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Naphthoquine phosphate and its combination with artemisinine
Acta Tropica 89 (2004) 375–381
Naphthoquine phosphate and its combination with artemisinine Jing-yan Wang∗ , Wu-chun Cao, Cheng-qi Shan, Min Zhang, Guo-fu Li, De-ben Ding, Yun-lin Shi, Bo-an Wu Institute of Microbiology and Epidemiology, Academy of Malitary Medical Sciences and Epidemiology, 20 Dongdajie Fengtai, Beijing 100071, China
Abstract Naphthoquine phosphate and artemisinine are two antimalarials developed in China. Both drugs have proven to be efficacious and well tolerated as monotherapy as well as in combination in patients suffering from malaria. The Co-naphthoquine, a novel antimalarial combination, is an oral fixed combination tablet of the naphthoquine phosphate and artemisinine. Artemisinin is characterised by a rapid onset of schizonticidal action and a short half-life. Parasite clearance is, however, often incomplete when it is employed as a single agent unless high dosages are used over several days, but such a regimen may reduce patient compliance and increase the danger of toxicity. Naphthoquine phosphate, by contrast, has a slower onset of action and a longer half-life, associated with a low recrudescence rate. The two components act synergistically in animal, and clinically provide more rapid relief of symptoms and a higher cure rate than either component alone. The combination tablet was initially developed by the Academy of Military Medical Sciences (AMMS), Beijing, China. © 2003 Elsevier B.V. All rights reserved. Keywords: Naphthoquine phosphate; Artemisinin; Combination; Antimalarial
1. Introduction With the current estimates of 300–500 million new malaria cases arising worldwide, each year, and the attendant 1.5–2.7 million annual deaths. Plasmodium falciparum malaria is still a major cause of mortality and morbidity in the tropical world. The development of drug resistance by P. falciparum has severely compromised our ability to treat this life threatening pathogen. Resistance has subsequently emerged to nearly all available antimalaria drugs. There is a ∗ Corresponding author. Tel.: +86-10-6381-3346; fax: +86-10-6381-3346. E-mail address: [email protected] (J.-y. Wang).
reported 100% resistence of P. falciparum to chloroquine and primaquine in Thailand, and partial resistance to all other antimalarials, for example up to 40% resistance to mefloquine (Looareesuwan et al., 1992). In particular it is the increasing drug resistance and widening geographic distribution of this most lethal of the malarial parasites, Plasmodium falciparum, that has directed attention towards the urgent need for development of novel antimalarial drugs. A recent approach has been to combine the proven efficacy of a newly discovered antimalarial agent, naphthoquine phosphate with artemisinine. The two components act synergistically in animal, and clinically provide more rapid relief of symptoms and a higher cure rate than either component alone.
0001-706X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2003.11.008
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Fig. 2. Naphthoquine phosphate.
Fig. 1. Artemisinine.
2. Artemisinine Facing the challenge of malaria great effects have been made in China since 1960s. Several new drugs have been developed. The most outstanding contribution was the discovery of artemisinine (Fig. 1). Its sesquiterpene lactone structure contains an endoperoxide group that appears essential for antimalarial activity. Artemisinine has been shown to be active against chloroquine-resistant falciparum malaria in China. It has been used in China for the treatment of uncomplicated malaria for over 20 years. It is a fast acting drug with rapid onset of its schizontocidal action on the parasite. The problem with the artemisinine is that when it is used alone over short periods, i.e. less than 5 days, clearance of parasitaemia from the blood is only temporary in up to 50% of patients (the recrudescence rates from 12% to as much as 55%). This high rate of recrudescence results from the rapid elimination of this drug and the need for the any antimalarial drug to be present in the blood at effective concentrations during four asexual cycles (>6 days) to ensure elimination of the parasite (White, 1997). Higher efficacy can be obtained by 5-and especially 7-day regimens but this is associated with reduced compliance in out-patients.
3. Naphthoquine phosphate Naphthoquine phosphate was registered in China in 1993. The chemical name is 4-(7-chloro-4-aminoquinoline)-2-tert-butylaminomethyl-5,6,7,8-4hydro-1-na-
phthol diphosphate. Molecular weight is 641.98 (Fig. 2). It has thorough killing function for various schizonts of plasmodia, including resistance of P. falciparum to chloroquine. It showed good antimalarial activity against Plasmodium berghei in mice. The ED50 and ED90 were 0.15 and 0.63 mg kg−1 , respectively. The curative dose in monkey infected by Plasmodium knowlesi was 30 mg kg−1 . Taken orally, it can be absorbed rapidly and perfectly. The drug concentration in the blood will reach maximum in 2–4 h after administration, the relative bioavailability is 96.4%. It is widely distributed, highest in liver then in kidney, lung and the blood cells’ concentration is higher than the blood plasma. Excreted mainly from urine, then in excrement. Hundred and one cases of falciparum infected patients in Hainan Island in China were treated with 1000 mg naphthoquine phosphate given two times (600 mg on 0 h and 400 mg on 24 h). All were cured clinically with the cure rate of 100% (Pang et al., 1999). In the comparative study of the therapeutic efficacy of naphthoquine phosphate 30 cases versus artesunate 30 cases on falciparum malarial, each group of P. falciparum infections was treated with naphthoquine phosphate (1000 mg) and artesunate (600 mg per 5 day) separately. The average time for fever clearance were 29.6 ± 16.4 and 18.4 ± 9.0 h, respectively. The average time for parasite clearance were 98.4 ± 27.7 and 43.0 ± 17.1 h, respectively. The 28-day cure rate for naphthoquine phosphate was 96.7%, for artesunate was 73.3% only (Table 1)
Table 1 Comparison of therapeutic efficacy between naphthoquine and artesunate in treatment of P. falciparum in Hainan Group
Case
Total dosage (mg)
Regimen
FCT (h)
PCT (h)
Cure rate (%)
Naphthoquine Artesunate
30 30
1000 600
2 5
29.6 ± 16.4 18.4 ± 9.0
98.4 ± 27.7 43.0 ± 17.1
96.7 73.3
PCT: parasites clearance time; FCT: fever clearance time.
J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381
(Guo et al., 2000). No distinctive adverse reaction is seen with total dosage under 1000 mg of naphthoquine phosphate. It may appear abdominal distention for individuals after administration, which disappears in 2–4 h. Naphthoquine phosphate, in contrast to artemisinine, has a much slower onset of action, with a longer half-life, and it can effect a complete and sustained parasite clearance.
4. The combination of naphthoquine phosphate and artemisine In 1990s the combination of the naphthoquine phosphate and artemisinine was developed in order to retain the strongpoint of the two drugs, to avert the shortcomings of them, to delay the emergence of drug resistance to them, and to prolong the useful life of the two drug. Co-naphthoquine is a new antimalarial combination comprising the naphthoquine phosphate and the artemisinin. The pharmacodynamic tests revealed that the combination of these two drugs was synergistic, the synergistic index was more than 4 or 8 that caculated from chloroquine-sensitive strain of P. berghei or chloroquine-resistent strain of P. berghei, respectively. The curative effects of the combination against P. berghei in mice were superior to the drug used in single. It was also revealed that the combination could delay the emergence of drug resistance and reduce its resistance level. The toxicity tests showed that the combination of naphthoquine and artemisinin was a low toxicity material with a wide safety range. 4.1. Pharmacodynamics tests 4.1.1. Potentiating effect The optimum proportion of naphthoquine phosphate and artemisinin was selected by orthogonal design in the test against P. berghei in mice by “4-day test”. The curative effect of combination on against P. berghei in mice was observed by “4-day test”. The results showed that the combination of naphthoquine phosphate and artemisinin had synergistic action and the optimum proportion of naphthoquine phosphate and artemisinin was 1:50. The synergistic index was more than 4 and 8 that calculated from chloroquine-sensitive strain of P. berghei or
377
Table 2 Comparison of rapidity of resistant development of naphthoquine, artemisinin and their combination on P. berghei K173 strain in mice by blood passage Drug
Development of drug resistance
Resistance index
Passage
Days through
ED90 (mg/kg per day)
QHS
20th 100th
140 700
153.32 194.85
4.4 5.6
NQ
20th 100th
140 700
24.94 152.23
32.8 200.3
Co-NQ
20th 100th
140 700
26.51 77.29
1.5 4.4
QHS: artemisinin; NQ: naphthoquine; and Co-NQ: Co-naphthoquine.
chloroquine-resistent strain of P. berghei, respectively. These results indicated that the rational combination of naphthoquine phosphate and artemisinin would exhibit a potential action against malaria. 4.1.2. Retarding effect on the development of drug resistance In these tests, drug resistant strains were developed by successive blood passages under increasing low doses of single or combination drug pressure. Parallel comparison of the rapidity of development of drug resistance was carried out through 100 passages for 700 days. It was found that naphthoquine single was easy to develop resistance. At 20th passage the resistance index (I90 ) of naphthoquine was more than 32.8, while the I90 of the combination was 1.5 only. After 100 passage the I90 of naphthoquine was more than 200.3. The I90 of combination was 4.4 only. This result indicated that the combination might delay the development of drug resistance and reducing resistance level (for result see Table 2). 4.1.3. Tests on P. knowlesi in monkey The tests were carried out in monkey against P. knowlesi for determining the appropriate proportion of naphthoquine and artemisinin and the appropriate treatment regimen. The results showed that the appropriate proportion of naphthoquine and artemisinin was 1:2.5, the appropriate treatment regimen was only one dose. The curative effect revealed that the combination was markedly superior to these of the single drug Table 3.
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Table 3 Comparison of therapeutic efficacy between artemisinin, naphthoquine and their combination against P. knowlesi in monkey Group
QHS + NQ QHS NQ
Dosage (mg/kg) QHS
NQ
25 31.6 –
10 – 10
Regimen
Total dosage (mg/kg)
Case
PCT (h)
Cure (105 day)
1 4 3
35 126.4 30
3 3 3
56.0 ± 8.0 60.0 ± 0.4 60.0 ± 12.0
3/3 0/3 3/3
4.2. Toxicity The Co-naphthoquine was given by intragastrical administration and by intraperitoneal administration, respectively. The results showed that the LD50 of combination was 2907 and 428 mg/kg given by intragastical administration and intraperitoneal administration, respectively. The chemotherapeutic index was 341 (LD50 /ED50 ). Long-term toxicity tests were carried out in rats and dogs. The Co-naphthoquine was given orally for 14 consecutive days. In rats, 70 MKD (mg/kg per day) was considered as safe dose, 140 MKD was considered the slight intoxication dose. In Beagle dogs the safe dose was 17.5 MKD, 87.5 MKD was considered the slight intoxication. According to the necropsy of rats and dogs, the toxicity target organs of this combination were erythroid series of bone marrow and liver. In the 140 MKD group, 1/4 beagle dog exhibited suppression of erythroid series of bone marrow in every phase and the total number decreased. However, statistical analysis showed no difference (Table 4). The GOT and GPT of serum increased after administration (Tables 5 and 6). After withdraw of drug, the changes were reversible. The general pharmacological tests were carried out in mice, rats and cats to observe the possible pharmacological activity of combination on central nervous Table 4 The effect of Co-naphthoquine on granuloid/erythroid of bone marrow of beagle dogs Dose (mg/kg per day)
D 14 (n = 4)
0 17.5 87.5 140
1.19 1.20 1.47 7.01
∗
P > 0.05.
± ± ± ±
0.17 0.16 0.42 11.06∗
D 42 (n = 2) 1.30 1.19 0.88 1.27
± ± ± ±
0.25 0.12 0.08 0.11
system, cardiovascular system and respiratory system. In these tests the combination was prepared in water suspension, 10 times as proposed daily clinical dosage were given by intragastical administration. The results showed that no general pharmacological activity was found in these observed systems in relating experimental animals by oral the combination at the dosage of 10 fold as proposed daily clinical dose. 4.3. Clinical efficacy of Co-naphthoquine The oral fixed combination tablet of naphthoquine and artemisinin (Co-naphthoquine) has been used as an antimalarial in clinical trials. The combination comparative with naphthoquine and artemisinin have been conducted. Overall 320 patients have been treated with Co-naphthoquine. The 28 day cure rate was 97.5% in Hainan China. After 24 h Co-naphthoquine eliminates more than 97% of the parasite present at baseline, with an overall time to parasite clearance of 30 h. Malaria symptoms resolve quickly, with a time to fever clearance of 17.5 h. Hundred and nine cases of vivax malaria in Hainan Island were treated with Co-naphthoquine. All of them were cured clinically, 98 of them cases were cured completely. The average time for fever clearance was 13.0 ± 5.5 h. The average time for parasite clearance was 18.1 ± 5.7 h. The 56-day cure rate was 90.0% Table 7. By the comparative studies, the curative effect, toleratility and safety of Co-naphthoquine were compared with artemisinin in single and naphthoquine in single, in treatment the P. falciparum patients (Table 8). The results showed that Co-naphthoquine has rapidly effect in clearing P. falciparum from peripheral blood and in resolving fever which is associated with acute malarai infection and was superior to these comparators in these respects. The therapeutic effect of Co-naphthoquine was superior to artemisinin
J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381
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Table 5 The changes of serum GOT of beagle dogs in long-term toxicity test of Co-naphthoquine Time (day)
n
GOT (U/l) 0
17.5
87.5
140 (mg/kg per day)
Before medication D7 D0
6 6
30.7 ± 7.5 39.3 ± 3.3
37.0 ± 8.5 43.0 ± 6.8
38.8 ± 7.1 34.5 ± 19.3
43.3 ± 17.6 38.0 ± 8.0
Medication period D7 D 14
6 6
35.5 ± 4.0 41.3 ± 5.2
37.5 ± 10.6 39.2 ± 5.2
52.0 ± 17.2 58.7 ± 36.5
74.8 ± 39.3 95.0 ± 21.5∗,#
Recovery period D 28 D 42
2 2
41.0 ± 1.4 51.0 ± 1.4
40.0 ± 0.0 40.5 ± 0.7
55.0 ± 7.1 42.5 ± 5.0
36.5 ± 2.1 25.5 ± 9.2
x¯ ± s. ∗ P < 0.05, compared with control of same period. # P < 0.05, compared with values before medication. Table 6 The changes of serum GPT of beagle dogs in long-term toxicity test of Co-naphthoquine Time (day)
n
GPT (U/l) 0
17.5
6 6
24.2 ± 7.9 37.8 ± 10.3
40.8 ± 19.3 43.5 ± 13.1
41.0 ± 16.2 35.8 ± 9.2
36.3 ± 8.0 45.8 ± 10.8
Medication period D7 D 14
6 6
56.5 ± 66.2 38.5 ± 13.0
37.8 ± 7.2 41.2 ± 9.8
57.5 ± 10.8# 108.2 ± 109.0
73.3 ± 24.1 117.3 ± 38.1∗,#
Recovery period D 28 D 42
2 2
37.0 ± 5.7 32.5 ± 7.8
35.0 ± 8.5 42.0 ± 1.4
87.0 ± 59.4 41.5 ± 5.0
Before medication D7 D0
87.5
140.0 (mg/kg per day)
39.0 ± 2.8 37.0 ± 9.9
x¯ ± s. ∗ P < 0.05, compared with control of same period. # P < 0.05, compared with values before medication. Table 7 Clinical pharmacodynamics of Co-naphthoquine Parasite
Case
Reduction in parasite in 24 h
PCT (h)
FCT (h)
Cure rate (%)
P. faciparum P. vivax
320 109
97.3 ± 8.8 99.99 ± 0.05
29.9 ± 8.7 18.1 ± 5.7
17.5 ± 11.4 13.0 ± 5.5
97.5 90
PCT: parasite clearance time; FCT: fever clearance time. Table 8 Comparison of therapeutic efficacy between Co-naphthoquine tablets and its components (aetemisinin tablets and naphthoquine tablets) in treatment P. faciparum in Hainan China Drug
Cases
Total dosage (mg)
PCT (h)
FCT (h)
28 day cure rate (%)
Co-NQ NQ QHS
100 100 30
1400 1000 2500
30.0 ± 8.8 45.5 ± 10.0 29.1 ± 6.0
17.5 ± 12.3 32.7 ± 17.7 18.1 ± 9.7
97 100 66.7
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Table 9 The number of patients of the increased GOT and GPT at different time after administration Drug
Co-NQ NQ QHS
Case
100 100 30
GOT
GPT
D3
D7
D 14
D3
D7
D 14
3 12 3
2 8 4
1 4 1
4 11 3
1 9 3
1 3 0
and naphthoquine. In the clinical trials no serious adverse experience was recorded and no unexpected hematology, biochemistry and ECG changes were found. The number of patients with increased GOT and GPT was listed in Table 9, which showed the Co-naphthoquine had not demonstrated more toxicity than naphthoquine and artemisinin respectively. Artemisinin was given at 0, 8, 24, 48 h, respectively with total dose 2500 mg. Naphthoquine was given at 0 and 24 h, respectively with total dose 1000 mg. Co-naphthoquine was given only one dose with total dose 1400 mg (Table 8).
5. Discussion and conclusion The spread of drug resistance of P. falciparum calls for a continuous effort to search for new antimalarial drugs that are effective against drug-resistant parasite. Combination therapy may provide a way in which resistance can be combated. Naphthoquine and artemisinin both are effective antimalarials that have been recently developed by Chinese scientists. These two drugs showed high in vivo and in vitro antimalarial activity and high clinical efficacy for treatment of multidrug-resistant P. falciparum. Artemisinin has been proven to be benefit with rapid onset action and produce a rapid and complete early reduction of parasites but recrudescence is common if it is given as a single agent for less than 5 days. The pharmacokinetics studies of naphthoquine phosphate revealed longer half-life than artemisinin. 14 healthy volunteers received a single dose 600 mg naphthoquine phosphate. The maximum plasma peak concentrations ranged from 98.9 to 245.2 ng/ml showed 2–4 h after administration. The elimination half-life was 41–57 h, and even 5 ng/ml in plasma could be detected up to 168 h. Compared with artemisinin, naphthoquine
phosphate proved to be more benefit with the longer duration action and higher cure rate, but with the disadvantages of slower action. The combination of naphthoquine and artemisinin may possess both desirable characteristics and overcome disadvantages of them. The pharmacodynamic tests confirmed that the combination with a appropriate proportion exhibited a synergistic activity against P. berghei in mice and the combination also showed a markedly effect on retarding the development of drug resistance and reducing the resistant level. The toxicity tests revealed that this combination is a low toxicity material and has a wide safety range in useing. An oral fixed combination tablet (Co-naphthoquine) has been used in clinical trials. The clinical trials confirmed that Co-naphthoquine is a safe, well tolerated, effective antimalarial including chloroquine resistant strains. The contribution of each active component to the efficacy of Co-naphthoquine tablet was amply demonstrated. Co-naphthoquine has been proven to be rapid effective in clearing parasites from peripheral blood and resolving fever which is associated with the fast onset action of artemisinin, meanwhile, it achieved a high 28 day cure rate which is associated with the long duration action of naphthoquine. In the clinical comparative studies, the results showed that Co-naphthoquine is superior to its components in single. In these clinical trials, no serious adverse effects were observed and no unexpected hematology, biochemistry and ECG changes were found. These clinical trials were carried out in Hainan of China. From the finished trial result, to administer only one dose, mean fever clearance time and mean parasite clearance time were 17.5 and 30.0 h, respectively, 28-day cure rate was more than 97%. It is effective against P. falciparum with resistance. The new complex shows as a promising drug with good efficacy and tolerability. No adverse effects associated with the combination tablet were observed in these clinical trials.
References Looareesuwan, S., Harinasuta, T., Chongsuphajaisiddhi, T., 1992. Drug resistant malaria with special reference in Thailand.
J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381 Southeast Asian J. Trop. Med. Public Health23, 621– 634. White, N.J., 1997. Assessment of the pharmacodytnamic properties of antimalarial drugs in vivo. Antimicrobial Agents Chemother. 41, 1413–1422. Pang, X., Wang, G., Xing, Q., et al., 1999. Hundred and one cases Plasmodium falciparum patients treated naphtho-
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quine phosphate. Chin. J. Parasitol. Parasitic Dis. 17 (1), 20. Guo, W., Zheng, Q., Li, G., et al., 2000. A randomized-controlled study of naphthoquine and artesunate in the treatment of falciparum malaria. J. Guangzhou Univ. Trad. Chin. Med. 17 (3), 235–237.
Naphthoquine phosphate and its combination with artemisinine Jing-yan Wang∗ , Wu-chun Cao, Cheng-qi Shan, Min Zhang, Guo-fu Li, De-ben Ding, Yun-lin Shi, Bo-an Wu Institute of Microbiology and Epidemiology, Academy of Malitary Medical Sciences and Epidemiology, 20 Dongdajie Fengtai, Beijing 100071, China
Abstract Naphthoquine phosphate and artemisinine are two antimalarials developed in China. Both drugs have proven to be efficacious and well tolerated as monotherapy as well as in combination in patients suffering from malaria. The Co-naphthoquine, a novel antimalarial combination, is an oral fixed combination tablet of the naphthoquine phosphate and artemisinine. Artemisinin is characterised by a rapid onset of schizonticidal action and a short half-life. Parasite clearance is, however, often incomplete when it is employed as a single agent unless high dosages are used over several days, but such a regimen may reduce patient compliance and increase the danger of toxicity. Naphthoquine phosphate, by contrast, has a slower onset of action and a longer half-life, associated with a low recrudescence rate. The two components act synergistically in animal, and clinically provide more rapid relief of symptoms and a higher cure rate than either component alone. The combination tablet was initially developed by the Academy of Military Medical Sciences (AMMS), Beijing, China. © 2003 Elsevier B.V. All rights reserved. Keywords: Naphthoquine phosphate; Artemisinin; Combination; Antimalarial
1. Introduction With the current estimates of 300–500 million new malaria cases arising worldwide, each year, and the attendant 1.5–2.7 million annual deaths. Plasmodium falciparum malaria is still a major cause of mortality and morbidity in the tropical world. The development of drug resistance by P. falciparum has severely compromised our ability to treat this life threatening pathogen. Resistance has subsequently emerged to nearly all available antimalaria drugs. There is a ∗ Corresponding author. Tel.: +86-10-6381-3346; fax: +86-10-6381-3346. E-mail address: [email protected] (J.-y. Wang).
reported 100% resistence of P. falciparum to chloroquine and primaquine in Thailand, and partial resistance to all other antimalarials, for example up to 40% resistance to mefloquine (Looareesuwan et al., 1992). In particular it is the increasing drug resistance and widening geographic distribution of this most lethal of the malarial parasites, Plasmodium falciparum, that has directed attention towards the urgent need for development of novel antimalarial drugs. A recent approach has been to combine the proven efficacy of a newly discovered antimalarial agent, naphthoquine phosphate with artemisinine. The two components act synergistically in animal, and clinically provide more rapid relief of symptoms and a higher cure rate than either component alone.
0001-706X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2003.11.008
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J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381
Fig. 2. Naphthoquine phosphate.
Fig. 1. Artemisinine.
2. Artemisinine Facing the challenge of malaria great effects have been made in China since 1960s. Several new drugs have been developed. The most outstanding contribution was the discovery of artemisinine (Fig. 1). Its sesquiterpene lactone structure contains an endoperoxide group that appears essential for antimalarial activity. Artemisinine has been shown to be active against chloroquine-resistant falciparum malaria in China. It has been used in China for the treatment of uncomplicated malaria for over 20 years. It is a fast acting drug with rapid onset of its schizontocidal action on the parasite. The problem with the artemisinine is that when it is used alone over short periods, i.e. less than 5 days, clearance of parasitaemia from the blood is only temporary in up to 50% of patients (the recrudescence rates from 12% to as much as 55%). This high rate of recrudescence results from the rapid elimination of this drug and the need for the any antimalarial drug to be present in the blood at effective concentrations during four asexual cycles (>6 days) to ensure elimination of the parasite (White, 1997). Higher efficacy can be obtained by 5-and especially 7-day regimens but this is associated with reduced compliance in out-patients.
3. Naphthoquine phosphate Naphthoquine phosphate was registered in China in 1993. The chemical name is 4-(7-chloro-4-aminoquinoline)-2-tert-butylaminomethyl-5,6,7,8-4hydro-1-na-
phthol diphosphate. Molecular weight is 641.98 (Fig. 2). It has thorough killing function for various schizonts of plasmodia, including resistance of P. falciparum to chloroquine. It showed good antimalarial activity against Plasmodium berghei in mice. The ED50 and ED90 were 0.15 and 0.63 mg kg−1 , respectively. The curative dose in monkey infected by Plasmodium knowlesi was 30 mg kg−1 . Taken orally, it can be absorbed rapidly and perfectly. The drug concentration in the blood will reach maximum in 2–4 h after administration, the relative bioavailability is 96.4%. It is widely distributed, highest in liver then in kidney, lung and the blood cells’ concentration is higher than the blood plasma. Excreted mainly from urine, then in excrement. Hundred and one cases of falciparum infected patients in Hainan Island in China were treated with 1000 mg naphthoquine phosphate given two times (600 mg on 0 h and 400 mg on 24 h). All were cured clinically with the cure rate of 100% (Pang et al., 1999). In the comparative study of the therapeutic efficacy of naphthoquine phosphate 30 cases versus artesunate 30 cases on falciparum malarial, each group of P. falciparum infections was treated with naphthoquine phosphate (1000 mg) and artesunate (600 mg per 5 day) separately. The average time for fever clearance were 29.6 ± 16.4 and 18.4 ± 9.0 h, respectively. The average time for parasite clearance were 98.4 ± 27.7 and 43.0 ± 17.1 h, respectively. The 28-day cure rate for naphthoquine phosphate was 96.7%, for artesunate was 73.3% only (Table 1)
Table 1 Comparison of therapeutic efficacy between naphthoquine and artesunate in treatment of P. falciparum in Hainan Group
Case
Total dosage (mg)
Regimen
FCT (h)
PCT (h)
Cure rate (%)
Naphthoquine Artesunate
30 30
1000 600
2 5
29.6 ± 16.4 18.4 ± 9.0
98.4 ± 27.7 43.0 ± 17.1
96.7 73.3
PCT: parasites clearance time; FCT: fever clearance time.
J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381
(Guo et al., 2000). No distinctive adverse reaction is seen with total dosage under 1000 mg of naphthoquine phosphate. It may appear abdominal distention for individuals after administration, which disappears in 2–4 h. Naphthoquine phosphate, in contrast to artemisinine, has a much slower onset of action, with a longer half-life, and it can effect a complete and sustained parasite clearance.
4. The combination of naphthoquine phosphate and artemisine In 1990s the combination of the naphthoquine phosphate and artemisinine was developed in order to retain the strongpoint of the two drugs, to avert the shortcomings of them, to delay the emergence of drug resistance to them, and to prolong the useful life of the two drug. Co-naphthoquine is a new antimalarial combination comprising the naphthoquine phosphate and the artemisinin. The pharmacodynamic tests revealed that the combination of these two drugs was synergistic, the synergistic index was more than 4 or 8 that caculated from chloroquine-sensitive strain of P. berghei or chloroquine-resistent strain of P. berghei, respectively. The curative effects of the combination against P. berghei in mice were superior to the drug used in single. It was also revealed that the combination could delay the emergence of drug resistance and reduce its resistance level. The toxicity tests showed that the combination of naphthoquine and artemisinin was a low toxicity material with a wide safety range. 4.1. Pharmacodynamics tests 4.1.1. Potentiating effect The optimum proportion of naphthoquine phosphate and artemisinin was selected by orthogonal design in the test against P. berghei in mice by “4-day test”. The curative effect of combination on against P. berghei in mice was observed by “4-day test”. The results showed that the combination of naphthoquine phosphate and artemisinin had synergistic action and the optimum proportion of naphthoquine phosphate and artemisinin was 1:50. The synergistic index was more than 4 and 8 that calculated from chloroquine-sensitive strain of P. berghei or
377
Table 2 Comparison of rapidity of resistant development of naphthoquine, artemisinin and their combination on P. berghei K173 strain in mice by blood passage Drug
Development of drug resistance
Resistance index
Passage
Days through
ED90 (mg/kg per day)
QHS
20th 100th
140 700
153.32 194.85
4.4 5.6
NQ
20th 100th
140 700
24.94 152.23
32.8 200.3
Co-NQ
20th 100th
140 700
26.51 77.29
1.5 4.4
QHS: artemisinin; NQ: naphthoquine; and Co-NQ: Co-naphthoquine.
chloroquine-resistent strain of P. berghei, respectively. These results indicated that the rational combination of naphthoquine phosphate and artemisinin would exhibit a potential action against malaria. 4.1.2. Retarding effect on the development of drug resistance In these tests, drug resistant strains were developed by successive blood passages under increasing low doses of single or combination drug pressure. Parallel comparison of the rapidity of development of drug resistance was carried out through 100 passages for 700 days. It was found that naphthoquine single was easy to develop resistance. At 20th passage the resistance index (I90 ) of naphthoquine was more than 32.8, while the I90 of the combination was 1.5 only. After 100 passage the I90 of naphthoquine was more than 200.3. The I90 of combination was 4.4 only. This result indicated that the combination might delay the development of drug resistance and reducing resistance level (for result see Table 2). 4.1.3. Tests on P. knowlesi in monkey The tests were carried out in monkey against P. knowlesi for determining the appropriate proportion of naphthoquine and artemisinin and the appropriate treatment regimen. The results showed that the appropriate proportion of naphthoquine and artemisinin was 1:2.5, the appropriate treatment regimen was only one dose. The curative effect revealed that the combination was markedly superior to these of the single drug Table 3.
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Table 3 Comparison of therapeutic efficacy between artemisinin, naphthoquine and their combination against P. knowlesi in monkey Group
QHS + NQ QHS NQ
Dosage (mg/kg) QHS
NQ
25 31.6 –
10 – 10
Regimen
Total dosage (mg/kg)
Case
PCT (h)
Cure (105 day)
1 4 3
35 126.4 30
3 3 3
56.0 ± 8.0 60.0 ± 0.4 60.0 ± 12.0
3/3 0/3 3/3
4.2. Toxicity The Co-naphthoquine was given by intragastrical administration and by intraperitoneal administration, respectively. The results showed that the LD50 of combination was 2907 and 428 mg/kg given by intragastical administration and intraperitoneal administration, respectively. The chemotherapeutic index was 341 (LD50 /ED50 ). Long-term toxicity tests were carried out in rats and dogs. The Co-naphthoquine was given orally for 14 consecutive days. In rats, 70 MKD (mg/kg per day) was considered as safe dose, 140 MKD was considered the slight intoxication dose. In Beagle dogs the safe dose was 17.5 MKD, 87.5 MKD was considered the slight intoxication. According to the necropsy of rats and dogs, the toxicity target organs of this combination were erythroid series of bone marrow and liver. In the 140 MKD group, 1/4 beagle dog exhibited suppression of erythroid series of bone marrow in every phase and the total number decreased. However, statistical analysis showed no difference (Table 4). The GOT and GPT of serum increased after administration (Tables 5 and 6). After withdraw of drug, the changes were reversible. The general pharmacological tests were carried out in mice, rats and cats to observe the possible pharmacological activity of combination on central nervous Table 4 The effect of Co-naphthoquine on granuloid/erythroid of bone marrow of beagle dogs Dose (mg/kg per day)
D 14 (n = 4)
0 17.5 87.5 140
1.19 1.20 1.47 7.01
∗
P > 0.05.
± ± ± ±
0.17 0.16 0.42 11.06∗
D 42 (n = 2) 1.30 1.19 0.88 1.27
± ± ± ±
0.25 0.12 0.08 0.11
system, cardiovascular system and respiratory system. In these tests the combination was prepared in water suspension, 10 times as proposed daily clinical dosage were given by intragastical administration. The results showed that no general pharmacological activity was found in these observed systems in relating experimental animals by oral the combination at the dosage of 10 fold as proposed daily clinical dose. 4.3. Clinical efficacy of Co-naphthoquine The oral fixed combination tablet of naphthoquine and artemisinin (Co-naphthoquine) has been used as an antimalarial in clinical trials. The combination comparative with naphthoquine and artemisinin have been conducted. Overall 320 patients have been treated with Co-naphthoquine. The 28 day cure rate was 97.5% in Hainan China. After 24 h Co-naphthoquine eliminates more than 97% of the parasite present at baseline, with an overall time to parasite clearance of 30 h. Malaria symptoms resolve quickly, with a time to fever clearance of 17.5 h. Hundred and nine cases of vivax malaria in Hainan Island were treated with Co-naphthoquine. All of them were cured clinically, 98 of them cases were cured completely. The average time for fever clearance was 13.0 ± 5.5 h. The average time for parasite clearance was 18.1 ± 5.7 h. The 56-day cure rate was 90.0% Table 7. By the comparative studies, the curative effect, toleratility and safety of Co-naphthoquine were compared with artemisinin in single and naphthoquine in single, in treatment the P. falciparum patients (Table 8). The results showed that Co-naphthoquine has rapidly effect in clearing P. falciparum from peripheral blood and in resolving fever which is associated with acute malarai infection and was superior to these comparators in these respects. The therapeutic effect of Co-naphthoquine was superior to artemisinin
J.-y. Wang et al. / Acta Tropica 89 (2004) 375–381
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Table 5 The changes of serum GOT of beagle dogs in long-term toxicity test of Co-naphthoquine Time (day)
n
GOT (U/l) 0
17.5
87.5
140 (mg/kg per day)
Before medication D7 D0
6 6
30.7 ± 7.5 39.3 ± 3.3
37.0 ± 8.5 43.0 ± 6.8
38.8 ± 7.1 34.5 ± 19.3
43.3 ± 17.6 38.0 ± 8.0
Medication period D7 D 14
6 6
35.5 ± 4.0 41.3 ± 5.2
37.5 ± 10.6 39.2 ± 5.2
52.0 ± 17.2 58.7 ± 36.5
74.8 ± 39.3 95.0 ± 21.5∗,#
Recovery period D 28 D 42
2 2
41.0 ± 1.4 51.0 ± 1.4
40.0 ± 0.0 40.5 ± 0.7
55.0 ± 7.1 42.5 ± 5.0
36.5 ± 2.1 25.5 ± 9.2
x¯ ± s. ∗ P < 0.05, compared with control of same period. # P < 0.05, compared with values before medication. Table 6 The changes of serum GPT of beagle dogs in long-term toxicity test of Co-naphthoquine Time (day)
n
GPT (U/l) 0
17.5
6 6
24.2 ± 7.9 37.8 ± 10.3
40.8 ± 19.3 43.5 ± 13.1
41.0 ± 16.2 35.8 ± 9.2
36.3 ± 8.0 45.8 ± 10.8
Medication period D7 D 14
6 6
56.5 ± 66.2 38.5 ± 13.0
37.8 ± 7.2 41.2 ± 9.8
57.5 ± 10.8# 108.2 ± 109.0
73.3 ± 24.1 117.3 ± 38.1∗,#
Recovery period D 28 D 42
2 2
37.0 ± 5.7 32.5 ± 7.8
35.0 ± 8.5 42.0 ± 1.4
87.0 ± 59.4 41.5 ± 5.0
Before medication D7 D0
87.5
140.0 (mg/kg per day)
39.0 ± 2.8 37.0 ± 9.9
x¯ ± s. ∗ P < 0.05, compared with control of same period. # P < 0.05, compared with values before medication. Table 7 Clinical pharmacodynamics of Co-naphthoquine Parasite
Case
Reduction in parasite in 24 h
PCT (h)
FCT (h)
Cure rate (%)
P. faciparum P. vivax
320 109
97.3 ± 8.8 99.99 ± 0.05
29.9 ± 8.7 18.1 ± 5.7
17.5 ± 11.4 13.0 ± 5.5
97.5 90
PCT: parasite clearance time; FCT: fever clearance time. Table 8 Comparison of therapeutic efficacy between Co-naphthoquine tablets and its components (aetemisinin tablets and naphthoquine tablets) in treatment P. faciparum in Hainan China Drug
Cases
Total dosage (mg)
PCT (h)
FCT (h)
28 day cure rate (%)
Co-NQ NQ QHS
100 100 30
1400 1000 2500
30.0 ± 8.8 45.5 ± 10.0 29.1 ± 6.0
17.5 ± 12.3 32.7 ± 17.7 18.1 ± 9.7
97 100 66.7
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Table 9 The number of patients of the increased GOT and GPT at different time after administration Drug
Co-NQ NQ QHS
Case
100 100 30
GOT
GPT
D3
D7
D 14
D3
D7
D 14
3 12 3
2 8 4
1 4 1
4 11 3
1 9 3
1 3 0
and naphthoquine. In the clinical trials no serious adverse experience was recorded and no unexpected hematology, biochemistry and ECG changes were found. The number of patients with increased GOT and GPT was listed in Table 9, which showed the Co-naphthoquine had not demonstrated more toxicity than naphthoquine and artemisinin respectively. Artemisinin was given at 0, 8, 24, 48 h, respectively with total dose 2500 mg. Naphthoquine was given at 0 and 24 h, respectively with total dose 1000 mg. Co-naphthoquine was given only one dose with total dose 1400 mg (Table 8).
5. Discussion and conclusion The spread of drug resistance of P. falciparum calls for a continuous effort to search for new antimalarial drugs that are effective against drug-resistant parasite. Combination therapy may provide a way in which resistance can be combated. Naphthoquine and artemisinin both are effective antimalarials that have been recently developed by Chinese scientists. These two drugs showed high in vivo and in vitro antimalarial activity and high clinical efficacy for treatment of multidrug-resistant P. falciparum. Artemisinin has been proven to be benefit with rapid onset action and produce a rapid and complete early reduction of parasites but recrudescence is common if it is given as a single agent for less than 5 days. The pharmacokinetics studies of naphthoquine phosphate revealed longer half-life than artemisinin. 14 healthy volunteers received a single dose 600 mg naphthoquine phosphate. The maximum plasma peak concentrations ranged from 98.9 to 245.2 ng/ml showed 2–4 h after administration. The elimination half-life was 41–57 h, and even 5 ng/ml in plasma could be detected up to 168 h. Compared with artemisinin, naphthoquine
phosphate proved to be more benefit with the longer duration action and higher cure rate, but with the disadvantages of slower action. The combination of naphthoquine and artemisinin may possess both desirable characteristics and overcome disadvantages of them. The pharmacodynamic tests confirmed that the combination with a appropriate proportion exhibited a synergistic activity against P. berghei in mice and the combination also showed a markedly effect on retarding the development of drug resistance and reducing the resistant level. The toxicity tests revealed that this combination is a low toxicity material and has a wide safety range in useing. An oral fixed combination tablet (Co-naphthoquine) has been used in clinical trials. The clinical trials confirmed that Co-naphthoquine is a safe, well tolerated, effective antimalarial including chloroquine resistant strains. The contribution of each active component to the efficacy of Co-naphthoquine tablet was amply demonstrated. Co-naphthoquine has been proven to be rapid effective in clearing parasites from peripheral blood and resolving fever which is associated with the fast onset action of artemisinin, meanwhile, it achieved a high 28 day cure rate which is associated with the long duration action of naphthoquine. In the clinical comparative studies, the results showed that Co-naphthoquine is superior to its components in single. In these clinical trials, no serious adverse effects were observed and no unexpected hematology, biochemistry and ECG changes were found. These clinical trials were carried out in Hainan of China. From the finished trial result, to administer only one dose, mean fever clearance time and mean parasite clearance time were 17.5 and 30.0 h, respectively, 28-day cure rate was more than 97%. It is effective against P. falciparum with resistance. The new complex shows as a promising drug with good efficacy and tolerability. No adverse effects associated with the combination tablet were observed in these clinical trials.
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