperaquine as a good companion drug for artemisinin derivatives and the fixed-dose combination produced in Myanmar to be of good quality. This is a collaborative project done with the University of Pharmacy (Yangon).

1.2. TUBERCULOSIS

1.2.1. Standardization and quality assurance of herbal drugs used in Multi-Drug Resistant Tuberculosis.

Herbal medicines, still remaining widely used, have increasingly become an important part of pharmacology research and drug development. However, little attention has been paid on quality assurance procedures such as plant identification, documentation, physicochemical characterization and methods of formulation, such that wide variations in results and conflicting outputs are not uncommon. In Myanmar, a preliminary screening on in vitro antimycobacterial activity and further clinical trials on Multi-drug Resistant Tuberculosis (MDR-TB) patients have identified five Myanmar medicinal plants as potential lead (hit) drugs for use in MDR-TB. However, in order to conduct a multi-center trial, quality assurance during mass production becomes a critical issue and the present study encompass the steps and validation methods taken to achieve this. Plants were collected and identified by the Department of Traditional Medicine and voucher specimens kept at the Museum cum Herbarium, DMR-LM. Laboratory-based quality assurance procedures conducted on raw materials, extracts and final product abiding with WHO Standard Guidelines (2000). Results indicated that all physico-chemical parameters, chromatographic finger-printing by HPTLC, heavy metals and bacteriological contamination, pesticide residues were within acceptable range. In bacteriology analysis, bacillus species (14x103 CFU/ml) and micrococcus species (1x103 CFU/ml) isolated in raw material extract and final herbal drug. All organisms are non-pathogenic. In pesticide residue determination, Aldrin (0.024mg/kg) benzene hexachloride (0.01mg/kg) and DDT (0.4mg/kg) were under WHO acceptable levels. Heavy metals like arsenic (0.1 mg/kg) lead (<10mg/kg), cadmium (<0.3mg/kg) and mercury (<0.02mg/kg) were also under the WHO acceptable limits. It was concluded that these methods can be used to test the quality assurance of batches herbal formulations in future. This is a collaborative project done with the Pharmacology Research Division, Bacteriology Research Division and Department of Traditional Medicine.

2. ENVIRONMENTAL HEALTH

2.1. PHARMACO EPIDEMIOLOGY OF POISONING

2.1.1. Monitoring of poisoning cases at New Yangon General Hospital (2010)

Epidemiological studies on poisoning are done with the aim to increase the awareness, understand the potential public health impact, establishing causal links between exposure and disease and preparedness of laboratory diagnostic methods and clinical management schemes. Registry from the Department of Hospital Records and Poison Treatment Center, New Yangon General Hospital, diagnosed and treated as poison cases, categorized under ICD-10, T36 to T65, basic codes 284 and 285 were collected and analyzed. Out of a total of 6112 admissions in the year 2010, 448 cases (7.3%) were identified as poisoning other than snake bite. In acute poisoning is mainly seen in the working age group (18-35 years), and is more prevalent in females 256(57.3%) than in males 192(42.6%). Drug overdose being the highest 229(51.1%) followed by chemical poisoning 92(20.54%), pesticides 68(15.1%)and food poisoning 35(7.8%). Drug overdose is mainly seen with antihistamines(mainly Chlorphenaramine meleate) 145(32.3%), sedatives and antipsychotics(mainly amitryptylline) 131(29.2%), analgesics (mainly paracetamol) 88(19.6%), unknown drug poisoning 45(10%) and unknown poison other than western medicine 36(8.2%). Although most patients recovered and were discharged without undue consequences 442(98.5%), 6(1.4%) expired in spite of treatment being due to drug poisoning 3(0.7%) and chemical poisoning 3(0.7%).

2.2 ANALYTICAL TOXICOLOGY

2.2.1. Detection of counterfeit and substandard drugs in the market using simple and available resources at the National Poison Control Centre (WHO/Patient Safety)

Counterfeit and substandard medicines are growing global problems affecting health care and patient safety. The project explored the problem of counterfeit/substandard antimicrobials in Myanmar under WHO-HQ/Patient Safety Program using simple techniques and available resources at National Poison Control Centre (NPCC), DMR-LM, with the Food and Drug Administration (FDA) acting as external quality control. Different brands of 81 antimicrobials including Amoxycillin (20), Cloxacillin (7), Ciprofloxacin (18), Norfloxacin (14), Erythromycin (7) and Metronidazole (13) were purchased by mystery clients from wellknown pharmacies in Yangon, Mandalay, Sagaing, Kachin and Mon States. The drugs collected had at least 1 year before the expiry date and both the name of pharmacy and brand name were coded. Quality assurance parameters such as uniformity in weight, pH value and disintegration test, and FT-IR spectrum and thin-layer chromatography (TLC), and presence of active compounds/impurities, assessed accordingly. Results indicated majority (35%) of samples being from Yangon Division with 9.9% being not registered at FDA. Variation in uniformity of weight (14.8%), prolong disintegration (6.2%), probable wrong ingredient/impurities (1.2%), and insufficient active ingredients (38.9%) were seen. Majority (85.7%) of cloxacillin capsules had ingredients damaged well before their expiratory dates. Suspected samples were sent to FDA for further investigation into counterfeiting. The findings indicated the existence of substandard drugs in Myanmar and the likely causes of low quality may result, both from improper manufacturing as well as deterioration from improper transport and storage conditions.

2.2.2 Application of Energy Dispersive X-ray Fluorescence (EDXRF) method for determination of multi-elements in cosmetics
(Please refer to annual report of Radiation Toxicology Research Division)

2.3. CLINICAL TOXICOLOGY

2.3.1 Blood lead levels in children with chronic lead poisoning.

Chronic exposure to lead often leads to negative mental and physical development especially in children. CDC has defined blood lead level (BLL) of 10μg/dL as threshold for antidotal therapy and D-Penicillamine has been the mainstay treatment at Yangon Children Hospital. However, even after successful treatment, patients often succumb to of chronic lead exposure when they return to their contaminated homes and environment. With the aim to determine BLL as indicator of severity of poisoning for antidotal therapy as well as detecting recurrence of chronic exposure on returning to their homes, a total of 117 children (4 months to 12 years) referred from YCH for BLL, together with their accompanying siblings (n=14) and parents (n=17), were recruited. Among them, 95 children (81.2%) had clinical features like pallor, fever, convulsions, epigastric pain, chest pain and diarrhea, with 26 (22.2%) giving history of living in environment with potentially high lead exposure. Mean BLL vary widely 37.1±26.3g/dl (range 10.1-81.2g/dl). All were treated by oral D-penicillamine (20mg/kg/day 4 divided doses), including 22 (18.8%) who had BLL of 10g/dl (range 1.6- 9.8g/dl). Rapid clinical improvement occurred in all subjects with disappearance of symptoms within 1 week and normalization of BLL within 15-30 days of treatment. To allow mobilization of lead from bones and tissues after normalization, the therapy was usually continued up to 60 days. However, despite full antidotal therapy and continued treatment, some coming from Myeik for follow-up showed gradual recurrence of BLL above 10μg/dL, 19 (16.2%) having 17.3±13.2g/dl after 3-4 months and 6 (5.1%) having 27.4±15.9g/dl after 6-8 months, respectively. It was concluded that with chronic lead poisoning, adequate treatment alone is insufficient. Contaminating sources must be eliminated and necessary remediation processes instituted to prevent further exposure after treatment. This is a collaborative project done with the Yangon Children Hospital and Occupational Health Division, Department of Health.

2.3.2 Factors influencing treatment outcome and antidotal therapy in organophosphate poisoning
( Please refer to annual report of Clinical Research Unit (Toxicology))

2.4. EXPERIMENTAL TOXICOLOGY

2.4.1. Study of hepatotoxic effect of single and multiple oral intake of paracetamol in animal model: subacute toxicity.

Paracetamol, a relatively safe painkiller and popular over-the-counter drug, can cause serious, dose-dependent, potentially fatal hepatic necrosis on acute ingestion in excess of 10- 15g (150-250mg/kg). In contrast, several therapeutic doses of 7.5-10g daily for 1-2 days, or multiple minor overdoses over a short period of time for dysmenorrhea or toothache, leading to development of severe hepatic damage, has also been reported. This multiple low-dose paracetamol was reported to exert similar or even greater hepatic damage than single dose of same magnitude in acute overdose. To test this hypothesis, graded doses of paracetamol, administered either as single or multiple doses, was conducted on 5 groups of mice (ddy strain) for acute toxicity (LD50) and 4 groups of rats (Wister strain) for subacute toxicity testing. In the acute toxicity study, the mice were challenged orally with graded single-dose paracetamol (0.5 to 0.6g/kg) and LD50 was found to be 0.525g/kg. In subacute toxicity study, the rats received paracetamol, orally, 0.25-0.5g/kg in 2-4 divided doses per day for one week. All rats survived but were sacrificed at the end of the study. Laboratory tests showed no significant difference except for liver function tests showing elevated SGOT and SGPT levels in the test (ranging 32.1±2.2 to 29.9±1.5U/L) when compared to the control group (12.3±0.4U/L). Histological examination of liver showed gradual increasing necrosis with dose in LD50 which gradually became apparent in the centrilobular hepatocytes as nuclear pyknosis, karyorrhexis and karyolysis with some cells showing loss of nucleus and nucleoi. Multiple-dosing showed extensive pyknotic changes in the nucleus of centrilobular hepatocytes similar to acute toxicity but without other gross changes. Other vital organs showed neither necrosis nor other specific changes. It was concluded that multiple low-dose intake can also lead to hepatic damage in rats.

SERVICES PROVIDED

TECHNICAL DEMONSTRATION AND TRAINING

LABORATORY SERVICES

Screening and analysis of drugs and other poisons from biological and non-biological samples in acute poisoning Respond to requests on screening and analysis of drug and poison has been provided to hospitals and includes drug poisoning (52.8%), lead poisoning (41.2%), unknown poisoning (5.8%) and others (0.2%). Requests for screening and identification were mainly from the Poison Treatment Centre, NYGH (32.4%), Medical Wards, YGH (4.6%) Yangon Children Hospital (14.8%) and Township Hospital, Myeik (40.6%). Others include No. 2 military Hospital, Insein General Hospital, North Okkalapa General Hospital and Thingangyun Sanpya General Hospital(7.4%).

POISON INFORMATION SERVICE

Information and advice were given to general hospitals, 24 hours a day, 7 days a week, under direct supervision of Deputy Director/Head of Division. Respond to 428 requests on drug and poison information has been provided to the public, hospitals and the media in 2010, and includes drug poisoning (48.7%), lead poisoning (28.1%), pesticide poisoning (12.8%) and others (10.4%).