Background It has recently been argued that unsafe medical injections are a major transmission route of HIV infection in the generalised epidemics of sub-Saharan Africa.
Background
It has recently been argued that unsafe medical injections are a major transmission route of HIV infection in the generalised epidemics of sub-Saharan Africa.Methods and Findings We have analysed the pattern of injections in relation to HIV incidence in a population cohort in Manicaland in a rural area of Zimbabwe. In Poisson regression models, injections were not found to be associated with HIV in males (rate ratio = 0.33; 95% confidence interval: 0.07 to 1.46) or females (rate ratio = 1.04; 95% confidence interval: 0.59 to 1.85).
Methods and Findings
We have analysed the pattern of injections in relation to HIV incidence in a population cohort in Manicaland in a rural area of Zimbabwe. In Poisson regression models, injections were not found to be associated with HIV in males (rate ratio = 0.33; 95% confidence interval: 0.07 to 1.46) or females (rate ratio = 1.04; 95% confidence interval: 0.59 to 1.85).Conclusion It is important that unsafe medical injections can be confidently excluded as a major source of HIV infection. In rural Zimbabwe the evidence is that they can.
Conclusion
It is important that unsafe medical injections can be confidently excluded as a major source of HIV infection. In rural Zimbabwe the evidence is that they can.

Introduction The widely held belief that heterosexual transmission is the driving force behind sub-Saharan Africa's (SSA's) HIV epidemic [ 1 ] has recently been questioned [ 2 ]. According to Gisselquist and colleagues, investigators have overlooked the importance, and indeed suppressed analysis, of unsafe medical injections as a route of transmission for HIV. Hitherto, assessments of this hypothesis have largely relied on ecological analyses—relating population-level data on unsafe injections to the distribution of HIV prevalence [ 3 , 4 ]. The absence of investigation into the role of unsafe injections, based on the assumed predominance of sexual transmission, has rightly been criticised. However, this criticism trivialises the difficulty of collecting and analysing relevant field data. Presently, the only published data on the possible contribution of injections to HIV transmission in SSA come from rural Uganda, where Kiwanuka et al. demonstrated no association of injections with HIV incidence [ 5 ]. Data from other SSA countries with generalised epidemics where spread has varied in scale and pattern are required to inform this debate. In this paper, we analyse data from a population cohort in Manicaland in rural Zimbabwe. We describe the determinants of injections in adults and then test the association between injections and incidence of HIV infection.
Introduction
The widely held belief that heterosexual transmission is the driving force behind sub-Saharan Africa's (SSA's) HIV epidemic [ 1 ] has recently been questioned [ 2 ]. According to Gisselquist and colleagues, investigators have overlooked the importance, and indeed suppressed analysis, of unsafe medical injections as a route of transmission for HIV. Hitherto, assessments of this hypothesis have largely relied on ecological analyses—relating population-level data on unsafe injections to the distribution of HIV prevalence [ 3 , 4 ]. The absence of investigation into the role of unsafe injections, based on the assumed predominance of sexual transmission, has rightly been criticised. However, this criticism trivialises the difficulty of collecting and analysing relevant field data.Presently, the only published data on the possible contribution of injections to HIV transmission in SSA come from rural Uganda, where Kiwanuka et al. demonstrated no association of injections with HIV incidence [ 5 ]. Data from other SSA countries with generalised epidemics where spread has varied in scale and pattern are required to inform this debate.In this paper, we analyse data from a population cohort in Manicaland in rural Zimbabwe. We describe the determinants of injections in adults and then test the association between injections and incidence of HIV infection.Methods Data were analysed from the baseline (1999/2000) and follow-up (2002/2003) rounds of a cohort of adults in the Manicaland HIV/STD Prevention Study. Eligible men and women aged 15 to 54 were recruited based on an initial household survey [ 6 ]. In response to the awakening controversy, a question exploring exposure to injection was added two-thirds of the way through follow-up. Thus, data were available from individuals from four of the 12 study sites. In these sites, 505 males and 1,342 females were interviewed, representing a follow-up of 69.7% of individuals interviewed at baseline. The subset of individuals who were HIV-negative at baseline ( n  = 1,606; 83.6%) was used for all analyses except for the examination of rates of injections stratified by HIV status at baseline. At follow-up, participants were asked whether they had received an injection or had been pricked by a needle since the baseline interview. A range of health and socio-demographic data were also collected, including self-reported history of sexually transmitted disease (STD) symptoms. Reports on STD symptoms were from the 1-y period before the follow-up interview, and thus did not correspond to the entire 3-y follow-up period. HIV serological testing was performed on dried blood spots using a highly sensitive and specific antibody dipstick assay [ 7 ]. Ethical approval for this study was granted by the Medical Research Council of Zimbabwe (MRCZ/A/681) and the Applied and Qualitative Research Ethics Committee, Oxford University, United Kingdom (N97.039). Written informed consent was sought from study participants. The objective of this analysis was to test the plausibility that injections are an important risk factor for HIV incidence. Having received an injection was modelled as a proximate cause with demographic variables, sexual behaviour, and STDs acting as potential confounders. Determinants of injections were analysed with univariable and multivariable Poisson regression models of incidence rate ratios (RRs). Attributes were retained in age-adjusted multivariable models if the stratum-specific RR differed from one and had a Wald-test  p -value ≤ 0.1. Using the same strategy, models were then fitted with HIV as the outcome variable.
Methods
Data were analysed from the baseline (1999/2000) and follow-up (2002/2003) rounds of a cohort of adults in the Manicaland HIV/STD Prevention Study. Eligible men and women aged 15 to 54 were recruited based on an initial household survey [ 6 ]. In response to the awakening controversy, a question exploring exposure to injection was added two-thirds of the way through follow-up. Thus, data were available from individuals from four of the 12 study sites. In these sites, 505 males and 1,342 females were interviewed, representing a follow-up of 69.7% of individuals interviewed at baseline. The subset of individuals who were HIV-negative at baseline ( n  = 1,606; 83.6%) was used for all analyses except for the examination of rates of injections stratified by HIV status at baseline.nAt follow-up, participants were asked whether they had received an injection or had been pricked by a needle since the baseline interview. A range of health and socio-demographic data were also collected, including self-reported history of sexually transmitted disease (STD) symptoms. Reports on STD symptoms were from the 1-y period before the follow-up interview, and thus did not correspond to the entire 3-y follow-up period. HIV serological testing was performed on dried blood spots using a highly sensitive and specific antibody dipstick assay [ 7 ].Ethical approval for this study was granted by the Medical Research Council of Zimbabwe (MRCZ/A/681) and the Applied and Qualitative Research Ethics Committee, Oxford University, United Kingdom (N97.039). Written informed consent was sought from study participants.The objective of this analysis was to test the plausibility that injections are an important risk factor for HIV incidence. Having received an injection was modelled as a proximate cause with demographic variables, sexual behaviour, and STDs acting as potential confounders. Determinants of injections were analysed with univariable and multivariable Poisson regression models of incidence rate ratios (RRs). Attributes were retained in age-adjusted multivariable models if the stratum-specific RR differed from one and had a Wald-test  p -value ≤ 0.1. Using the same strategy, models were then fitted with HIV as the outcome variable.pResults Overall, 744 out of 1,847 individuals (40.3%) reported having received an injection or needle prick during the 3-y follow-up period. Females reported more injections than males (RR = 1.93). Rates were not significantly higher for individuals who were HIV positive at baseline (RR = 1.07,  p  = 0.81 for males; RR = 1.13,  p  = 0.28 for females) ( Table 1 ). Table 1 Univariable and Multivariable Poisson Regression Models of Incidence of Injections and Needle Pricks—Presented Separately for Males and Females Reported injections and person years may not add up across different attributes because of missing data
 a  Adjusted for age and attributes significant at  p  ≤ 0.1 (Wald test)
 b  Exposure between baseline and follow-up (approximately 3 y)
 c  Exposure in the 1 y prior to follow-up
 d  Not significant but shown to demonstrate the direction of effect CI, confidence interval; PYAR, person years at risk Being widowed, separated, or divorced was the only attribute associated with increased rates of injections for males ( Table 1 ). For females, STD symptoms and childbearing/pregnancy were significant in adjusted models ( Table 1 ). There were 67 HIV seroconversions (48 females and 19 males); 13 (19%) of those seroconverting reported not having had sex in the inter-survey period, and 40 (60%) reported not having had an injection during the period ( Table 2 ). Table 2 Univariable and Multivariable Poisson Regression Models of HIV Incidence—Presented Separately for Males and Females Seroconversions and person years may not add up across different attributes because of missing data
 a  Adjusted for age and attributes significant at  p  ≤ 0.1 (Wald test)
 b  Exposure between baseline and follow-up (approximately 3 y)
 c  Not significant but shown to demonstrate the direction of effect
 d  Exposure in the 1 y prior to follow-up CI, confidence interval; PYAR, person years at risk There was no significant association between injections and HIV incidence among either males or females—in either unadjusted or adjusted models. For males, HIV seroconversion rates were elevated amongst 25- to 44-y-olds, sexually active individuals, and those who had suffered STD symptoms, though none of these attributes reached levels of statistical significance ( Table 2 ). For females, having multiple sexual partners, having STD symptoms, and being widowed/separated/divorced were associated with increased HIV incidence. Childbearing/pregnancy, which was strongly associated with injections (see  Table 1 ), had no association with HIV incidence ( Table 2 ).
Results
Overall, 744 out of 1,847 individuals (40.3%) reported having received an injection or needle prick during the 3-y follow-up period. Females reported more injections than males (RR = 1.93). Rates were not significantly higher for individuals who were HIV positive at baseline (RR = 1.07,  p  = 0.81 for males; RR = 1.13,  p  = 0.28 for females) ( Table 1 ).ppUnivariable and Multivariable Poisson Regression Models of Incidence of Injections and Needle Pricks—Presented Separately for Males and Females
Reported injections and person years may not add up across different attributes because of missing dataa  Adjusted for age and attributes significant at  p  ≤ 0.1 (Wald test)pb  Exposure between baseline and follow-up (approximately 3 y)c  Exposure in the 1 y prior to follow-upd  Not significant but shown to demonstrate the direction of effectCI, confidence interval; PYAR, person years at riskBeing widowed, separated, or divorced was the only attribute associated with increased rates of injections for males ( Table 1 ). For females, STD symptoms and childbearing/pregnancy were significant in adjusted models ( Table 1 ).There were 67 HIV seroconversions (48 females and 19 males); 13 (19%) of those seroconverting reported not having had sex in the inter-survey period, and 40 (60%) reported not having had an injection during the period ( Table 2 ).Univariable and Multivariable Poisson Regression Models of HIV Incidence—Presented Separately for Males and Females
Seroconversions and person years may not add up across different attributes because of missing dataa  Adjusted for age and attributes significant at  p  ≤ 0.1 (Wald test)pb  Exposure between baseline and follow-up (approximately 3 y)c  Not significant but shown to demonstrate the direction of effectd  Exposure in the 1 y prior to follow-upCI, confidence interval; PYAR, person years at riskThere was no significant association between injections and HIV incidence among either males or females—in either unadjusted or adjusted models. For males, HIV seroconversion rates were elevated amongst 25- to 44-y-olds, sexually active individuals, and those who had suffered STD symptoms, though none of these attributes reached levels of statistical significance ( Table 2 ). For females, having multiple sexual partners, having STD symptoms, and being widowed/separated/divorced were associated with increased HIV incidence. Childbearing/pregnancy, which was strongly associated with injections (see  Table 1 ), had no association with HIV incidence ( Table 2 ).Discussion These data, from a population cohort in rural Zimbabwe, suggest that—at the population level—injections are not a major route of HIV transmission. There was a very slight, non-significant association between injections and HIV amongst females. Could this association achieve significance with a greater sample size or more events? Given the strong association between STD symptoms and both injections and HIV incidence, this is possible, but would likely be a result of residual confounding. In other words, both HIV and injections have a common association with STDs. It has been argued that the association between sexual activity and HIV is confounding between STDs and pregnancy and injections and that it is the injections that are causal. Our analysis does indeed find these associations—but finds STD symptoms the strongest predictor of new HIV infections. Our measure of injection risk is unambiguous but lacks many dimensions relating to unsafe injections. Presently, we have collected data about the receipt of injections and other needle pricks. Thus, the exposures in these analyses are not restricted to injections received from the health-care sector—the source that Gisselquist et al. originally hypothesized as a major route of transmission [ 2 ]. Also, these data reflect only whether an individual had an injection or not—rather than the number of injections received. World Health Organization estimates suggest that the number of injections people receive is not evenly distributed in SSA populations [ 8 ]. Thirteen of 67 individuals seroconverting in this study reported no sexual partners in the inter-survey period. Only four of these 13 reported never having had sex. This leads us to suspect that incorrect categorisation of HIV status at baseline in addition to misreporting of sexual behaviour may explain some of these infections: it is possible that a proportion of the nine individuals who reported having had sex in their lifetime but not in the inter-survey period had been recently infected but had not yet seroconverted at the time of the baseline testing [ 9 ]. In this scenario, their exposure would have occurred prior to baseline, rather than in the follow-up period. Recall biases may also play a role, given the relatively long follow-up of 3 y [ 10 ]. Non-regular partners, especially those from the beginning of the recall period, may have been under-reported. Eliciting accurate reporting of sexual activity is notoriously difficult in Africa and elsewhere, though the use of informal confidential voting interviews has decreased social desirability biases in this cohort [ 11 ]. Nonetheless, the cases ( n  = 4) where individuals seroconverted who had reported never having had sex may still be a product of social desirability reporting bias. Clearly, incidence data offer the most explanatory power in elucidating the determinants of the HIV epidemic, but these anomalous cases also highlight the difficulties of collecting time-varying sexual behaviour and serostatus information. Interestingly, in light of expected HIV-associated disease and care, individuals who were HIV positive at baseline did not have higher rates of injections than individuals who were HIV negative. Injections were found to be highly associated with childbirth/pregnancy. HIV-positive women—and especially those at advanced stages of infection—are known to experience reduced fertility [ 12 ]. Therefore, a reduction in use of maternal health services may partially explain why injections were not more common in the HIV-positive population. A more discriminating measure of exposure, including the reason for injection, could help to explain this observation. Had injections proven to be a risk factor for HIV incidence, further investigations would have been needed to determine the source and types of risky needle pricks. However, no such association was found. Unsafe injections are unacceptable, but this evidence suggests that they do not play a major role in the transmission of HIV in rural Zimbabwe. Patient Summary Background There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important. What Did the Authors Do? In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period. What Did They Find? 744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection). What Do These Findings Mean? These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex. Where Can I Get More Information? Information on safe sex:  http://www.thebody.com/safesex.html
 World Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously:  http://www.who.int/hiv/topics/harm/reduction/en/
 Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe:  http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm
Discussion
These data, from a population cohort in rural Zimbabwe, suggest that—at the population level—injections are not a major route of HIV transmission. There was a very slight, non-significant association between injections and HIV amongst females. Could this association achieve significance with a greater sample size or more events? Given the strong association between STD symptoms and both injections and HIV incidence, this is possible, but would likely be a result of residual confounding. In other words, both HIV and injections have a common association with STDs. It has been argued that the association between sexual activity and HIV is confounding between STDs and pregnancy and injections and that it is the injections that are causal. Our analysis does indeed find these associations—but finds STD symptoms the strongest predictor of new HIV infections.Our measure of injection risk is unambiguous but lacks many dimensions relating to unsafe injections. Presently, we have collected data about the receipt of injections and other needle pricks. Thus, the exposures in these analyses are not restricted to injections received from the health-care sector—the source that Gisselquist et al. originally hypothesized as a major route of transmission [ 2 ]. Also, these data reflect only whether an individual had an injection or not—rather than the number of injections received. World Health Organization estimates suggest that the number of injections people receive is not evenly distributed in SSA populations [ 8 ].Thirteen of 67 individuals seroconverting in this study reported no sexual partners in the inter-survey period. Only four of these 13 reported never having had sex. This leads us to suspect that incorrect categorisation of HIV status at baseline in addition to misreporting of sexual behaviour may explain some of these infections: it is possible that a proportion of the nine individuals who reported having had sex in their lifetime but not in the inter-survey period had been recently infected but had not yet seroconverted at the time of the baseline testing [ 9 ]. In this scenario, their exposure would have occurred prior to baseline, rather than in the follow-up period. Recall biases may also play a role, given the relatively long follow-up of 3 y [ 10 ]. Non-regular partners, especially those from the beginning of the recall period, may have been under-reported. Eliciting accurate reporting of sexual activity is notoriously difficult in Africa and elsewhere, though the use of informal confidential voting interviews has decreased social desirability biases in this cohort [ 11 ]. Nonetheless, the cases ( n  = 4) where individuals seroconverted who had reported never having had sex may still be a product of social desirability reporting bias. Clearly, incidence data offer the most explanatory power in elucidating the determinants of the HIV epidemic, but these anomalous cases also highlight the difficulties of collecting time-varying sexual behaviour and serostatus information.nInterestingly, in light of expected HIV-associated disease and care, individuals who were HIV positive at baseline did not have higher rates of injections than individuals who were HIV negative. Injections were found to be highly associated with childbirth/pregnancy. HIV-positive women—and especially those at advanced stages of infection—are known to experience reduced fertility [ 12 ]. Therefore, a reduction in use of maternal health services may partially explain why injections were not more common in the HIV-positive population. A more discriminating measure of exposure, including the reason for injection, could help to explain this observation.Had injections proven to be a risk factor for HIV incidence, further investigations would have been needed to determine the source and types of risky needle pricks. However, no such association was found. Unsafe injections are unacceptable, but this evidence suggests that they do not play a major role in the transmission of HIV in rural Zimbabwe.Patient Summary Background There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important. What Did the Authors Do? In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period. What Did They Find? 744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection). What Do These Findings Mean? These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex. Where Can I Get More Information? Information on safe sex:  http://www.thebody.com/safesex.html
 World Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously:  http://www.who.int/hiv/topics/harm/reduction/en/
 Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe:  http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm
Patient Summary
Background There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important. What Did the Authors Do? In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period. What Did They Find? 744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection). What Do These Findings Mean? These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex. Where Can I Get More Information? Information on safe sex:  http://www.thebody.com/safesex.html
 World Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously:  http://www.who.int/hiv/topics/harm/reduction/en/
 Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe:  http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm

Background There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important.
Background
There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important.What Did the Authors Do? In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period.
What Did the Authors Do?
In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period.What Did They Find? 744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection).
What Did They Find?
744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection).What Do These Findings Mean? These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex.
What Do These Findings Mean?
These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex.Where Can I Get More Information? Information on safe sex:  http://www.thebody.com/safesex.html
 World Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously:  http://www.who.int/hiv/topics/harm/reduction/en/
 Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe:  http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm
Where Can I Get More Information?
Information on safe sex:  http://www.thebody.com/safesex.htmlWorld Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously:  http://www.who.int/hiv/topics/harm/reduction/en/Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe:  http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm


Background Reserpine, an alkaloid from  Rauwolfia serpentina  was widely used for its antihypertensive action in the past. In later years, its use has been reduced because of precipitation of depression and extra pyramidal symptoms due to its central action. In the present investigation, reserpine methonitrate (RMN), a novel quaternary analogue of reserpine was synthesised and evaluated biochemically for its central and peripheral amine depleting actions in rats while its influence on the blood pressure was measured in anaesthetized rats in comparison with reserpine
Background
Reserpine, an alkaloid from  Rauwolfia serpentina  was widely used for its antihypertensive action in the past. In later years, its use has been reduced because of precipitation of depression and extra pyramidal symptoms due to its central action. In the present investigation, reserpine methonitrate (RMN), a novel quaternary analogue of reserpine was synthesised and evaluated biochemically for its central and peripheral amine depleting actions in rats while its influence on the blood pressure was measured in anaesthetized rats in comparison with reserpineRauwolfia serpentinaResults Reserpine treatment (5 mg/kg) produced a significant increase in the urinary excretion of VMA, 5-HIAA and HVA while RMN at doses of equal to and double the equimolar doses of reserpine (5 and 10 mg/kg) produced significant increase in VMA and 5-HIAA excretion without producing any effect on HVA excretion compared to control animals. Reserpine in the dose range of 0.5 to15 μg/kg produced significant reduction in blood pressure compared to control. RMN was also found to produce significant decrease in blood pressure at doses of 10, 25 and 50 μg/kg body weight in comparison to control. The results indicated peripheral depletion of biogenic amines by RMN without affecting the central stores of the amines.
Results
Reserpine treatment (5 mg/kg) produced a significant increase in the urinary excretion of VMA, 5-HIAA and HVA while RMN at doses of equal to and double the equimolar doses of reserpine (5 and 10 mg/kg) produced significant increase in VMA and 5-HIAA excretion without producing any effect on HVA excretion compared to control animals. Reserpine in the dose range of 0.5 to15 μg/kg produced significant reduction in blood pressure compared to control. RMN was also found to produce significant decrease in blood pressure at doses of 10, 25 and 50 μg/kg body weight in comparison to control. The results indicated peripheral depletion of biogenic amines by RMN without affecting the central stores of the amines.Conclusions The present study clearly indicated that the quaternization of reserpine restricts its transfer across the blood-brain barrier and could be the reason for its selective peripheral action. It is also clear that the hypotensive actions of RMN could be due to peripheral depletion of catecholamines.
Conclusions
The present study clearly indicated that the quaternization of reserpine restricts its transfer across the blood-brain barrier and could be the reason for its selective peripheral action. It is also clear that the hypotensive actions of RMN could be due to peripheral depletion of catecholamines.

Background Reserpine, an alkaloid isolated from  Rauwolfia  species, was introduced for the treatment of hypertension and schizophrenia in 1950's but was replaced by more effective drugs by the end of 1970's [ 1 - 6 ]. Reserpine is known to act centrally as well as peripherally by depletion of biogenic amines like noradrenaline, dopamine and serotonin. Mostly, its peripheral depletion of amines is responsible for its antihypertensive effect while its central depletion of amines is responsible for its antipsychotic action [ 7 - 13 ]. However, because of its central action it produces sedation and Parkinsonism when used for the management of hypertension for prolonged periods [ 14 - 17 ]. As a result it has reduced usage for chronic treatment in hypertensive patients and its use is limited to selective patient population only [ 18 , 19 ]. Hence there is a need for structural modification of the drug to make it more acceptable therapeutically for the treatment of hypertension. Attempts were made in the past to synthesize derivatives of reserpine with possibly higher and/or modified activities or with fewer side effects [ 20 - 23 ]. Compared to reserpine itself, a number of reserpine analogues were found to exert a stronger influence on the amine concentration in the periphery than in brain [ 24 - 26 ]. Based on the poor ability of quaternary compounds to penetrate the blood-brain barrier, a great deal of research has been devoted towards quaternization of existing drugs to achieve preferential peripheral action [ 27 - 32 ]. Earlier reports have demonstrated the synthesis of quaternary derivatives of reserpine and isoreserpine, however their pharmacology was not studied [ 33 , 34 ]. Previous studies by our group also revealed that reserpine methiodide produced selective depletion of peripheral biogenic amines without affecting their central stores [ 35 ]. In the present investigation, a quaternary analogue of reserpine  viz ., reserpine methonitrate (RMN), which was synthesized in our laboratory was evaluated in rats for its amine depleting actions compared to reserpine. For this, the urinary levels of vanillylmandelic acid (VMA), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) which are the respective metabolites of noradrenaline, serotonin and dopamine were estimated after reserpine or RMN treatment in rats. The change in the blood pressure response of anaesthetized rats after treatment with RMN was also evaluated in comparison to reserpine.
Background
Reserpine, an alkaloid isolated from  Rauwolfia  species, was introduced for the treatment of hypertension and schizophrenia in 1950's but was replaced by more effective drugs by the end of 1970's [ 1 - 6 ]. Reserpine is known to act centrally as well as peripherally by depletion of biogenic amines like noradrenaline, dopamine and serotonin. Mostly, its peripheral depletion of amines is responsible for its antihypertensive effect while its central depletion of amines is responsible for its antipsychotic action [ 7 - 13 ]. However, because of its central action it produces sedation and Parkinsonism when used for the management of hypertension for prolonged periods [ 14 - 17 ]. As a result it has reduced usage for chronic treatment in hypertensive patients and its use is limited to selective patient population only [ 18 , 19 ]. Hence there is a need for structural modification of the drug to make it more acceptable therapeutically for the treatment of hypertension.RauwolfiaAttempts were made in the past to synthesize derivatives of reserpine with possibly higher and/or modified activities or with fewer side effects [ 20 - 23 ]. Compared to reserpine itself, a number of reserpine analogues were found to exert a stronger influence on the amine concentration in the periphery than in brain [ 24 - 26 ].Based on the poor ability of quaternary compounds to penetrate the blood-brain barrier, a great deal of research has been devoted towards quaternization of existing drugs to achieve preferential peripheral action [ 27 - 32 ]. Earlier reports have demonstrated the synthesis of quaternary derivatives of reserpine and isoreserpine, however their pharmacology was not studied [ 33 , 34 ]. Previous studies by our group also revealed that reserpine methiodide produced selective depletion of peripheral biogenic amines without affecting their central stores [ 35 ]. In the present investigation, a quaternary analogue of reserpine  viz ., reserpine methonitrate (RMN), which was synthesized in our laboratory was evaluated in rats for its amine depleting actions compared to reserpine. For this, the urinary levels of vanillylmandelic acid (VMA), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) which are the respective metabolites of noradrenaline, serotonin and dopamine were estimated after reserpine or RMN treatment in rats. The change in the blood pressure response of anaesthetized rats after treatment with RMN was also evaluated in comparison to reserpine.vizResults Biochemical estimations The main aim of the study was to determine whether RMN was able to deplete the central and peipheral biogenic amines to the same extent as produced by reserpine. Reserpine at a dose of 5 mg/kg body weight produced significant increase in the urinary excretion profile of VMA compared to control animals. The analogue at doses equimolar to reserpine of 5 and 10 mg/kg body weight produced more significant increase in VMA excretion compared to controls and that observed with reserpine (Fig  1 ). However, the higher dose (10 mg/kg body weight) of RMN did not further enhance the excretion of VMA produced by 5 mg/kg body weight dose. Figure 1 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of VMA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: #p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate Significant increase in 5-HIAA excretion was observed with reserpine at a dose of 5 mg/kg body weight and with the equivalent dose of RMN (Fig  2 ). The amount of 5-HIAA excreted in animals treated with the analogue/reserpine was found to be more than in the control. However the effect was found to be more with analogue compared to reserpine. The enhancement in dose to 10 mg/kg body weight of RMN did not produce any further increase in 5-HIAA excretion. Figure 2 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of 5-HIAA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: # p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate A marked increase in the HVA excretion was observed in animals treated with reserpine compared to controls while minor change was observed in animals treated with RMN at doses of 5 and 10 mg/kg body weight compared to control (Fig  3 ). Figure 3 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of HVA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: # p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate Effect on normal blood pressure of anaesthetized rats The effect of reserpine and RMN on the normal blood pressure of anaesthetized rats was shown in Table  1 . Dose dependent hypotension was observed with reserpine as well as with RMN. However, the vehicle (DMSO) also produced hypotension which was approximately 15 mm Hg from basal level. Reserpine at doses of 0.5, 1, 5, 10 and 15 μg/kg produced significant (p < 0.01) reduction in blood pressure compared to control. RMN was also found to produce significant (p < 0.01) decrease in blood pressure at doses of 10, 25 and 50 μg/kg body weight compared to control Table 1 Effect of reserpine and reserpine methonitrate on the mean arterial pressure of anaesthetized rats. Drug Dose (μg/kg) Mean arterial pressure (mmHg, n = 6) Reduction due to drug Before drug After drug Mean reduction Vehicle 0.05 ml 126.2 ± 2.8 110.0 ± 4.1 16.2 ± 1.4 --- Reserpine 0.25 138.3 ± 4.6 119.5 ± 4.2 18.8 ± 1.4 NS 2.6 0.50 134.1 ± 5.5 91.2 ± 5.0 42.9 ± 0.9** 26.5 1 135.2 ± 4.7 80.5 ± 3.8 54.8 ± 1.6** 38.6 5 130.5 ± 5.1 69.0 ± 3.5 61.5 ± 2.8** 45.3 10 130.0 ± 4.2 58.7 ± 3.9 71.2 ± 1.3** 55.0 15 131.5 ± 5.1 41.1 ± 2.9 90.4 ± 3.1** 74.2 Reserpine methonitrate equivalent to reserpine 10 128.8 ± 6.4 90.0 ± 9.1 38.8 ± 3.6** 22.6 25 135.0 ± 8.5 73.0 ± 7.4 62.0 ± 2.8** 45.8 50 136.8 ± 5.3 42.5 ± 3.7 93.0 ± 8.0** 76.8 Significant difference from DMSO treated group: **p < 0.01 NS – No significant difference from DMSO treated group.
Results
Biochemical estimations The main aim of the study was to determine whether RMN was able to deplete the central and peipheral biogenic amines to the same extent as produced by reserpine. Reserpine at a dose of 5 mg/kg body weight produced significant increase in the urinary excretion profile of VMA compared to control animals. The analogue at doses equimolar to reserpine of 5 and 10 mg/kg body weight produced more significant increase in VMA excretion compared to controls and that observed with reserpine (Fig  1 ). However, the higher dose (10 mg/kg body weight) of RMN did not further enhance the excretion of VMA produced by 5 mg/kg body weight dose. Figure 1 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of VMA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: #p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate Significant increase in 5-HIAA excretion was observed with reserpine at a dose of 5 mg/kg body weight and with the equivalent dose of RMN (Fig  2 ). The amount of 5-HIAA excreted in animals treated with the analogue/reserpine was found to be more than in the control. However the effect was found to be more with analogue compared to reserpine. The enhancement in dose to 10 mg/kg body weight of RMN did not produce any further increase in 5-HIAA excretion. Figure 2 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of 5-HIAA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: # p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate A marked increase in the HVA excretion was observed in animals treated with reserpine compared to controls while minor change was observed in animals treated with RMN at doses of 5 and 10 mg/kg body weight compared to control (Fig  3 ). Figure 3 Diagram illustrating the effect of reserpine and reserpine methonitrate on the 24 h urinary excretion of HVA in rats.  Each bar indicates the mean excretion of six animals. Significant difference from control group: *p < 0.05 Significant difference from reserpine treated group: # p < 0.05 NS-No significant difference between 5 and 10 mg/kg treated groups of reserpine methonitrate
Biochemical estimations
The main aim of the study was to determine whether RMN was able to deplete the central and peipheral biogenic amines to the same extent as produced by reserpine. Reserpine at a dose of 5 mg/kg body weight produced significant increase in the urinary excretion profile of VMA compared to control animals. The analogue at doses equimolar to reserpine of 5 and 10 mg/kg body weight produced more significant increase in VMA excretion compared to controls and that observed with reserpine (Fig  1 ). However, the higher dose (10 mg/kg body weight) of RMN did not further enhance the excretion of VMA produced by 5 mg/kg body weight dose.Significant increase in 5-HIAA excretion was observed with reserpine at a dose of 5 mg/kg body weight and with the equivalent dose of RMN (Fig  2 ). The amount of 5-HIAA excreted in animals treated with the analogue/reserpine was found to be more than in the control. However the effect was found to be more with analogue compared to reserpine. The enhancement in dose to 10 mg/kg body weight of RMN did not produce any further increase in 5-HIAA excretion.A marked increase in the HVA excretion was observed in animals treated with reserpine compared to controls while minor change was observed in animals treated with RMN at doses of 5 and 10 mg/kg body weight compared to control (Fig  3 ).Effect on normal blood pressure of anaesthetized rats The effect of reserpine and RMN on the normal blood pressure of anaesthetized rats was shown in Table  1 . Dose dependent hypotension was observed with reserpine as well as with RMN. However, the vehicle (DMSO) also produced hypotension which was approximately 15 mm Hg from basal level. Reserpine at doses of 0.5, 1, 5, 10 and 15 μg/kg produced significant (p < 0.01) reduction in blood pressure compared to control. RMN was also found to produce significant (p < 0.01) decrease in blood pressure at doses of 10, 25 and 50 μg/kg body weight compared to control Table 1 Effect of reserpine and reserpine methonitrate on the mean arterial pressure of anaesthetized rats. Drug Dose (μg/kg) Mean arterial pressure (mmHg, n = 6) Reduction due to drug Before drug After drug Mean reduction Vehicle 0.05 ml 126.2 ± 2.8 110.0 ± 4.1 16.2 ± 1.4 --- Reserpine 0.25 138.3 ± 4.6 119.5 ± 4.2 18.8 ± 1.4 NS 2.6 0.50 134.1 ± 5.5 91.2 ± 5.0 42.9 ± 0.9** 26.5 1 135.2 ± 4.7 80.5 ± 3.8 54.8 ± 1.6** 38.6 5 130.5 ± 5.1 69.0 ± 3.5 61.5 ± 2.8** 45.3 10 130.0 ± 4.2 58.7 ± 3.9 71.2 ± 1.3** 55.0 15 131.5 ± 5.1 41.1 ± 2.9 90.4 ± 3.1** 74.2 Reserpine methonitrate equivalent to reserpine 10 128.8 ± 6.4 90.0 ± 9.1 38.8 ± 3.6** 22.6 25 135.0 ± 8.5 73.0 ± 7.4 62.0 ± 2.8** 45.8 50 136.8 ± 5.3 42.5 ± 3.7 93.0 ± 8.0** 76.8 Significant difference from DMSO treated group: **p < 0.01 NS – No significant difference from DMSO treated group.
Effect on normal blood pressure of anaesthetized rats
The effect of reserpine and RMN on the normal blood pressure of anaesthetized rats was shown in Table  1 . Dose dependent hypotension was observed with reserpine as well as with RMN. However, the vehicle (DMSO) also produced hypotension which was approximately 15 mm Hg from basal level. Reserpine at doses of 0.5, 1, 5, 10 and 15 μg/kg produced significant (p < 0.01) reduction in blood pressure compared to control. RMN was also found to produce significant (p < 0.01) decrease in blood pressure at doses of 10, 25 and 50 μg/kg body weight compared to controlEffect of reserpine and reserpine methonitrate on the mean arterial pressure of anaesthetized rats.DrugDose (μg/kg)Mean arterial pressure (mmHg, n = 6)Reduction due to drugBefore drugAfter drugMean reductionSignificant difference from DMSO treated group: **p < 0.01NS – No significant difference from DMSO treated group.Discussion The structural modification of existing drugs to achieve selective action is not uncommon in providing better pharmaceutical care to the needy patients. It has been well established that the antihypertensive and tranquilizing actions of reserpine are mediated through the depletion of biogenic amines in the body [ 12 , 13 , 36 ]. The peripheral depletion of amines is responsible for its antihypertensive effect [ 11 , 37 ] while their central depletion plays a role in sedation and depression of reserpine [ 38 , 39 ]. Reserpine exerts its depleting effect by specifically inhibiting the adenosine triphosphate-Mg 2+ -dependent incorporation of biogenic amines into their storage vesicles [ 40 , 41 ]. Since reserpine depletes noradrenaline, 5-HT and dopamine from their storage sites, this results in a consequent increase in their metabolite levels in urine. Previous investigators have demonstrated a marked increase in the urinary excretion of peripheral and central metabolites of biogenic amines in animals treated with reserpine [ 8 , 42 - 44 ]. In the present investigation, a non-invasive biochemical approach was followed to determine the 24 h urinary excretion of VMA, 5-HIAA and HVA in rats treated with reserpine or RMN. Moreover, VMA, the peripheral metabolite of noradrenaline; 5-HIAA, the main metabolite of serotonin; and HVA, the predominant metabolite of dopamine were selected as the biomarkers for evaluation since noradrenaline exists both centrally and peripherally, serotonin exists mainly peripherally while majority of dopamine exists centrally. Since 99% of the total body's content of serotonin is present in the periphery, it is considered that the major part of the excreted 5-HIAA is from the peripheral release [ 45 , 46 ]. Similarly, high levels of dopamine are found in the centre rather than periphery, and any change in the HVA excretion in urine was considered as a corresponding change in dopamine levels at the central regions [ 47 ]. These indices provide an indirect evidence for the peripheral and central monoamine depleting effects of reserpine and its quaternary analogue. The results showed that reserpine increased the urinary excretion of VMA, 5-HIAA and HVA indicating the depletion of peripheral as well as central biogenic amines. These are in agreement with the results observed by previous investigators [ 8 , 35 , 42 - 44 ]. The increase in the urinary excretion of VMA and 5-HIAA with RMN is higher than with reserpine at equimolar dose of 5 mg/kg body weight. The localized distribution of the analogue in the periphery could led to higher level of depletion of peripheral noradrenaline and serotonin hence their metabolite levels were found to be increased much more which also substantiate our previous studies [ 35 ]. The inability of the analogue to increase HVA excretion unlike reserpine could be due to its non-entry across the blood-brain barrier and into the central nervous system to deplete dopamine which is present predominantly in mesolimbic, nigrostriatal and tuberoinfendibular systems [ 48 ]. The increased urinary levels of 5-HIAA observed with RMN could be due to the peripheral release of 5-HT as it is found predominantly at the periphery in enterochromaffin cells. The higher dose (10 mg/kg) of RMN did not produce any further increase in the VMA and 5-HIAA excretion compared to lower dose. The possible reason for this effect could be that 5 mg/kg dose was sufficient to deplete the amines completely from the storage sites. In order to evaluate whether the quaternary analogue of reserpine (RMN) still retains the peripheral blood pressure lowering activity, further experiments were carried out on the blood pressure of anaesthetized rats. Thus far, the results of RMN on the blood pressure response of anaesthetized rats confirmed that the peripheral actions of reserpine molecule are not affected by quaternization. However, in the present study the vehicle (DMSO) also produced minor hypotensive effect on blood pressure of rats when administered alone with the dose used for the administration of the drugs. Earlier workers [ 49 ] also reported hypotension with DMSO supporting the present observations. Reserpine produced dose dependent reduction in blood pressure as demonstrated by previous investigators [ 50 , 51 ]. As indicated in earlier reports [ 9 , 11 , 40 , 52 - 54 ] the hypotensive effect of reserpine observed in rats is due to the depletion of catecholamines from the peripheral stores. The effect of equimolar doses of RMN also indicated hypotension however, with higher doses compared to reserpine. It is further indicated that quaternization of reserpine not only restricted the entry of RMN to central nervous system but also reduced to the target tissue in the periphery. Hence relatively higher doses were required to produce reserpine like effect. Mechanistically, the hypotensive actions of RMN could also be due to peripheral depletion of catecholamines as evident from the positive correlation with the results of previous section on the peripheral depletion of monoamines.
Discussion
The structural modification of existing drugs to achieve selective action is not uncommon in providing better pharmaceutical care to the needy patients. It has been well established that the antihypertensive and tranquilizing actions of reserpine are mediated through the depletion of biogenic amines in the body [ 12 , 13 , 36 ]. The peripheral depletion of amines is responsible for its antihypertensive effect [ 11 , 37 ] while their central depletion plays a role in sedation and depression of reserpine [ 38 , 39 ]. Reserpine exerts its depleting effect by specifically inhibiting the adenosine triphosphate-Mg 2+ -dependent incorporation of biogenic amines into their storage vesicles [ 40 , 41 ].Since reserpine depletes noradrenaline, 5-HT and dopamine from their storage sites, this results in a consequent increase in their metabolite levels in urine. Previous investigators have demonstrated a marked increase in the urinary excretion of peripheral and central metabolites of biogenic amines in animals treated with reserpine [ 8 , 42 - 44 ].In the present investigation, a non-invasive biochemical approach was followed to determine the 24 h urinary excretion of VMA, 5-HIAA and HVA in rats treated with reserpine or RMN. Moreover, VMA, the peripheral metabolite of noradrenaline; 5-HIAA, the main metabolite of serotonin; and HVA, the predominant metabolite of dopamine were selected as the biomarkers for evaluation since noradrenaline exists both centrally and peripherally, serotonin exists mainly peripherally while majority of dopamine exists centrally. Since 99% of the total body's content of serotonin is present in the periphery, it is considered that the major part of the excreted 5-HIAA is from the peripheral release [ 45 , 46 ]. Similarly, high levels of dopamine are found in the centre rather than periphery, and any change in the HVA excretion in urine was considered as a corresponding change in dopamine levels at the central regions [ 47 ]. These indices provide an indirect evidence for the peripheral and central monoamine depleting effects of reserpine and its quaternary analogue.The results showed that reserpine increased the urinary excretion of VMA, 5-HIAA and HVA indicating the depletion of peripheral as well as central biogenic amines. These are in agreement with the results observed by previous investigators [ 8 , 35 , 42 - 44 ]. The increase in the urinary excretion of VMA and 5-HIAA with RMN is higher than with reserpine at equimolar dose of 5 mg/kg body weight. The localized distribution of the analogue in the periphery could led to higher level of depletion of peripheral noradrenaline and serotonin hence their metabolite levels were found to be increased much more which also substantiate our previous studies [ 35 ]. The inability of the analogue to increase HVA excretion unlike reserpine could be due to its non-entry across the blood-brain barrier and into the central nervous system to deplete dopamine which is present predominantly in mesolimbic, nigrostriatal and tuberoinfendibular systems [ 48 ].The increased urinary levels of 5-HIAA observed with RMN could be due to the peripheral release of 5-HT as it is found predominantly at the periphery in enterochromaffin cells. The higher dose (10 mg/kg) of RMN did not produce any further increase in the VMA and 5-HIAA excretion compared to lower dose. The possible reason for this effect could be that 5 mg/kg dose was sufficient to deplete the amines completely from the storage sites.In order to evaluate whether the quaternary analogue of reserpine (RMN) still retains the peripheral blood pressure lowering activity, further experiments were carried out on the blood pressure of anaesthetized rats. Thus far, the results of RMN on the blood pressure response of anaesthetized rats confirmed that the peripheral actions of reserpine molecule are not affected by quaternization. However, in the present study the vehicle (DMSO) also produced minor hypotensive effect on blood pressure of rats when administered alone with the dose used for the administration of the drugs. Earlier workers [ 49 ] also reported hypotension with DMSO supporting the present observations. Reserpine produced dose dependent reduction in blood pressure as demonstrated by previous investigators [ 50 , 51 ]. As indicated in earlier reports [ 9 , 11 , 40 , 52 - 54 ] the hypotensive effect of reserpine observed in rats is due to the depletion of catecholamines from the peripheral stores.The effect of equimolar doses of RMN also indicated hypotension however, with higher doses compared to reserpine. It is further indicated that quaternization of reserpine not only restricted the entry of RMN to central nervous system but also reduced to the target tissue in the periphery. Hence relatively higher doses were required to produce reserpine like effect. Mechanistically, the hypotensive actions of RMN could also be due to peripheral depletion of catecholamines as evident from the positive correlation with the results of previous section on the peripheral depletion of monoamines.Conclusion In conclusion, the present study indicated that the quaternization of reserpine molecule prevents its access into the central nervous system and thereby produces selective peripheral depletion of biogenic amines. Furthermore, the study indicated that quaternization of reserpine had not abolished the hypotensive response but only higher doses were required.
Conclusion
In conclusion, the present study indicated that the quaternization of reserpine molecule prevents its access into the central nervous system and thereby produces selective peripheral depletion of biogenic amines. Furthermore, the study indicated that quaternization of reserpine had not abolished the hypotensive response but only higher doses were required.Methods Chemistry The synthesis of RMN was done as follows: The solution of reserpine (2 g, 3.3 mmols) in dichloromethane (20 ml) was added to methyl iodide (11 ml, 176 mmols) and the resulting mixture was kept for two days in dark. The solid was filtered and washed with a little cold dichloromethane and dried under vacuum at 70°C for 2 h to yield reserpine methiodide (RMI) [ 33 , 34 ]. Then, to a solution of RMI (0.25 gm, 0.67 mmols) in a mixture of dichloromethane (3 ml) and aqueous ethanol (90%, 2 ml) was added a solution of silver nitrate (56 mg, 0.67 mmols) in aqueous ethanol (90%, 2 ml). The reaction mixture was stirred overnight at room temperature. The solution was filtered and washed thoroughly with chloroform : methanol (1:1). The solid obtained after evaporation of the solvent was passed through a silica gel column and eluted with chloroform : methanol (80:20) to yield RMN, m.p. 292–294°C. Chemicals used Reserpine and thiopentone were generous gift samples from Novartis India Limited and Abbott Laboratories, Mumbai respectively. The standard samples of VMA, 5-HIAA, HVA and iso-VMA (internal standard) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals used were of HPLC or analytical grade as appropriate. The solutions of reserpine and RMN under study were prepared in DMSO and the volume of each dose was adjusted to 0.1 ml/100 gm body weight as suggested by Varma et al., [ 49 ]. The doses of RMN were calculated on equimolar basis of reserpine. Animal experiments Albino rats of either sex weighing between 100–150 gm (Charkaborty Enterprise, Kolkata) were used in the study. They were acclimatized to the laboratory conditions for at least 10 days prior to the experiment and were provided with standard diet and water  ad libitum  with 12 h light and dark cycle. The animal experiments conducted in this research work were approved by the Institutional Animal Ethics Committee and by the government regulatory body for animal research (Regd. No. 516/01/A/CPCSEA). Biochemical estimations Animals were divided into 4 groups of six each and were housed individually in metabolic cages. Funnels of suitable size were arranged at the bottom of the metabolic cages for collection of urine. Perforated plastic discs were arranged in the funnels to retain fecal matter. The animals were maintained at room temperature and acclimatized to metabolic cages for few days prior to drug administration. The treatment given to the groups of animals was as follows: Group 1: Control animals treated with DMSO intraperitoneally at a dose of 0.1 ml/100 gm body weight. Group 2: Animals administered intraperitoneally with reserpine at a dose of 5 mg/kg body weight. Group 3: Animals administered intraperitoneally with RMN at a dose equivalent to 5 mg/kg body weight of reserpine. Group 4: Animals administered intraperitoneally with RMN at a dose equivalent to 10 mg/kg body weight of reserpine. In each group, animals were placed individually in metabolic cages after drug administration and were allowed access to water. The 24 h urine samples from the point of drug administration was collected for each animal in a beaker containing 5 ml of 6  M  HCl arranged at the bottom of the funnel. The volumes of the 24 h urine samples collected in the beakers were noted individually and about 2 ml of urine (mixture) from each animal was taken into sample tubes and centrifuged at 3000 rpm for 10 minutes. The supernatants were transferred into another set of clean and dry tubes and stored at -20°C until analysis by HPLC. Simultaneous HPLC determination of VMA, 5-HIAA and HVA in urine The procedure described by Wako-chem. Co.,[ 55 ] was used for the simultaneous determination of the above metabolites. The urine samples were thawed before analysis. To 0.2 ml of each sample, 0.1 ml of internal standard (iso-VMA, 1000 ηg) and 0.7 ml of mobile phase were added. The solutions were mixed well and filtered through 0.4 μm membrane filter. The filtrate (20 μL) was injected into the column (RP C-18, 250 mm × 4.6 mm I.D; particle size 5 μm; YMC Inc., USA). The mobile phase (filtered through 0.4 μm membrane filter) comprised of 10:90 v/v of acetonitrile and 0.1  M  KH 2 PO 4  and the flow rate of the mobile phase was maintained at 0.8 ml/min, which yields a column back pressure of 220–230 kgf/cm 2 . Detection was done by UV absorption at 230 ηm. The range of the detector was set at 0.001 a.u.f.s. The peak area ratios of VMA, 5-HIAA and HVA to that of internal standard were calculated and substituted in the respective regression equations to estimate the amount of the metabolite present in the sample. Effect on normal blood pressure of anaesthetized rats The procedure described by Noble [ 56 ] was followed to evaluate the effect of RMN on normal blood pressure of anaesthetized rats in comparison with reserpine. Groups of rats of six each were anaesthetized with an intraperitoneal injection of thiopentone (40 mg/kg body weight). The femoral vein was cannulated for administration of supplementary doses of anaesthetic (if required) and drug solutions. Haemodynamic setup was used to record the blood pressure of rats. The blood pressure of each animal was recorded from left common carotid artery connected to a mercury manometer on kymograph paper. The normal blood pressure of rats was recorded after stabilization for 30 minutes. The different doses of reserpine (0.25, 0.50, 1, 5, 10 and 15 μg/kg body weight) or RMN (10, 25 and 50 μg/kg body weight) were studied in separate groups (n = 6) to determine the change in blood pressure response. Statistical analysis Data are expressed as mean ± standard error of means. Statistical analysis was done using one-way analysis of variance (ANOVA). Post-hoc comparisons were done by using Dunnet's  t -test. In all the cases, p < 0.05 was considered statistically significant.
Methods
Chemistry The synthesis of RMN was done as follows: The solution of reserpine (2 g, 3.3 mmols) in dichloromethane (20 ml) was added to methyl iodide (11 ml, 176 mmols) and the resulting mixture was kept for two days in dark. The solid was filtered and washed with a little cold dichloromethane and dried under vacuum at 70°C for 2 h to yield reserpine methiodide (RMI) [ 33 , 34 ]. Then, to a solution of RMI (0.25 gm, 0.67 mmols) in a mixture of dichloromethane (3 ml) and aqueous ethanol (90%, 2 ml) was added a solution of silver nitrate (56 mg, 0.67 mmols) in aqueous ethanol (90%, 2 ml). The reaction mixture was stirred overnight at room temperature. The solution was filtered and washed thoroughly with chloroform : methanol (1:1). The solid obtained after evaporation of the solvent was passed through a silica gel column and eluted with chloroform : methanol (80:20) to yield RMN, m.p. 292–294°C.
Chemistry
The synthesis of RMN was done as follows: The solution of reserpine (2 g, 3.3 mmols) in dichloromethane (20 ml) was added to methyl iodide (11 ml, 176 mmols) and the resulting mixture was kept for two days in dark. The solid was filtered and washed with a little cold dichloromethane and dried under vacuum at 70°C for 2 h to yield reserpine methiodide (RMI) [ 33 , 34 ]. Then, to a solution of RMI (0.25 gm, 0.67 mmols) in a mixture of dichloromethane (3 ml) and aqueous ethanol (90%, 2 ml) was added a solution of silver nitrate (56 mg, 0.67 mmols) in aqueous ethanol (90%, 2 ml). The reaction mixture was stirred overnight at room temperature. The solution was filtered and washed thoroughly with chloroform : methanol (1:1). The solid obtained after evaporation of the solvent was passed through a silica gel column and eluted with chloroform : methanol (80:20) to yield RMN, m.p. 292–294°C.Chemicals used Reserpine and thiopentone were generous gift samples from Novartis India Limited and Abbott Laboratories, Mumbai respectively. The standard samples of VMA, 5-HIAA, HVA and iso-VMA (internal standard) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals used were of HPLC or analytical grade as appropriate. The solutions of reserpine and RMN under study were prepared in DMSO and the volume of each dose was adjusted to 0.1 ml/100 gm body weight as suggested by Varma et al., [ 49 ]. The doses of RMN were calculated on equimolar basis of reserpine.
Chemicals used
Reserpine and thiopentone were generous gift samples from Novartis India Limited and Abbott Laboratories, Mumbai respectively. The standard samples of VMA, 5-HIAA, HVA and iso-VMA (internal standard) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals used were of HPLC or analytical grade as appropriate.The solutions of reserpine and RMN under study were prepared in DMSO and the volume of each dose was adjusted to 0.1 ml/100 gm body weight as suggested by Varma et al., [ 49 ]. The doses of RMN were calculated on equimolar basis of reserpine.Animal experiments Albino rats of either sex weighing between 100–150 gm (Charkaborty Enterprise, Kolkata) were used in the study. They were acclimatized to the laboratory conditions for at least 10 days prior to the experiment and were provided with standard diet and water  ad libitum  with 12 h light and dark cycle. The animal experiments conducted in this research work were approved by the Institutional Animal Ethics Committee and by the government regulatory body for animal research (Regd. No. 516/01/A/CPCSEA). Biochemical estimations Animals were divided into 4 groups of six each and were housed individually in metabolic cages. Funnels of suitable size were arranged at the bottom of the metabolic cages for collection of urine. Perforated plastic discs were arranged in the funnels to retain fecal matter. The animals were maintained at room temperature and acclimatized to metabolic cages for few days prior to drug administration. The treatment given to the groups of animals was as follows: Group 1: Control animals treated with DMSO intraperitoneally at a dose of 0.1 ml/100 gm body weight. Group 2: Animals administered intraperitoneally with reserpine at a dose of 5 mg/kg body weight. Group 3: Animals administered intraperitoneally with RMN at a dose equivalent to 5 mg/kg body weight of reserpine. Group 4: Animals administered intraperitoneally with RMN at a dose equivalent to 10 mg/kg body weight of reserpine. In each group, animals were placed individually in metabolic cages after drug administration and were allowed access to water. The 24 h urine samples from the point of drug administration was collected for each animal in a beaker containing 5 ml of 6  M  HCl arranged at the bottom of the funnel. The volumes of the 24 h urine samples collected in the beakers were noted individually and about 2 ml of urine (mixture) from each animal was taken into sample tubes and centrifuged at 3000 rpm for 10 minutes. The supernatants were transferred into another set of clean and dry tubes and stored at -20°C until analysis by HPLC. Simultaneous HPLC determination of VMA, 5-HIAA and HVA in urine The procedure described by Wako-chem. Co.,[ 55 ] was used for the simultaneous determination of the above metabolites. The urine samples were thawed before analysis. To 0.2 ml of each sample, 0.1 ml of internal standard (iso-VMA, 1000 ηg) and 0.7 ml of mobile phase were added. The solutions were mixed well and filtered through 0.4 μm membrane filter. The filtrate (20 μL) was injected into the column (RP C-18, 250 mm × 4.6 mm I.D; particle size 5 μm; YMC Inc., USA). The mobile phase (filtered through 0.4 μm membrane filter) comprised of 10:90 v/v of acetonitrile and 0.1  M  KH 2 PO 4  and the flow rate of the mobile phase was maintained at 0.8 ml/min, which yields a column back pressure of 220–230 kgf/cm 2 . Detection was done by UV absorption at 230 ηm. The range of the detector was set at 0.001 a.u.f.s. The peak area ratios of VMA, 5-HIAA and HVA to that of internal standard were calculated and substituted in the respective regression equations to estimate the amount of the metabolite present in the sample. Effect on normal blood pressure of anaesthetized rats The procedure described by Noble [ 56 ] was followed to evaluate the effect of RMN on normal blood pressure of anaesthetized rats in comparison with reserpine. Groups of rats of six each were anaesthetized with an intraperitoneal injection of thiopentone (40 mg/kg body weight). The femoral vein was cannulated for administration of supplementary doses of anaesthetic (if required) and drug solutions. Haemodynamic setup was used to record the blood pressure of rats. The blood pressure of each animal was recorded from left common carotid artery connected to a mercury manometer on kymograph paper. The normal blood pressure of rats was recorded after stabilization for 30 minutes. The different doses of reserpine (0.25, 0.50, 1, 5, 10 and 15 μg/kg body weight) or RMN (10, 25 and 50 μg/kg body weight) were studied in separate groups (n = 6) to determine the change in blood pressure response.
Animal experiments
Albino rats of either sex weighing between 100–150 gm (Charkaborty Enterprise, Kolkata) were used in the study. They were acclimatized to the laboratory conditions for at least 10 days prior to the experiment and were provided with standard diet and water  ad libitum  with 12 h light and dark cycle. The animal experiments conducted in this research work were approved by the Institutional Animal Ethics Committee and by the government regulatory body for animal research (Regd. No. 516/01/A/CPCSEA).ad libitumBiochemical estimations Animals were divided into 4 groups of six each and were housed individually in metabolic cages. Funnels of suitable size were arranged at the bottom of the metabolic cages for collection of urine. Perforated plastic discs were arranged in the funnels to retain fecal matter. The animals were maintained at room temperature and acclimatized to metabolic cages for few days prior to drug administration. The treatment given to the groups of animals was as follows: Group 1: Control animals treated with DMSO intraperitoneally at a dose of 0.1 ml/100 gm body weight. Group 2: Animals administered intraperitoneally with reserpine at a dose of 5 mg/kg body weight. Group 3: Animals administered intraperitoneally with RMN at a dose equivalent to 5 mg/kg body weight of reserpine. Group 4: Animals administered intraperitoneally with RMN at a dose equivalent to 10 mg/kg body weight of reserpine. In each group, animals were placed individually in metabolic cages after drug administration and were allowed access to water. The 24 h urine samples from the point of drug administration was collected for each animal in a beaker containing 5 ml of 6  M  HCl arranged at the bottom of the funnel. The volumes of the 24 h urine samples collected in the beakers were noted individually and about 2 ml of urine (mixture) from each animal was taken into sample tubes and centrifuged at 3000 rpm for 10 minutes. The supernatants were transferred into another set of clean and dry tubes and stored at -20°C until analysis by HPLC.
Biochemical estimations
Animals were divided into 4 groups of six each and were housed individually in metabolic cages. Funnels of suitable size were arranged at the bottom of the metabolic cages for collection of urine. Perforated plastic discs were arranged in the funnels to retain fecal matter. The animals were maintained at room temperature and acclimatized to metabolic cages for few days prior to drug administration.The treatment given to the groups of animals was as follows:Group 1: Control animals treated with DMSO intraperitoneally at a dose of 0.1 ml/100 gm body weight.Group 2: Animals administered intraperitoneally with reserpine at a dose of 5 mg/kg body weight.Group 3: Animals administered intraperitoneally with RMN at a dose equivalent to 5 mg/kg body weight of reserpine.Group 4: Animals administered intraperitoneally with RMN at a dose equivalent to 10 mg/kg body weight of reserpine.In each group, animals were placed individually in metabolic cages after drug administration and were allowed access to water. The 24 h urine samples from the point of drug administration was collected for each animal in a beaker containing 5 ml of 6  M  HCl arranged at the bottom of the funnel. The volumes of the 24 h urine samples collected in the beakers were noted individually and about 2 ml of urine (mixture) from each animal was taken into sample tubes and centrifuged at 3000 rpm for 10 minutes. The supernatants were transferred into another set of clean and dry tubes and stored at -20°C until analysis by HPLC.MSimultaneous HPLC determination of VMA, 5-HIAA and HVA in urine The procedure described by Wako-chem. Co.,[ 55 ] was used for the simultaneous determination of the above metabolites. The urine samples were thawed before analysis. To 0.2 ml of each sample, 0.1 ml of internal standard (iso-VMA, 1000 ηg) and 0.7 ml of mobile phase were added. The solutions were mixed well and filtered through 0.4 μm membrane filter. The filtrate (20 μL) was injected into the column (RP C-18, 250 mm × 4.6 mm I.D; particle size 5 μm; YMC Inc., USA). The mobile phase (filtered through 0.4 μm membrane filter) comprised of 10:90 v/v of acetonitrile and 0.1  M  KH 2 PO 4  and the flow rate of the mobile phase was maintained at 0.8 ml/min, which yields a column back pressure of 220–230 kgf/cm 2 . Detection was done by UV absorption at 230 ηm. The range of the detector was set at 0.001 a.u.f.s. The peak area ratios of VMA, 5-HIAA and HVA to that of internal standard were calculated and substituted in the respective regression equations to estimate the amount of the metabolite present in the sample.
Simultaneous HPLC determination of VMA, 5-HIAA and HVA in urine
The procedure described by Wako-chem. Co.,[ 55 ] was used for the simultaneous determination of the above metabolites. The urine samples were thawed before analysis. To 0.2 ml of each sample, 0.1 ml of internal standard (iso-VMA, 1000 ηg) and 0.7 ml of mobile phase were added. The solutions were mixed well and filtered through 0.4 μm membrane filter. The filtrate (20 μL) was injected into the column (RP C-18, 250 mm × 4.6 mm I.D; particle size 5 μm; YMC Inc., USA). The mobile phase (filtered through 0.4 μm membrane filter) comprised of 10:90 v/v of acetonitrile and 0.1  M  KH 2 PO 4  and the flow rate of the mobile phase was maintained at 0.8 ml/min, which yields a column back pressure of 220–230 kgf/cm 2 . Detection was done by UV absorption at 230 ηm. The range of the detector was set at 0.001 a.u.f.s. The peak area ratios of VMA, 5-HIAA and HVA to that of internal standard were calculated and substituted in the respective regression equations to estimate the amount of the metabolite present in the sample.MEffect on normal blood pressure of anaesthetized rats The procedure described by Noble [ 56 ] was followed to evaluate the effect of RMN on normal blood pressure of anaesthetized rats in comparison with reserpine. Groups of rats of six each were anaesthetized with an intraperitoneal injection of thiopentone (40 mg/kg body weight). The femoral vein was cannulated for administration of supplementary doses of anaesthetic (if required) and drug solutions. Haemodynamic setup was used to record the blood pressure of rats. The blood pressure of each animal was recorded from left common carotid artery connected to a mercury manometer on kymograph paper. The normal blood pressure of rats was recorded after stabilization for 30 minutes. The different doses of reserpine (0.25, 0.50, 1, 5, 10 and 15 μg/kg body weight) or RMN (10, 25 and 50 μg/kg body weight) were studied in separate groups (n = 6) to determine the change in blood pressure response.
Effect on normal blood pressure of anaesthetized rats
The procedure described by Noble [ 56 ] was followed to evaluate the effect of RMN on normal blood pressure of anaesthetized rats in comparison with reserpine. Groups of rats of six each were anaesthetized with an intraperitoneal injection of thiopentone (40 mg/kg body weight). The femoral vein was cannulated for administration of supplementary doses of anaesthetic (if required) and drug solutions.Haemodynamic setup was used to record the blood pressure of rats. The blood pressure of each animal was recorded from left common carotid artery connected to a mercury manometer on kymograph paper. The normal blood pressure of rats was recorded after stabilization for 30 minutes. The different doses of reserpine (0.25, 0.50, 1, 5, 10 and 15 μg/kg body weight) or RMN (10, 25 and 50 μg/kg body weight) were studied in separate groups (n = 6) to determine the change in blood pressure response.Statistical analysis Data are expressed as mean ± standard error of means. Statistical analysis was done using one-way analysis of variance (ANOVA). Post-hoc comparisons were done by using Dunnet's  t -test. In all the cases, p < 0.05 was considered statistically significant.
Statistical analysis
Data are expressed as mean ± standard error of means. Statistical analysis was done using one-way analysis of variance (ANOVA). Post-hoc comparisons were done by using Dunnet's  t -test. In all the cases, p < 0.05 was considered statistically significant.tAbreviations RMN: Reserpine methonitrate VMA: Vanillylmandelic acid 5-HIAA: 5-Hydroxyindoleacetic acid HVA: Homovanillic acid DMSO: Dimethyl sulfoxide
Abreviations
RMN: Reserpine methonitrateVMA: Vanillylmandelic acid5-HIAA: 5-Hydroxyindoleacetic acidHVA: Homovanillic acidDMSO: Dimethyl sulfoxideAuthors' contributions SN made significant contribution in designing the studies, conducting the experiments, interpretation of the data, conceptualization of statistical analyses and drafting the final manuscript. KMB assisted in experimental work, data analysis and writing of the manuscript. SS conceived the study, made substantial contributions in data analysis, data interpretation, writing of the manuscript and in coordination of the experiments. All authors read and approved the final manuscript.
Authors' contributions
SN made significant contribution in designing the studies, conducting the experiments, interpretation of the data, conceptualization of statistical analyses and drafting the final manuscript. KMB assisted in experimental work, data analysis and writing of the manuscript. SS conceived the study, made substantial contributions in data analysis, data interpretation, writing of the manuscript and in coordination of the experiments. All authors read and approved the final manuscript.