Pharmacological Sanctuary Research Articles

Le tractus génital masculin

Despite semen being the main vector of human immunodeficiency virus (HIV) dissemination worldwide, the origin of the virus in this bodily fluid remains unknown. Of particular significance is the persistence of virus release in the semen of a subset of HIV-infected men under antiretroviral therapy, who otherwise show an undetectable blood viral load. It is therefore considered critical to identify the sources of virus shedding in semen for the more efficient control of HIV transmission. Our recent findings indicate HIV infection of several semen-producing organs, including the testis (which represents a pharmacological sanctuary for several antiretroviral drugs). This reinforces phylogenetic observations suggesting that the free viral particles and infected cells contaminating semen are produced within the male genital tract. The fact that HIV replicates within the male genital organs raises several questions: Is one or several of the male genital tract organs responsible for the persistence of HIV in semen despite efficient antiviral therapies? What is the nature of HIV interactions with spermatozoa and testicular germ cells? Recent results established that semen from HIV negative men modifies HIV infectivity: does the seminal fluid from HIV+ men enhance or inhibit the efficiency of HIV sexual transmission?

Identification of a Potential Pharmacological Sanctuary for HIV Type 1 in a Fraction of CD4+Primary Cells

We have identified a subset of HIV-susceptible CD4(+)CCR5(+) cells in human PBMCs that can efficiently exclude protease inhibitors (PI) due to high P-glycoprotein (P-gp) efflux activity. Phenotypically these cells are heterogeneous, include both T and non-T cells, and some display markers of memory cells. Cells with high P-gp represent 16-56% (median = 37.3) of all CD4(+)CCR5(+) cells in healthy donors, and are selectively depleted in HIV-1-infected individuals (4.1-33%, median = 10.1). A fraction of primary cells productively infected by HIV-1, in vitro, have high P-gp pump activity, demonstrating that infection does not inhibit P-gp function. In agreement with these data, HIV-susceptible cells expressing high levels of P-gp require higher levels of PI for complete inhibition of virus spread. We conclude that the PI concentrations achieved in plasma could be suboptimal for full inhibition of virus spread in high P-gp cells, indicating that they may represent a pharmacological sanctuary for HIV-1.

Le tractus génital masculin : un hôte pour le VIH

Despite semen being the main vector of human immunodeficiency virus (HIV) dissemination worldwide, the origin of the virus in this bodily fluid remains unknown. Of particular significance is the persistence of virus release in the semen of HIV-infected men under antiretroviral therapy, who otherwise show an undetectable blood viral load. It is therefore considered critical to identify the sources of virus shedding in semen for the more efficient control of HIV transmission. A number of studies indirectly suggest that the free viral particles and infected cells contaminating semen are produced within the male genital tract. Our recent findings indicate HIV infection of several semen-producing organs, including the testis (which represents a pharmacological sanctuary for several antiretroviral drugs), thus reinforcing the hypothesis of the local origin of the seminal contamination. Whether one or several of these organs constitute a viral reservoir seeding semen despite antiviral therapies, remains to be determined. In addition, the detection of virus within the testicular germ cells should be taken into account in the context of assisted reproductive techniques using these cells from HIV positive men.

P-Glycoprotein and Breast Cancer Resistance Protein: Two Dominant Transporters Working Together in Limiting the Brain Penetration of Topotecan

The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(-/-) and Bcrp1(-/-), and compound Mdr1a/b(-/-)Bcrp1(-/-) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(-/-) and Bcrp1(-/-) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(-/-)Bcrp1(-/-) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was approximately 0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice, respectively, resulting in 2.0-fold (P < 0.01), 0.65-fold (P, not significant), and 3.2-fold (P < 0.01), respectively, higher brain-to-plasma AUC ratios. Results using Mrp4(-/-) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp-mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. Our results using Mdr1a/b(-/-)Bcrp1(-/-) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.

Effect of Transporter Modulation on the Emergence of Nelfinavir Resistance In Vitro

Background HIV drug resistance is of increasing concern and could result from inadequate drug potency, poor therapy adherence and the existence of pharmacological sanctuary sites for viral replication. One contributing factor to the generation of such sites could be drug efflux transporters, which have been shown capable of effluxing HIV protease inhibitors from cells. Methods In this ‘proof-of-concept’ study, the ability of the efflux transport inhibitor verapamil to modulate the intracellular accumulation of radiolabeled nelfinavir (NFV) and the antiviral effect of NFV was assessed in MT4 cells. Wild-type virus was then serially passaged with increasing concentrations of NFV with and without verapamil and resistance mutations monitored by sequencing of the viral protease gene. Results The cellular accumulation ratio of 3H-NFV was 116.8 ±9.7 in controls and was significantly increased to 149.8 ±24.5 following incubation with verapamil (P&lt;0.05, n=4). The EC50 of NFV was decreased in MT4 cells in the presence of verapamil from 8.5 ±1.3 nM to 4.4 ±0.8 nM ( P&lt;0.001, n=6). Of the 24 isolates passaged without verapamil, 21 carried the D30N mutation at detectable levels. Of the 23 passaged with verapamil, 14 carried the mutation (odds ratio=4.5; P&lt;0.05). Conclusions These results suggest that ‘intracellular boosting’ of PIs is achievable through inhibition of drug efflux proteins in vitro and that such boosting has the ability to enhance suppression of viral replication, slowing the emergence of resistance mutations.

Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein

If tuberculosis therapy is to be shortened it is imperative that the sterilising activity of current and future anti-tuberculosis drugs is enhanced. Intracellular Mycobacterium tuberculosis (MTB) phagocytosed by macrophages may be a key subpopulation of bacteria that are less readily eliminated by therapy. Here we investigate whether macrophages provide MTB with a pharmacological sanctuary site, making them less susceptible to chemotherapy than extracellular bacilli. Intracellular drug activity was determined by a novel colorimetric method that measures the ability of a drug to protect A-THP1 cells from infection-mediated cell death by H37Rv. Extracellular bactericidal activity was determined by the microplate alamar blue assay (MABA). Further, the effect of P-glycoprotein (P-gp) expressed on macrophages on the intracellular kill of H37Rv was assessed. To screen the anti-tuberculosis drugs for P-gp substrate specificity, their toxicity and cellular accumulation were determined in CEM and CEM(VBL100) cells. Intracellular and extracellular anti-tuberculosis drug activity following 7-day treatment with isoniazid (mean EC(50)+/-SD: 36.7+/-2.2 and 57.2+/-2.5 ng/mL, respectively) and ethambutol (243+/-95 and 263+/-12 ng/mL, respectively) were similar. However, for rifampicin a higher concentration was required to kill intracellular (148+/-32 ng/mL) versus extracellular (1.27+/-0.02 ng/mL) bacilli. The P-gp inhibitor tariquidar, significantly increased intracellular kill of H37Rv by ethambutol and rifampicin and both of these drugs were shown to be substrates for P-gp using the P-gp overexpressing CEM(VBL100) cells. We observed a large discrepancy between intracellular and extracellular activity of rifampicin (but not with isoniazid or ethambutol). Several factors could have accounted for this including inoculum size, media and cell-mediated metabolism. These factors make the comparison of intracellular and extracellular drug activity complex. However, the intracellular assay described here has potential for studying the impact of host proteins (such as drug transporters) on the intracellular activity of drugs, and has been used successfully here to demonstrate that both rifampicin and ethambutol are substrates for P-gp.

Susceptibility of Human Testis to Human Immunodeficiency Virus-1 Infection in Situ and in Vitro

Semen represents the main vector for human immunodeficiency virus (HIV) dissemination worldwide and has been shown to harbor replication-competent virus despite otherwise effective highly active anti-retroviral therapy, which achieves undetectable viral load in plasma. Despite this, the origin of seminal HIV particles remains unclear, as does the question of whether the male genital tract organs contribute virus to semen. Here we investigated the presence of HIV receptors within the human testis using immunohistochemistry and quantitative real-time polymerase chain reaction. We also analyzed the infectivity of a dual tropic HIV-1 strain in an organotypic culture, as well as the impact of viral exposure on testosterone production. Our study establishes that CXCR4+, CCR5+, CD4+, and DC-SIGN+ cells are present within the interstitial tissue of human testis and that these molecules persist throughout our organotypic culture. Our data also reveal that the human testis is permissive to HIV-1 and supports productive infection, leaving testosterone production apparently unaffected. Infected cells appeared to be testicular macrophages located within the interstitial tissue. That the testis itself represents a potential source of virus in semen could play a role in preventing viral eradication from semen because this organ constitutes a pharmacological sanctuary for many current antiretrovirals.

Estimating seminal vesicle (SV) and prostate (PR) gland concentrations of drugs using linear regression of gland-specific biochemical markers in split ejaculate fractions

Background The male genital tract (MGT) has been implicated as a pharmacologic sanctuary for HIV due to the potential for differential penetration of antiretroviral drugs into the varied environments in each MGT gland. Fructose and prostate specific antigen (PSA) are unique to the SV and PR, respectively, the two largest MGT glands. Using the heterogeneity of these markers in incompletely mixed fractions of the ejaculate, we propose to estimate the SV and PR concentrations of fructose and PSA. This will enable drug measurement in the SV and PR based on drug concentrations in each fraction of a single ejaculate. Methods 12 healthy volunteers submitted a single ejaculate collected in 3 to 5 fractions in a multi-chamber tray. Each fraction was assayed for PSA and fructose. We calculated fructose and PSA concentrations in the SV and PR using linear regression of fructose on PSA where the axis intercepts estimate concentrations of fructose in the SV and PSA in the PR. Results All subjects provided 3 to 5 fractions of ejaculate. We observed a linear relationship (R2 >0.7) between PSA and fructose in 10 of 12 subjects. In these, the median fructose concentration in the SV was 16 mM (IQR 14 - 22) and median PSA concentration in the PR was 95× 105 ng/mL (IQR 48 - 125). The 2 non-linear subjects had the lowest fructose fraction heterogeneity. Conclusions We estimated the level of PSA and fructose in the SV and PR using a split ejaculate-linear regression method, thus enabling non-invasive measurement of drug levels in these glands. Clinical Pharmacology & Therapeutics (2005) 77, P20–P20; doi: 10.1016/j.clpt.2004.11.078

Sequence heterogeneity and viral dynamics in cerebrospinal fluid and plasma during antiretroviral therapy.

Understanding of human immunodeficiency virus (HIV) neuropathogenesis has been greatly enhanced by careful analyses of postmortem brain tissue, as well as by primate models of infection with related lentiviruses. However, brain tissue from persons living with HIV infection is rarely available for study, and elegant observations from primate systems may require confirmation in humans. Because of these inherent limitations, it is important to learn as much as possible by studying cerebrospinal fluid (CSF) during HIV infection. The present discussion considers selected issues relevant to the study of CSF from HIV-infected individuals. These include a strategy to intensely sample CSF to better understand viral dynamics and the role of the brain as a pharmacologic sanctuary site, evidence for HIV sequence diversity in CSF and plasma during HIV therapy and the implications of such diversity, and the importance of host genetics relevant to studying HIV neuropathogenesis.

Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview

Active drug efflux transporters of the ATP binding cassette (ABC)-containing family of proteins have a major impact on the pharmacological behavior of most of the drugs in use today. Pharmacological properties affected by ABC transporters include the oral bioavailability, hepatobiliary, direct intestinal, and urinary excretion of drugs and drug-metabolites and -conjugates. Moreover, the penetration of drugs into a range of important pharmacological sanctuaries, such as brain, testis, and fetus, and the penetration into specific cell- and tissue compartments can be extensively limited by ABC transporters. These interactions with ABC transporters determine to a large extent the clinical usefulness, side effects and toxicity risks of drugs. Many other xenotoxins, (pre-)carcinogens and endogenous compounds are also influenced by the ABC transporters, with corresponding consequences for the well-being of the individual. We aim to provide an overview of properties of the mammalian ABC transporters known to mediate significant transport of clinically relevant drugs.

Human Leydig cells are productively infected by some HIV-2 and SIV strains but not by HIV-1.

With the use of highly active antiretroviral therapy, the identification of HIV reservoirs within the body has become an important issue. However, the testis has been largely ignored despite representing a pharmacologic sanctuary which could act as a viral reservoir. Because alterations in testosterone production have frequently been reported in HIV-infected individuals, we investigated whether the testosterone-producing Leydig cells could become directly infected by HIV-1, HIV-2 or SIV. Purified Leydig cells were infected with a panel of HIV-1, HIV-2 and SIV strains and examined for expression of HIV/SIV receptors. Additionally, the impact of CD4 transduction on Leydig cell infection was determined. Leydig cells were unable to support productive infection of the seven HIV-1 isolates tested. No CD4, CXCR4 or CCR5 expression was evident on the surface of Leydig cells and transduction with a CD4 expressing adenovirus did not induce HIV-1 infection. In contrast, some primary and laboratory adapted CD4-independent HIV-2 and SIV strains were able to enter and replicate productively in Leydig cells. Our results suggest that Leydig cells do not represent a target for HIV-1 infection within the testis. In contrast, Leydig cells support HIV-2 and SIV infection and thus represent a potential target for infection. Receptor use and significance of HIV-2/SIV infection of Leydig cells remain to be determined.

Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs.

Various drug transporters of the ATP-binding cassette (ABC) family restrict the oral bioavailability and cellular, brain, testis, cerebrospinal fluid and fetal penetration of substrate drugs. MDRI P-glycoprotein (P-gp) has been demonstrated to transport most HIV protease inhibitors (HPI) and to reduce their oral bioavailability and lymphocyte, brain, testis and fetal penetration, possibly resulting in major limiting effects on the therapeutic efficacy of these drugs. To investigate whether the ABC transporters MRP1, MRP2, MRP3, MRP5 and breast cancer resistance protein 1 (Bcrp1) are efficient transporters of the HPI saquinavir, ritonavir and indinavir. Polarized epithelial non-human (canine) cell lines transduced with human or murine complementary DNA (cDNA) for each of the transporters were used to study transepithelial transport of the HPI. MRP2 efficiently transported saquinavir, ritonavir and indinavir and this transport could be enhanced by probenecid. Sulfinpyrazone was also able to enhance MRP2-mediated saquinavir transport. In contrast, MRP1, MRP3, MRP5, or Bcrp1 did not efficiently transport the HPI tested. Human MRP2 actively transports several HPI and could, based on its known and assumed tissue distribution, therefore reduce HPI oral bioavailability. It may also limit brain and fetal penetration of these drugs and increase their hepatobiliary, intestinal and renal clearance. MRP2 function and enhancement of its activity could adversely affect the therapeutic efficacy, including the pharmacological sanctuary penetration, of HPI. In vivo inhibition of MRP2 function might, therefore, improve HIV/AIDS therapy.

Successful Laparoscopic Removal of a Solitary Adrenal Metastasis from Ovarian Carcinoma: A Case Report

Background. The adrenal gland is a site of metastatic spread of some tumors. Usually the presence of adrenal metastasis signals widespread disease; however, isolated adrenal metastases have been reported. The adrenal gland, along with certain other organs, is considered a pharmacological sanctuary. Several reports describe long-term survival after adrenalectomy due to isolated adrenal metastases, in particular from non-small-cell lung cancer. There are several reports of laparoscopic resection of isolated adrenal metastases.Case. We present a rare case of a laparoscopically resected solitary adrenal metastasis originating from ovarian carcinoma. To the best of our knowledge there was no report in the literature of an isolated adrenal metastasis from ovarian carcinoma. The patient described developed an isolated left adrenal metastasis 11 months after completion of adjuvant chemotherapy for stage III ovarian cancer. The serum CA125 dropped from 365 to 35 postoperation.Conclusion. Isolated adrenal metastasis from ovarian cancer is a rare event, never before described in the literature. This case report describes a laparoscopic resection of an adrenal metastasis from ovarian cancer, and the drop of CA125 levels indicated complete resection of the metastasis.

P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir.

The low oral bioavailability of the HIV protease inhibitor (HPI) saquinavir is dramatically increased by coadministration of the HPI ritonavir. Because saquinavir and ritonavir are substrates and inhibitors of both the drug transporter P-glycoprotein (P-gp) and of the metabolizing enzyme CYP3A4, we wanted to sort out whether the ritonavir effect is primarily mediated by inhibition of CYP3A4 or P-gp or both. P-gp is known to limit the bioavailability, brain, testis, and fetal penetration of its substrates, so effective inhibition of P-gp by ritonavir in vivo might open up pharmacological sanctuary sites for saquinavir, with the potential of beneficial effects on therapy, but also of increased toxicity. In vitro, P-gp-mediated transport of saquinavir and ritonavir was only moderately inhibited by both HPIs compared with the potent P-gp inhibitor PSC833. When [(14)C]saquinavir was orally coadministered with a maximum tolerated dose of ritonavir to wild-type and P-gp-deficient mice, saquinavir bioavailability was dramatically increased in both strains, but P-gp still limited the oral bioavailability of saquinavir, and its penetration into brain and fetus. These data indicate that in vivo, ritonavir is a relatively poor P-gp inhibitor. The highly increased bioavailability of saquinavir because of ritonavir coadministration most likely results from reduced saquinavir metabolism. Importantly, our data indicate that it is unlikely that ritonavir coadministration will substantially affect the contribution of P-gp to pharmacological sanctuary sites such as brain, testis, and fetus. Thus, if one wanted to effectively open these sites for therapeutic purposes, more efficient P-gp inhibitors should be applied.

Demographics of Brain Metastasis

Brain metastases are the most common intracranial tumor, significantly out-numbering primary brain tumors. The apparent increase in the ratio of brain metastases to primary tumors may be the result of a number of factors, including the possibility of a CNS "pharmacologic sanctuary," an aging population, and improved imaging studies. Among adults, the most common origins of brain metastasis include primary tumors of the lung, breast, skin (melanoma), and gastrointestinal tract. Among patients under 21 years of age, brain metastases most often arise from the sarcomas and germ cell tumors.

A critique of the role of the blood-brain barrier in the chemotherapy of human brain tumors

There is general agreement that most chemotherapy agents achieve only relatively low concentrations in the normal central nervous system, that the blood-brain barrier is variably disrupted in malignant brain tumors, and that the concentration of chemotherapy drugs in the brain adjacent to tumor is intermediate between concentrations achieved in brain tumors vs normal brain. However, there is substantial controversy regarding the role of the blood-brain barrier in resistance to chemotherapy of intracerebral tumors. Many chemotherapy agents achieve concentrations in brain tumors that are comparable to those in extracerebral tumors, and drugs that cross the intact blood-brain barrier only poorly may be active against intracerebral tumors. Furthermore, the hypothesis that the brain is a pharmacological sanctuary where metastases may grow while tumor is responding in other parts of the body may be flawed: there are only 2 or 3 types of malignancies (out of all those that are sensitive to chemotherapy) in which the risk of isolated central nervous system relapse is moderately high, and even in these 2 or 3, effective central nervous system prophylaxis has minimal or no impact on overall survival. Furthermore, drugs that cross the BBB do not appear to be more effective than other drugs at reducing the risk of brain metastases, and brain metastases at the time of diagnosis do not necessarily convey a worse prognosis than metastases to various other sites. While average drug concentrations in brain adjacent to tumor are lower than those within brain tumors, very small numbers of tumor cells may be capable of inducing local leakiness in blood vessels, and there is little information on drug concentrations achieved in individual tumor cells within the brain adjacent to tumor. Furthermore, any limitation of uptake of drugs into brain tumors could be at least partially due to increased tissue pressure within tumors rather than being due to blood-brain barrier phenomena. This distinction could be important, since strategies that one might use to increase drug delivery to brain tumors might differ depending on whether the reduced delivery were due to barrier phenomena vs blood flow phenomena. The role of the blood-brain barrier in resistance of intracerebral tumors to chemotherapy remains unclear: while it may well play some role (and perhaps even a major one), self-fulfilling prophecies and unintentional bias in data selection and interpretation may have previously made it appear more important than it actually is.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Model Challenges Breast Cancer Treatment Strategy

The breast cancer treatment failure rate remains unacceptably high. The current breast cancer treatment paradigm, based primarily on Gompertzian kinetics and animal models, advocates short-course, intensive chemotherapy subsequent to tumor debulking, citing drug resistance and host toxicity as the primary reasons for treatment failure. To better understand treatment failure, we have studied breast cancer from the perspective of computer modeling. Our results demonstrate breast cancers grow in an irregular fashion; this differs from the Gompertzian mode of animal models and thus challenges the validity of the current paradigm. Clinical and laboratory data support the concept of irregular growth rather than the common claim that human tumors grow in a Gompertzian fashion. Treatment failure mechanisms for breast cancer appear to differ from those for animal models, and thus treatments optimize on animal models may not be optimal for breast cancer. A failure mechanism consistent with our results involves temporarily dormant tumor cells in anatomical or pharmacological sanctuary, which eventually result in aggressive metastatic disease.

The role of prophylactic cranial irradiation in the management of small cell lung cancer

Those patients who benefit most in terms of increased survival from the current treatment of small cell lung cancer (SCLC) are also those who run the greatest risk of developing brain metastases. In a compilation of 11 studies with 1688 patients, Pedersen (1) calculated that about 10% had central nervous system (CNS) metastases at diagnosis of SCLC, while a further 20% (range 7-30%) developed CNS metastases during therapy. At autopsy the frequency was found to be about 50% (1, 2). An increase in frequency of brain metastases in SCLC with increased survival was predicted as early as 1973 (3), and the prediction was confirmed in 1979 by Nugent et al. (4), who demonstrated an actuarial probability of developing brain metastases of 80% 2 years after diagnosis. Similar tendencies were subsequently demonstrated by others (5), and in a series from the Finsen Institute in Copenhagen the cumulative risk of brain metastases reached 50% at 2 years survival (1). An early and widely disseminated hypothesis explaining the increase in CNS metastases in patients with prolonged survival describe the brain as a pharmacologic sanctuary, where metastases can grow shielded from cytostatic agents by the blood-brain-barrier (BBB). This hypothesis has to some degree been corroborated by the clinical experience with childhood leukemia, where prophylactic cranial irradiation (PCI) is an integrated part of most treatment programmes. When CNS metastases are diagnosed by intravenous iodine contrast-enhanced CT-scan, the BBB is not intact in the tumor areas, and such areas therefore should be accessible to drugs according to the same principles that govern extracranial metastases. During the last decade, several reports about the effect of systemic chemotherapy on brain metastases from SCLC have been published, see (6, 7) for a review. Thus, the concept of the brain as a pharmacologic sanctuary site for metastases is inconsistent with newer clinical observations. In spite of the fact that clinical brain metastasis is an ominous prognostic indicator, it is disputable what influence the presence of brain metastases per se, if actively

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues.

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.

Central Nervous System Pharmacology of Antileukemic Drugs

The blood-brain barrier provides a pharmacologic sanctuary for leukemic cells within the central nervous system (CNS), protecting them from the cytotoxic effects of systemic antileukemic therapy. Attempts to overcome this problem have included specific CNS-directed treatment in the form of direct intrathecal drug injection, cranial irradiation, and alteration in the dose and schedule of systemic agents to enhance their CNS penetration. Use of these treatments and strategies has led to the effective prevention and control of meningeal leukemia. Intrathecal therapy, primarily with methotrexate or cytosine arabinoside, is a form of regional chemotherapy that can achieve very high drug concentrations at the target site [i.e., in the meninges and cerebrospinal fluid (CSF)] with a low total dose. Therefore, there is minimal systemic toxicity. The dose and schedules, clinical pharmacology, and toxicities of the commonly used intrathecal agents are discussed in detail in this article. Another approach to overcoming the limited penetration of antileukemic drugs into the CNS has been the use of high-dose systemic therapy. Methotrexate and cytosine arabinoside in high doses have produced favorable clinical responses in patients with overt meningeal disease, and pharmacokinetic studies have documented cytotoxic concentrations of these drugs within the cerebrospinal fluid. A clear understanding of the CNS pharmacology of the antileukemic drugs is required in order to use these agents in the safest and most efficacious manner for the treatment of meningeal leukemia.